Sound-based amplification of microfluidics

Abstract

Systems, apparatus, and techniques have been developed that utilize sound-based microfluidic amplification for the detection of biological substances such as misfolded proteins associated with diseases of incorrect protein folding. In one embodiment, the system has a housing that defines a main channel configured to contain a fluid solution containing a sample comprising multiple proteins, and one or more side channels that are in fluid communication with the main channel, each of the one or more side channels configured to establish a liquid-gas interface between the fluid solution in the main channel and the air contained in the one or more side channels. The system may also have at least one sound-generating element configured to generate sound waves in the air of the one or more side channels, causing vibrations at the liquid-gas interface and mixing of the fluid solution in the main channel.

Description

[Technical Field]

[0001] This application is an international patent application claiming priority and benefit to U.S. Provisional Patent Application No. 63 / 497,907, filed on 24 April 2023, the entire contents of which are incorporated herein by reference.

[0002] This disclosure relates to a fluid device, and more specifically to a microfluidic device for sound-based amplification of biological materials. [Background technology]

[0003] Prion diseases (PDs) are a type of deadly neurodegenerative disease collectively known as infectious spongiform encephalopathy (TSEs), and exist in several forms, including Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, and chronic wasting disease (CWD) in deer. These diseases are caused by cellular prion proteins (PrP C Formation of abnormally folded protease resistance (PrP Sc This is caused by the accumulation of ) in the brain. Once formed, PrP Sc PrP C Combined, incorrect folding and further PrP Sc It can act as a seed that triggers the formation of PrP, ultimately leading to aggregation into subsequent fibers and plaques. After initiation, aggregated PrP Sc It spreads throughout the central nervous system, damaging tissues and ultimately leading to death. PD has a long incubation period of at least 16 months before infected animals show signs of infection. However, throughout this incubation period, PD-infected individuals can spread PrP within the population through both direct and indirect contact. ScCWD has spread throughout North America since its first reported case in 1967, infecting both captive and free-ranging deer populations in the United States and Canada. Currently, spontaneous cases of CWD are confirmed in Scandinavia, and the disease was introduced to South Korea in 1997 through the shipment of infected moose from Canada. Animals infected with CWD release infectious prions through saliva, urine, and feces, which spread rapidly among deer and can potentially spread to other animals. [Overview of the Initiative]

[0004] This disclosure describes systems, apparatus, and techniques configured to utilize sound-based microfluidic amplification for the detection of biological substances, such as misfolded proteins, associated with diseases of incorrect protein folding. The system may have sound-generating elements, such as piezoelectric elements, photoacoustic mixers, or speakers, in contact with the housing of the apparatus and causing vibrations at the liquid-gas interface or thin film within the chamber or flow path of the apparatus. The gas may contain multiple types of molecules (e.g., air, including molecules such as nitrogen, oxygen, and carbon dioxide) or only one type of molecule (e.g., nitrogen). The vibration of this liquid-gas (or air-fluid) interface or thin film can cause mixing within the fluid in the apparatus. This mixing can bring certain biological substances into contact with each other and amplify those biological substances. In the case of chronic wasting disease, which is a disease of incorrectly folded proteins, the incorrectly folded proteins associated with this disease can cause normal proteins to also fold, effectively amplifying the number of incorrectly folded proteins in a sample through sound-based mixing. By amplifying the amount of misfolded proteins in the sample, the system can detect the presence of misfolded proteins and determine whether the sample is positive for the relevant disease.

[0005] In one embodiment, the system comprises a housing that defines a main channel configured to contain a fluid solution containing a sample comprising a plurality of proteins, and one or more side channels fluidly communicating with the main channel, each of which is configured to establish a liquid-gas interface between the fluid solution in the main channel and the gas contained in the one or more side channels, and at least one sound generating element configured to generate sound waves in at least one of the gas in the one or more side channels or the fluid solution in the main channel, thereby causing vibration of the liquid-gas interface and mixing of the fluid solution in the main channel. Alternatively, one or more thin film structures are configured to be extruded into the main channel, and at least one sound generating element is configured to generate sound waves that induce vibration of the thin film and mixing of the fluid solution in the main channel.

[0006] In one embodiment, the method comprises including a fluid solution containing a sample comprising a plurality of proteins in a main channel of a housing, wherein the housing comprises at least one of one or more side channels or one or more thin films that are in fluid communication with the main channel, and each of the one or more side channels or thin films is configured to establish a liquid-gas interface between the fluid solution or direct contact in the main channel and the gas contained in the one or more side channels, and generating sound waves in the gas of the one or more side channels or in at least one gas of the fluid solution in the main channel via at least one sound generating element, thereby causing vibration of the liquid-gas interface and mixing of the fluid solution in the main channel.

[0007] Details of one or more embodiments of this disclosure are described in the accompanying drawings and the following description. Other features, objects, and advantages of this disclosure are evident from the specification and drawings and from the claims. [Brief explanation of the drawing]

[0008] [Figure 1A]Figure 1A is a conceptual diagram of protein amplification resulting from the acoustic amplification of a sample protein in a fluid, as described herein. [Figure 1B] Figure 1B is a schematic side cross-sectional view and diagram illustrating the sound-based amplification device shown in the example of Figure 1A. [Figure 1C] Figure 1C is a perspective view of the exemplary sound-based amplification device shown in Figure 1A. [Figure 2A-2D] Figure 2A is an image of exemplary fluorescent particles moving using the sound vibrations described herein, Figure 2B is an image of exemplary fluorescent particles moving using the sound vibrations described herein, Figure 2C is a graph of particle velocity for different driving voltages, and Figure 2D is a graph of particle velocity for different frequencies of the piezoelectric transducer. [Figure 3] Figure 3 includes fluorescence images at different amplification times. [Figure 4A-4B] Figure 4A is a graph of fluorescence intensity after different amplification times, and Figure 4B is a graph of fluorescence intensity after different amplification times. [Figure 4C] Figure 4C is a transmission electron microscope (TEM) image of a prion fibril amplified as described herein. [Figure 4D] Figure 4D is a transmission electron microscope (TEM) image of a prion fibril amplified as described herein. [Figure 5A-5B] Figure 5A is a graph of the fluorescence intensity of positive and negative samples, and Figure 5B includes images of positive and negative samples with different colors, using gold nanoparticle aggregation analysis samples. [Figure 6] Figure 6 is a flowchart illustrating an embodiment of the technique for amplifying biological substances using the sound-based amplification device described herein. [Modes for carrying out the invention]

[0009] This disclosure describes systems, apparatus, and techniques configured to amplify proteins in a sample using sound-based amplification for the detection of one or more types of proteins, such as misfolded prions associated with various diseases. This sound-based amplification may utilize a liquid-gas interface to induce the amplification. Various diseases are caused by the accumulation of abnormally folded or misfolded proteins. In one embodiment, the protein is a misfolded prion protein associated with CWD. Recent studies have shown that infected populations decreased by approximately 10.4% in white-tailed deer in southeastern Wyoming and by 21% in sympatric mule deer populations. Furthermore, the social cost of CWD is very high, as Wisconsin reported spending over $32.3 million on CWD monitoring and control in 2001 and 2006. Therefore, there is an urgent need for a reliable in-situ diagnostic platform for the management of CWD in wildlife.

[0010] The current ultimate standard for diagnosing TSE heavily relies on enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) techniques based on analytical samples, which are expensive, time-consuming, and require considerable training and expertise to perform. Furthermore, diagnostic sensitivity is significantly hampered by the inability of antibodies to distinguish between PrPC and PrPSc, and the need for enzymatic, chemical, and / or thermal immersion to enrich PrPSc. Therefore, a definitive diagnosis of TSE often requires subsequent histopathological examination. Recently, novel, ultra-sensitive seeding assays based on intravitreal (in vitro) amplification of PrPCWD have made a significant improvement in the diagnosis of CWD. Both real-time vibration-induced conversion (RT-QuIC) and periodic amplification of protein misfolding (PMCA) utilize PrPSc's ability to periodically induce misfolding of PrPC to form aggregates of PrPSc fibrils. Real-time vibration-induced conversion (RT-QuIC) utilizes shear force, while periodic amplification of protein misfolding (PMCA) uses sonic processing to mechanically break down PrPSc fibrils into smaller nucleation sites (fragmentation). This results in exponential growth of PrPSc, which can be monitored in real time by its ability to bind a fluorescent dye, namely thioflavin T. This process can take up to 30-90 hours and requires periodic vibration to induce fibril formation. While RT-QuIC boasts high sensitivity, its application in field diagnostics is limited by several factors. For example, it requires bulky and expensive equipment. Furthermore, manual handling of reagents increases the risk of contamination as well as reagent costs. Thus, there is a critical need to develop a field analysis platform that provides ultrafast CWD diagnostics in a fully automated manner.

[0011] Microfluidics offer numerous advantages in diagnostic applications over macroscopic platforms, including low reagent requirements, high specific surface area-to-volume ratios, biohazard containment, and high heat and mass transfer rates. However, conventional microfluidic devices suffer from inherently low Reynolds numbers, which limit mass transfer to a diffusion-limited form. Furthermore, the use of sophisticated pumps limits their application in field diagnostics. Both active and passive micromixers have been developed, including inertial, acoustic, electrokinetic, and magnetofluidic micromixers. Among these, acoustic micromixers can be a powerful tool due to their high mixing index, low operating cost, biocompatibility, portability, and non-contact technology.

[0012] As described herein, the apparatus, systems, and techniques involve the use of acoustic amplification of biological elements such as proteins. While numerous acoustic fluid micromixers can be used, lateral cavity acoustic transducers (liquid-gas interfaces) can be used for biological applications. For example, the shear stress induced by the liquid-gas interface can be sufficient to fragment 50 kbp DNA into 5 kbp fragments. Acoustic fluid microfluidics can provide sufficient shear stress to fragment non-covalent interactions between amyloid fibril subunits. It is important to note that the operating mixing frequencies of these apparatuses are versatile, allowing for effective mixing at frequencies that may or may not coincide with the acoustic resonances of the liquid-gas interface, mechanical configuration, or fluid channel. This flexibility significantly enhances the usefulness of the apparatus across a variety of experimental conditions and sample types.

[0013] The liquid-gas interface described herein may refer to any liquid phase of a substance, e.g., a fluid that may contain one or more biological components, and the gaseous phase of a substance, e.g., an atmosphere that may contain one or more different types of gas molecules. The apparatus may be configured to establish a liquid-gas interface that transfers energy perpendicularly from the liquid-gas interface in substantially two dimensions. This two-dimensional interface may exist, for example, in a channel having a width substantially greater than its height. Such a channel can establish a relatively planar or curved liquid-gas interface, the curve being generally limited to two dimensions. In other embodiments, the apparatus may be configured to establish a liquid-gas interface that transfers energy in three dimensions. This three-dimensional liquid-gas interface may be established in a channel having a relatively large cross-sectional area that facilitates a substantially three-dimensionally curved liquid-gas interface. In some embodiments, the three-dimensional liquid-gas interface may even be a partial or complete bubble (e.g., a sealed liquid-gas interface). In this way, the three-dimensional liquid-gas interface can transfer energy substantially in three dimensions.

[0014] Figure 1A is a conceptual diagram of protein amplification resulting from acoustic amplification of a sample protein in a fluid, as described herein. As described herein, system 100 has an RT-QuIC integrated on a PDMS-coated glass slide, together with a microfluidic apparatus based on acoustic microfluidics, and can mix PrPSc and PrPC. Mixing PrPSc and PrPC is described as one embodiment of amplification of misfolded proteins or other biological substances, for which system 100 may be utilized. A sample containing proteins such as PrPSc and PrPC can be contained in the fluid of the main channel 104. Air (or some gas) is contained in air cavities 110 of one or more side channels 106. A liquid-gas interface 108 is provided to transmit vibrations to the fluid. The substrate 102 may be at least part of a housing containing the main channel 104 and one or more side channels 106.

[0015] In some embodiments, reagent mixing (e.g., in a fluid contained within system 100) is achieved in a lateral cavity sound transducer (liquid-gas interface) where an array of terminal channels (e.g., one or more of the side channels 106) captures bubbles that function as a vibrating membrane. In some embodiments, reagent mixing can be performed at the liquid-gas interface, or at a mechanical configuration, or at the acoustic resonance of a fluid channel. The liquid-gas interface is one embodiment of the sound-generating element. In some embodiments, the liquid-gas interface may be in a two-dimensional (2D) or three-dimensional (3D) configuration as described herein. For example, the liquid-gas interface may be the interface between a liquid and one or more bubbles. This vibrating membrane is also called the liquid-gas interface or fluid-air interface. The application of high-frequency sound waves (e.g., 4.6 kHz) causes one or more liquid-gas interfaces 108 to resonate, and the resulting sound field biases the bulk liquid and generates a net force from the ends of cavities (e.g., one or more air cavities 110 in the side channel 106) perpendicular to the bubble interface. The vibration of the sound transducer can resonate the liquid-gas interface 108 and / or other structures of the apparatus, imparting energy to the liquid in the channel. The resulting acoustic flow enhances collisions between PrPSc and PrPC, as well as fragmentation of PrPSc into small pieces, resulting in the proliferation of active species for PrPSc nucleation. Liquid-gas interfaces are well-suited for this application due to their simplicity in manufacturing, controllability of mixing rate, and high mixing index. In some embodiments, controllability of mixing rate may refer to the amplitude of the applied energy, the resonant frequency of one or more fluids or structures, or other selectable parameters.

[0016] By combining reagent microfluidics induced by an acoustic fluidic micromixer with vibrations based on prion fibril amplification, this method was able to dramatically reduce the amplification time from 48 hours using other techniques to less than 3 hours using the acoustic amplification described herein. Furthermore, this amplification technique can be made compatible with an aggregation analysis sample based on gold nanoparticles that eliminates the need for a bulky auxiliary detection module. In some embodiments, this amplification technique can be adapted for capillary height measurements, colorimetric analysis (e.g., gold nanoparticle colorimetry, silver nanoparticle calorimetry, or other colorimetry using other particles or dyes such as one or more organic dyes), or fluorescence imaging. In some embodiments, detection of a target biological material that can be amplified as described can occur using the naked eye instead of a microscope or other instrument. In some embodiments, detection of a target biological material that can be amplified as described can be performed using a microscope or camera or other image capture or observation device. Thus, ultrafast prion amplification in this acoustically-driven microfluidic platform can reduce the amplification time and the costs associated with detecting any amplification results. Micro QuIC devices such as system 100 are characterized by advantages such as simplicity of use, automation, low cost, and portability. These advantages are ideal for the future development of an automated all-in-one, on-chip prion amplification tool kit for point-of-care diagnostics.

[0017] 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. Here, we discuss misfolded proteins for CWD, but this method can also be used to detect other diseases or conditions of misfolded proteins. In one embodiment, tissue is added to a solution containing a high concentration of healthy, unmisfolded prions and injected into a microfluidic channel. The sample is then vibrated for a period of time, for example, 4 hours. Applying an AC bias to the transducer will cause sound waves generated by the piezoelectric transducer to vibrate at the liquid-gas interface. In some embodiments, the liquid-gas interface can be in a two-dimensional or three-dimensional configuration. For example, the liquid-gas interface can be the interface between the liquid and the gas. This can result in a flow of reagent solution that actively mixes the prion proteins. If the sample has misfolded prions (as in CWD-positive animals), the misfolded prions will misfold healthy prions, resulting in a significant increase in the number of misfolded prions. If there are no misfolded prions in the sample (as in CWD-negative animals), then misfolding of the healthy prion protein will not occur. After mixing, the microfluidic channels are observed under a fluorescence microscope or through other visualization methods, such as using gold nanoparticles. If the solution exhibits high fluorescence intensity or a specific color, the sample is CWD-positive; if the solution exhibits very low fluorescence intensity or a specific color, the sample is CWD-negative.

[0018] In some embodiments, system 100 may include a housing defining a main flow path 104 configured to contain fluid solution including a sample containing multiple proteins, and one or more side flow paths 106 in fluid communication with the main flow path, each of the one or more side flow paths being configured to establish a liquid-gas interface 108 between the fluid solution in the main flow path and air contained in the one or more side flow paths 106, and at least one sound generating element (not shown in FIG. 1A) configured to generate sound waves in the air of the one or more side flow paths 106 or in the fluid in the main flow path 104 to cause vibration of the liquid-gas interface 108 and mixing of the fluid solution in the main flow path 104. The liquid-gas interface 108 is shown in FIG. 1A as a two-dimensional interface because energy is generally transmitted within the plane of the substrate 102. However, if the thickness of the substrate 102 can be increased such that the liquid-gas interface is similar to the width of the side flow path 106, the liquid-gas interface 108 can form more three-dimensional interfaces that transmit energy at different angles within the plane of the substrate 102 and also within the height of the substrate 102.

[0019] The main channel and side channels may have a variety of different dimensions. These dimensions may be selected according to one or more properties of the target sample, biological substances in the sample, fluid viscosity, frequency, or any other properties. In some embodiments, the main channel has a width of 100 to 500 micrometers. In the embodiment of Figure 1A, the width of the main channel 104 is about 300 micrometers. In some embodiments, each of the side channels 106 has a width of 50 to 500 micrometers. In one embodiment, the width of the side channel 106 is about 100 micrometers. In some embodiments, the width of each side channel 106 is smaller than the width of the main channel. The side channels 106 are described to establish a liquid-gas interface 108, but any structure or modification of the main channel 104 that can maintain an interface between bubbles or air and liquid in order to carry sound waves into the fluid may be used. For example, the side channel may take the form of a bulge, gap, crack, or any other structure that is in fluid communication with the main channel 104.

[0020] The housing defining the main channel 104 and one or more side channels 106 may, in some embodiments, have a portion of the substrate 102. In some embodiments, the housing may have a glass slide as the substrate 102 and a first polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), or glass layer, disposed on the glass slide and at least partially defining the main channel between the glass slide and the first housing layer. In some embodiments, the system may have a first tube fluid-connected to the inlet of the main channel and a second tube fluid-connected to the outlet of the main channel through which a sample can be added to the main channel. In some embodiments, the housing has a second PDMS, PMMA, or glass layer disposed around at least a portion of the first housing layer and defining the space between the first and second housing layers, and a hydrophobic fluid disposed within the space between the first and second housing layers. In some embodiments, the housing may be made of any polymer. One embodiment of the housing may be made of CD-ROM material.

[0021] System 100 may have one or more sound-generating elements. In one embodiment, the sound-generating element is a piezoelectric transducer. In another embodiment, the sound-generating element is a lateral cavity sound transducer (liquid-gas interface). The sound-generating element may be in contact with a portion of the housing and / or located at one or more open ends of several side passages exposed to air. System 100 may also have a control device configured to energize the sound-generating element for one or more periods. This control device can turn the voltage from the power supply on or off, or, in some embodiments, the control device can adjust the current or voltage to adjust the frequency and / or amplitude of the sound-generating element to generate a desired sound wave in the main chamber.

[0022] In some embodiments, at least one sound-generating element is configured to generate sound waves in the air of one or more side channels so that a certain amount of misfolded protein is amplified to a detectable level by contacting other proteins in the multiple proteins. The system 100 can also facilitate the detection of any amplified biological material. In one embodiment, the amplification can be visualized with the naked eye through the transparent portion of the housing and the color change of the sample. In some embodiments, at least a portion of the housing is transparent, allowing for the measurement of the fluorescence intensity of at least some of the multiple proteins in the sample in the main channel after mixing. In some embodiments, the outlet of the housing is connected to a capillary tube to measure the viscosity change. In some embodiments, an additional capillary channel is configured in relation to the main channel to measure the viscosity change of the amplified reagent. The substrate 102 is generally shown as a flattened or planar substrate, but the apparatus 100 may instead be configured to create a three-dimensional liquid-gas interface. In some embodiments, the three-dimensional liquid-gas interface can form one or more partial or complete bubbles.

[0023] Figure 1B is a schematic and lateral cross-sectional view showing an exemplary sound-based amplification system 120 that may be similar to system 100 in Figure 1A. System 120 in Figure 1B may also represent a cross-sectional view of system 100. System 120 may have a substrate 122 on which a first layer 126 is partially placed to form a channel 124. A second layer 130 surrounds at least some of the first layer 126, and a substance 128 such as mineral oil may be placed between the first layer 126 and the second layer 130. Tubes 132A and 132B (collectively "tube 132") are configured so that the tube 132 moves through the first layer 126 and the second layer 130, allowing a sample to be injected into and removed from the channel 124. One or more piezoelectric transducers 134 may be positioned in contact with a portion of system 120, such as in contact with the underside of the substrate 120. The piezoelectric transducer 134 is just one example and can be used to generate vibrational energy and transmit it to a part of the system 120 at a target resonant frequency.

[0024] As shown in the example in Figure 1B, the sample may be delivered through the tube 132 into a main channel 124 defined by a first layer 126 (e.g., PDMS, PMMA, or glass) on a substrate 122 connected to a piezoelectric transducer 134. A second layer 130 (e.g., PDMS, PMMA, or glass) may define a channel that may contain another substance 122, such as mineral oil or other fluid, which may be provided to stabilize the sample and fluid in the main channel 122.

[0025] System 120 in Figure 1B is an embodiment of a microfluidic vibration-induced conversion (Micro-QuIC) apparatus. As described herein, this apparatus can combine the active mixed benefits of sound technology with vibration-induced prion amplification. For this purpose, Figures 1A and 1B provide a schematic diagram and a photograph of an assembled embodiment of the Micro-QuIC apparatus, respectively. In one embodiment, the base of the sound-fluid apparatus (e.g., substrate 122) is a thin glass coverslip. This glass coverslip plays a role in transferring vibrational energy from the sound transducer 134 (shown in Figure 1B) to the PDMS chip via bent waves that exit the sound transducer and propagate along the glass coverslip. The main design of the microfluidic channel geometry may have lateral cavity structures (e.g., one or more side channels, such as channel 106 shown in Figure 1A) that trap air cavities when reagents are injected. The resulting one or more liquid-gas interfaces act as vibrating membranes in response to vibrations from the transducer, generating acoustic flow within the channel to agitate the sample. As shown in the embodiment in Figure 1A, the lateral cavity structure (e.g., main channel 104) is designed with a channel width of 300 μm and a height of 100 μm. In these embodiments, there are 16 sets of lateral cavity structures in total along the length of the main channel (e.g., channel 104 or channel 124).

[0026] A second layer 130, which has an outer channel made of PDMS, PMMA, or glass, is attached to the top of the first layer 126 (e.g., a microfluidic PDMS, PMMA, or glass layer) and can create a void filled with a substance 128 (e.g., mineral oil) to prevent heat-mediated evaporation of the reagent. The sample reagent can be introduced into the main channel 104 or 124 through tube 132A or 132B (e.g., inlet or outlet) while the entire apparatus is incubated at 45°C. A piezoelectric transducer 143, speaker, or other vibration generator can be attached to the back of the substrate 122 (e.g., a glass slide) to provide a sound field for inducing vibrations at the liquid-gas interface. This generates microvortex flow within the main channel 104 or 124, accelerating both collisions between PrP particles and shear-induced fragmentation of PrPSc fibrils. Collisions between PrP particles accelerate the conversion of PrPc to PrPSc. In other words, misfolded proteins come into contact with normally folded proteins, causing further misfolding of the proteins to amplify the misfolded proteins in the sample. Fragmented PrPSc acts as a nucleation site for further fibril formation. As the number of fibrils increases, the fluorescent dye, thioflavin T, specifically binds to amyloid aggregates that can be observed under a fluorescence microscope. While fluorescence can be used in some embodiments, other types of visualization can be used. For example, gold nanoparticles can be used to bind to aggregates that change the visible color of the fluid sample after amplification, provided that enough misfolded proteins are present.

[0027] In some embodiments, misfolded chronic wasting disease (CWD) prion seeds derived from biological samples are added to an rPrP solution and injected into a microfluidic apparatus. This fluid creates a liquid-gas interface between the main channel and the side channels. By applying high-frequency sound waves (e.g., 4.56 kHz), vibrations at the liquid-gas interface can be induced, creating vortices. These solutions can then be cultured by agitation at 45 degrees Celsius for about 3 hours, although shorter or longer periods or higher or lower temperatures are also possible. If present, PrPCWD induces a conformational change of rPrP through contact during mixing induced by sound waves. The resulting product can be labeled with thioflavin T, which can be detected under a fluorescence microscope (e.g., excitation wavelengths in the range of about 300 nanometers (nm) to about 480 nm and emission wavelengths in the range of about 395 nm to about 500 nm). These excitation and emission wavelengths are merely examples, and other wavelengths may be used in other embodiments. As shown in Figure 1B, the double layers of the PDMS apparatus (e.g., a first layer 126 and a second layer 130) 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 the PDMS to prevent evaporation of the solution. A piezoelectric transducer 134 or other sound-generating element can be mounted on the back of the substrate 122 (e.g., the glass slide). These materials described with respect to Figures 1A and 1B are merely examples, and other materials can be used to construct systems 100 and 120 in other embodiments.

[0028] Figure 1C is a perspective view of an exemplary sound-based amplification device 160, which may be similar to system 100 in Figure 1A and / or system 120 in Figure 1B. As shown in Figure 1C, the device 160 has a housing 164 in which a piezoelectric transducer 162 is mounted. A liquid containing a sample biological material, such as a protein that may or may not have misfolded proteins, can be injected into the main channel in the housing 164 via a tube 166. The fluid can be extracted from the main channel via an outlet tube 168.

[0029] Figures 2A and 2B are images of exemplary fluorescent microparticles moving using the sound vibrations described herein. The apparatus (e.g., System 100, System 120, or Apparatus 160) can be characterized in different ways. The ability of this apparatus to generate microvortex flow was characterized using fluorescent microparticles as shown in Figures 2A and 2B. The fluorescent microparticles exhibited vigorous circular motion when an input bias voltage of 10 Vpp at 4.6 kHz was applied, as shown by the circular arrows in Figure 2B. The flow velocity is highly dependent on two parameters: frequency and voltage.

[0030] In one embodiment, the AC frequency was swept from 1 kHz to 100 kHz in 50 Hz increments to determine the frequency at which the liquid-gas interface produced the strongest sonic fluid effect. The experimental results showed that the strongest sonic fluid effect occurred when the device was excited at 4.6 kHz, which coincided with the resonant frequency of the piezoelectric transducer. Outside the resonant frequency of the piezoelectric transducer, the fluid velocity was dramatically attenuated, as shown in Figure 2D. Therefore, the AC bias was maintained at the resonant frequency for all our experiments. The device was further characterized by applying different drive voltages to the piezoelectric transducer. Figure 2C shows the mixing performance at different drive voltages at a frequency of 4.6 kHz. The results showed that as the drive voltage of the piezoelectric transducer increased, the mixing efficiency increased. These voltages and frequencies are specific to one particular 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. While this embodiment used the resonant frequency of the piezoelectric transducer, other systems may use other resonances as additions or substitutions. For example, energy may be transmitted through resonance at the liquid-gas interface, resonance in the flow path, resonance in the structure of the apparatus, and / or resonance in any other element that is part of the apparatus including the liquid-gas interface.

[0031] Figure 3 includes fluorescence images after different amplification times. Using the apparatus described herein, the technique can achieve on-chip prion amplification by microQuIC. Next, experimental conditions for microQuIC were established. In some embodiments, real-time prion amplification can occur in a microfluidic channel. In one embodiment, a solution containing a CWD-positive sample was injected and acoustically mixed at 45°C with an on / off interval of 30 seconds. When an AC bias is applied, the sound waves cause active vibrations at the liquid-gas interface, mixing the prion proteins. When the misfolded protein collides with the unfolded protein, conversion of the unfolded protein into the misfolded protein occurs. The misfolded protein begins to form a fibril structure, enabling thioflavin T binding, and therefore fluorescence binding.

[0032] In this example, spontaneously misfolded rHaPrP (positive control) was first spiked into a master mix consisting of 1X PBS, 1 mM EDTA, 170 mM NaCl, 10 micron M ThT, and rHaPrP in the range of 0.1 mg / mL to 0.4 mg / mL. The use of high concentrations of rHaPrP can accelerate the reaction rate; therefore, unless otherwise noted, the rHaPrP concentration was always maintained at 0.4 mg / mL. The master mixes seeded to both positive and negative were injected into a microQuIC instrument and amplified for 180 minutes. Here, an AC bias of 10 Vpp at a frequency of 4.6 kHz was applied at 30-minute intervals.

[0033] Fluorescence images were taken every 30 minutes, followed by a 180-minute amplification process, as shown in Figure 3. For the quantification of fluorescence intensity, fluorescent particles were analyzed within the field of view by thresholding. Both the number and size of fluorescent particles increased from the positive seed sample, indicating successful prion amplification, while there was no measurable change in fluorescence intensity from the negative seed sample. Based on these results, prion amplification was achieved in 180 minutes, 16 times faster than conventional RT-QuIC. To verify the amplified prion fibrils, the dimensions and morphology of amyloid fibrils amplified by both RT-QuIC and microQuIC were compared using transmission electron microscopy (TEM) (Figures 4C and 4D). As previously reported, the fibrils from both samples had a helical shape with an average width of 25 nm (however, in other examples, they can be smaller or larger), further verifying that the amplification products are equivalent in both methods.

[0034] Figures 4A and 4B are graphs of fluorescence intensity after different amplification times. These graphs illustrate examples of real-sample amplification and optical detection based on AuNP. Since microQuIC can significantly increase rHaPrP misfolding and amplification dynamics, the apparatus and technique can be utilized for potential microQuIC for CWD diagnosis using PrPCWD-positive and negative white-tailed deer lymphoid tissue as one example. Homogenates of independently confirmed CWD-positive and negative white-tailed deer medial retropharyngeal lymph nodes (RPLNs) can be prepared. The microQuIC technique facilitated rapid misfolding and amplification of rHaPrP solutions inoculated with CWD-positive tissue samples. In one example, the microfluidic apparatus described herein was prepared and injected with a master mix seeded with positive (n=5) and negative (n=5) tissue samples. Each sample was cultured at 40°C for 3 hours with periodic sound mixing. As shown in Figure 4A, fluorescence measurement, an evaluation criterion for microfluidic channels, demonstrated that this technique can distinguish between CWD-positive and negative samples through differences in fluorescence intensity. For comparison, Figure 4B shows real-time prion amplification in a conventional RT-QuIC reaction, which can take 34 hours to reach maximum amplification for positive samples.

[0035] Figure 5A shows graphs of fluorescence intensity for positive and negative samples. As shown in the graph in Figure 5A, the fluorescence intensity of the positive sample, which contained misfolded proteins amplified by the sound amplification technique, was detectably different from that of the negative sample. The significant reduction in analysis time (approximately 3 hours in some examples) makes MicroQuIC an attractive alternative to the current ultimate standard for CWD diagnosis.

[0036] In several embodiments, an optically-based false-folding PrP detection system using gold nanoparticles (MN-QuIC) can eliminate the need for bulky fluorescence detection modules. In conjunction with microQuIC, an AuNP-based detection system would provide a valuable tool toward a field-deployable CWD testing platform. In one embodiment, microQuIC and MN-QuIC were used in combination. The tissue sample was first amplified in a microfluidic channel at 40 degrees Celsius with periodic sound mixing for 3 hours, as described herein. The amplified mixture was withdrawn from the microfluidic channel and cultured with gold nanoparticle reagent for 10 minutes. Upon addition of the positive sample, the absorbance peak remained unchanged at 515 nm, while the addition of the negative sample absorbance peak shifted to a longer wavelength of approximately 560 nm.

[0037] Figure 5B includes images of positive and negative samples with different colors, using gold nanoparticle agglutination analysis samples, showing that the wavelength of the negative tissue sample is longer. This detectable color difference demonstrates a portable detection method for field applications. Both CWD-positive and negative samples were amplified using microfluidic channels as described herein. Gold nanoparticle agglutination analysis samples can detect CWD-positive / negative samples. Simply put, CWD-negative samples allow for the agglutination of AuNPs, resulting in a blue shift in their absorption spectra, while CWD-positive samples inhibit AuNPs, and therefore their absorption spectra remain unaffected. This eliminates the need for bulky and expensive fluorescence detection systems, making it more suitable for field applications.

[0038] CWD is merely one example of the use of the apparatus, systems, and technologies described herein. Given the continued prevalence of CWD among deer populations throughout North America and Northern Europe, there is an urgent need to develop novel diagnostic tools for CWD. Here, RT-QuIC amplification of CWD prions can be combined with an acoustic fluid micromixer for ultrafast amplification of positive and negative CWD samples. The acoustic fluid micromixer will exert high shear stress between PrPSc fibrils, actively fragmenting them and subsequently serving as new nucleation sites. This technique significantly reduced the lag phase time to approximately 3 hours, at least an order of magnitude faster than current ultimate standards such as RT-QuIC and PMCA. This amplification strategy is also compatible with other non-fluorescence-based detection methods, such as AuNP agglutination analysis, which demonstrates the potential for in-situ diagnosis.

[0039] Therefore, the microQuIC analytical samples described herein can be used for the visual identification of positive samples of specific biological substances, such as CWD-positive and negative lymphoid tissues that follow QuIC amplification. In some embodiments, RPLN and palatine tonsils collected from white-tailed deer were the basis for the analyses performed here, as these tissues are ideal for the early and accurate identification of CWD infection. In other embodiments, the technique can be utilized for pre-mortem CWD diagnosis. RT-QuIC amplification tests using samples collected from living deer can be readily combined with microQuIC, providing field-deployable pre-mortem testing for wild and agricultural deer. Furthermore, microQuIC can have utility as a food safety test, providing recent information management of RT-QuIC-based detection of CWD in white-tailed deer muscle used for human and animal consumption. More broadly, microQuIC analysis samples have the potential to become a versatile platform for detecting various TSEs and proteopathies for which RT-QuIC and PMCA are currently used, such as sheep scrapie, bovine BSE, human CJD, Parkinson's disease, and Alzheimer's disease.

[0040] Figure 6 is a flowchart illustrating one embodiment of the technique for amplifying a biological substance using a sound-based amplification device of one embodiment described herein. The technique in Figure 6 is described in relation to system 100, but can be performed using any of the sound amplification systems and devices described herein, such as system 120 and device 160.

[0041] As shown in the embodiment of Figure 6, a user can inject a sample containing multiple biological substances, such as proteins, into the main channel 104 of the sound-based amplification device of system 100 (600). For example, the sample may be in a fluid injected into the main channel 104 via one or more tubes. System 100 then contains the fluid sample in the main channel 104 (602). System 100 then generates sound waves in the air of one or more side channels 106 via at least one sound-generating element, causing vibrations at the fluid-gas interface and mixing of proteins in the fluid in the main channel 104 (604). This mixing process can take place for several minutes or several hours, usually less than four hours. In some embodiments, the mixing process can take less than three hours, less than two hours, or less than one hour. During this period of mixing, misfolded proteins come into contact with normal proteins, causing the normal proteins to also misfold.

[0042] After the amplification process is complete, the user can determine an image of the sample in the main channel 104 (606). In other embodiments, the sample may be extracted for visualization. Changes in the amplified protein (if present in the sample) can be detected using fluorescence imaging of gold nanoparticles, other optical techniques, or viscosity measurement. The user or an automated optical detection device (e.g., a camera and visualization detection software) can then determine from the image the state of the misfolded protein in the sample, which may indicate the presence of a positive or negative result for the misfolded protein (608). For example, fluorescence intensity exceeding a positive threshold in gold nanoparticle visualization, or the absence of a blue shift, may indicate that the sample is positive for misfolded protein and diseases such as CWD.

[0043] The folding method and materials used in the above embodiments are different from those used in other embodiments, which may use other methods and materials to achieve similar results. Materials include SU8 2050 (Microchem), silicon wafers (Siegert wafers), SU8 developer (Microchem), developer cover glass (24x50mm, Globe Scientific), piezoelectric transducer (7BB-27-4L0, Mouser Electronics), Thioflavin T (ThT), 100kDa Pall MWCO filter, ethylenediaminetetraacetic acid (EDTA), sodium chloride (NaCl), phosphate buffered silane (PBS), and trimethoxysilane. Fluorescent microspheres (FCDG006) were purchased from Bans Laboratories, and polydimethylsiloxane (PDMS) and curing agent were obtained from Dow Corning as SYLGARD® 184 silicone elastomer kit.

[0044] The following are examples of techniques for constructing and using the sound amplification system described in this specification.

[0045] Preparation of the reconfigurable circuit board The synthesis and purification of recombinant Chinese hamster PrP (HaPrP90-231) followed the method of Schwabenlander et al. In summary, a truncated form of the Syrian hamster PRNP gene (amino acids 90-231) was cloned into a pD431-SR expression vector (Atom, Newark, California, USA), expressed in Rosetta (DE3)E E. coli, and the substrate was synthesized.

[0046] RT-QuIC for spontaneous misfolding of recombinant prion proteins For QuIC analysis, the master mix was prepared according to the following specifications. 1X PBS, 1 mM EDTA, 170 mM NaCl, 10 μM thioflavin T (ThT), and 0.4 mg / mL rHaPrP. A 10% tissue homogenate was further diluted 100-fold to 0.1% SDS / PBS / N2 (final tissue dilution: 0.1%), and 2 μL of the dilution was added to each well containing 98 μL of RT-QuIC master mix. Spontaneous misfolding of recombinant prion proteins was similarly performed, but using unfiltered recombinant proteins and reagents. Infectious seeds were not required for these reactions. Reactions for both MicroQuIC and RT-QuIC were seeded using spontaneously misfolded material. Plates were amplified on a FLUOstar® Omega Plate Reader (BMG Labotec, Carrie, North Carolina, USA) (42°C, 700 rpm, dual orbital, 57 seconds of vibration, 83 seconds of rest). Fluorescence readouts were performed at intervals of less than 45 minutes.

[0047] Thermomixer-based amplification Prion amplification based on QuIC can be generated using a standard benchtop vibrating incubator (thermomixer). Plates prepared for amplification on the thermomixer were prepared in the same way as those amplified on a plate reader. The reaction was carried out for 24 hours at 48°C and 600 RPM (60 seconds of vibration and 60 seconds of stillness) on a ThermoMixer® C equipped with SmartBlock plates and Thermotop (Eppendorf, Enfield, Connecticut, USA). Run times of 24 hours based on independent RT-QuIC results for lymph nodes and palatine tonsils from CWD-positive white-tailed deer, including those examined here, show significant dissemination activity within 9–24 hours.

[0048] Preparation and operation of microfluidic devices An example of PDMS-based microfluidic device fabrication was carried out using soft lithography as described above. A master mold was prepared on an approximately 10 cm (4 inch) silicon wafer using SU8 2050 spin-coated to a height of 50 μm (Brewer Sciences Model CEE-100). Following the coating process, a pre-sintering step was performed on the SU8-coated silicon wafer for a duration of 12 seconds before exposure to UV light through a film mask with the required design (MA6, Carl Sass). A post-sintering step was performed before SU8 development. SU8 development was performed by gently washing the wafer in the developer solution for 3 minutes. Finally, the Si wafer was silylated with trimethoxysilane for 30 minutes. PDMS-based microfluidic chips were fabricated by thermo-curing (3 hours at 90 degrees Celsius) PDMS with a curing agent mixture (10:1) on the Si wafer of the master mold. The cured PDMS was peeled off and cut into individual chips. Before bonding the PDMS to the microscope cover glass, holes were punched at the entrance and exit using a 1mm bio-punch (Acuderm). The bonding procedure involved treating both the cover glass and the PDMS with a high-frequency generator (Electrotechnic BD-10A) for 1 minute. The microfluidic apparatus was heated at 65 degrees Celsius for 2 hours to aid in the bonding process. To fabricate the outer PDMS case, a rectangular mold (3cm × 1.5cm × 0.5cm) was mounted on a silicon wafer of approximately 10cm (4 inches), and then silanized with trimethoxysilane for 30 minutes to create a master mold.

[0049] An outer PDMS case was fabricated by heat-curing a PDMS master mix at 90°C for 3 hours. After cutting to individual chip sizes, holes were punched at the inlet and outlet using a 1 mm biological punch. The outer PDMS case was bonded to the microfluidic apparatus by aligning the inlet and outlet. Mineral oil was injected between the microfluidic PDMS channel and the outer PDMS case. Finally, the piezoelectric transducer was attached to the back of the apparatus using epoxy adhesive. A photograph of the final assembled apparatus can be seen in Figure 1C. Before conducting amplification experiments with recombinant PrP samples, all apparatuses were experimentally tested with 0.005% fluorescent polystyrene microspheres (1 μm) to determine the optimized operating frequency of the piezoelectric transducer for achieving uniform mixing. Based on these tests, 4.6 kHz was determined as the operating frequency for all microQuIC apparatuses. All amplification experiments were performed at this frequency. To track aggregated PrP, fluorescence images were obtained every 30 minutes for approximately 3 hours using laser excitation at 445 nm. Fluorescence intensity was measured by correcting the whole-cell fluorescence measurements.

[0050] Detection based on gold nanoparticles Prion detection based on gold nanoparticles followed the method of Christenson et al. Briefly, gold nanoparticles (usaNanopartz®, Loveland, Colorado, USA) capped with 2.45 nM citrate were exchanged in a low-concentration phosphate buffer (10 mM Na2HPO4 (anhydrous), 2.7 mM KCl, 1.8 mM KH2PO4 (single nucleotide)). After amplification, the protein solution was diluted 2-fold with 1X PBS by adding a final concentration of 1 mM EDTA, 170 mM NaCl, and 1.266 mM sodium phosphate. Finally, 40 μl of the diluted protein solution was added to 360 μl of AuNP solution and reacted at room temperature (RT) for 30 minutes. Color changes were observed visually and with a colorimeter (FLUOstar® omega plate reader, BMG Laboratory, Carrie, North Carolina, USA) at wavelengths of 400-800 nm.

[0051] The tissue preparation followed the method of Christenson et al. (Christenson, PR, Li, M., Roden, G., Schwabenland, MD, Wolf, TM, Oh, SH, Larsen, PA et al., Field-Deployable Diagnostic Analysis for Visual Detection of Misfolded Prions, Scientific Reports, 12(1), 12246, 2022). Briefly, eight white-tailed deer tissues (4 CWD negative and 4 CWD positive) were obtained from white-tailed deer through the Minnesota Department of Natural Resources Cooperation, and their CWD status was identified using BioRad TeE's Short Assay Protocol (SAP) combo kit (BioRad Laboratory, California, USA). White-tailed deer retropharyngeal lymph nodes (RPLNs) and palatine tonsils were homogenized in PBS (10 wt% W / v) using a bed bug homogenizer (Benchmark Scientific, New Jersey, USA) with 1.5 mm zirconium beads at maximum speed for 90 seconds.

[0052] Statistical analysis was performed using GraphPad Prism version 9.0 for Windows® (GraphPad Software, San Diego, California, USA, www.graphpad.com). The potential use of AuNP on spontaneously misfolded rHaPrP was demonstrated using three technical replicates. CWD prions were tested for each animal using AuNP and RT-QuIC using three and four technical replicates. RPLN and / or palatine tonsil tissue from 10 positive and 14 negative animals were included in the examples described here. Mean differences of all parameters of interest between samples were tested using a unilateral Mann-Whitney unpaired u test (α=0.05).

[0053] The following exemplary processes may be used to fabricate sound-based amplification devices, such as systems 100 and 120 or apparatus 160, and fluids for testing samples. These are merely examples, and other techniques may vary, for example, depending on materials, time, or the order of steps.

[0054] the purpose MicroQuIC is a method for amplifying and detecting prion gene fragments in a sample. It can function by amplifying fibril formation through sound-driven fluid mixing of the photosensitive form of prions, usually HaPrP, within a microfluidic channel. After mixing, the microfluidic channel containing the post-amplification solution is observed under a fluorescence microscope. The difference between samples with fibril formation and those without can be easily distinguished via the fluorescence intensity measured from the microfluidic channel. Furthermore, this system was compatible with the previously reported MN-QuIC system, which can distinguish the presence of misfolded PrP using gold nanoparticle aggregation analysis samples.

[0055] Device identifier Hot plate, AC function generator, vacuum chamber, MA6 aligner, BD-10A high-frequency generator.

[0056] material MilliQ DI Water ·5X PBS buffer • 0.1% SDS in PBS • 10% (W:v) tissue homogenate (-80°C) • 10 mM Thioflavin T (optional) HaPrP(90-231)(-80℃) 100kDa Pall MWCO filter 100mM EDTA 2M NaCl 1X PBS • 96-well plate • SYGARD 184 Silicone Elastomer Kit (PDMS) • 20 x 50 mm microscope cover glass • Piezoelectric converter (4.6kHz) SU-8 photoresist • Microchem's NANO(trademark) SU-8 2050 photoresist Microchem SU-8 Developer • 10mm silicon wafer H2SO4 ·H2O2 • 1mm biological punch • Photomask for microfluidic channels Epoxy adhesive

[0057] procedure <Mold making (1) - Microfluidic PDMS channel> (1) Place a 10mm silicon wafer in piranha solution (H2SO4:H2O2=3:1) for 5 minutes, then wash thoroughly with DI water. (2) A silicon wafer is placed on top of the spin coater, and 5 mL of SU-8 photoresist is supplied. (3) Rotate at 500 rpm for 10 seconds, then at 1250 rpm for 30 seconds. (4) The silicon wafer is baked at 65°C for 5 minutes and then at 95°C for 20 minutes. (5) Place the photolithography mask and silicon wafer on the aligner and expose to ultraviolet light (240 mJ / cm²). 2 Expose the area to light. (6) Bake the silicon wafer at 65°C for 2 minutes and then at 95°C for 10 minutes. (7) Immerse the silicon wafer in SU-8 developer and gently vibrate it for 10 minutes. (8) Wash with DI water and dry to complete the mold making process. (9) Place the mold in a sealed container containing 100 μL of trimethoxysilane. (10) Wait for the trimethoxysilane to evaporate. <Mold making (2) - Outer PDMS case> (1) Use epoxy adhesive to attach a 3cm x 1.5cm rectangular mold to the silicon wafer. (2) Place the mold in a sealed container containing 100 μL of trimethoxysilane. (3) Wait for the trimethoxysilane to evaporate. <Device fabrication> (1) Mix 30 mL of SYLGARD-184 elastomer base with 3 mL of hardener. (2) Degas under vacuum for 20 minutes. (3) Pour the SYLGARD-184 mixture (PDMS) into the molds for the microfluidic PDMS channels and outer PDMS cases, and bake at 65°C for 2 hours. (4) Cut out the microfluidic PDMS channel and the outer PDMS case, and puncture the entrance and exit using a 1 mm biological punch. (5) The surface of the microfluidic PDMS channel and the 20mm x 50mm microscope cover glass are treated with a high-frequency generator for 5 minutes. (6) The microfluidic PDMS channel is mounted on a microscope cover glass and fired at 65°C for 30 minutes. (7) The surface of the outer PDMS case and the glass portion of the microfluidic channel are treated with a high-frequency generator for 5 minutes. (8) By aligning the inlet and outlet, the outer PDMS case is attached to the top of the microfluidic channel. (9) Bake at 65°C for 30 minutes. (10) Use epoxy adhesive to attach the piezoelectric transducer to the glass side of the microfluidic chip. (11) Connect the PTFE tube to the inlet and outlet of the PDMS chip. (12) Inject mineral oil into the chamber between the microfluidic PDMS channel and the outer PDMS case. <Preparation of sample solution> This procedure includes dilutions for lymphatic and nerve tissues. Extraction protocols for other sample types include the correct dilutions for those samples. For other sample types, begin from step 4. (1) Remove the sample from the freezer and place it in the biosafety cabinet. (2) Cut the tissue for secondary sample collection. (3) Add 100 mg of tissue to a labeled bead tube in 900 μL of 1X PBS (1.5 mm zirconium bead tube). (4) Use a bead beater to homogenize at maximum speed for 90 seconds. (5) Bring the sample to -80 °C to make it ready for execution. (6) (Optional) Weigh ThT (0.3 mg) and add it to 1 mL of filtered water. This will result in 1 mM ThT. It can be done up to 5 days in advance. (7) Remove a sufficient number of tubes of HaPrP from -80 °C and thaw them. Do not vortex. (8) While the HaPrP is thawing, prepare the reaction cocktail as follows, but do not add the HaPrP yet. · The reaction cocktail is for one full plate (scale down as needed for less than a full plate) · All components need to be filtered through a 0.22 μm filter within one month (Master mix / Reaction cocktail) · Can vary between batches · Vary depending on PrP concentration · 5X PBS: 2000 μL · 2M NaCl: 850 μL · 100 mM EDTA: 100 μL · 1 mM ThT: 100 μL (optional, if not using ThT, just add 100 μL of water) (9) Vortex the cocktail without using HaPrP. · If contamination is an issue, this cocktail can be filtered through a 0.2 micron syringe filter. (10) Make the tissue dilution buffer 0.1% SDS in PBS. (11) Once thawed, filter the HaPrP through a 100 kDa spin column at 3000 x g for 15 minutes. The filter can be used twice. (Tubes are usually 500 μL aliquots, and the maximum capacity of the filter is 500 μL). (12) While the HaPrP is sedimenting, appropriately dilute the tissue homogenate in the prepared solution (0.1% SDS in PBS) to 10 -10 (or the necessary dilution). The original homogenate is considered 10 -1 and so on. · For lymph and brain tissue, 10-3 That is the best option. • Control is 10 -3 It should be diluted to (1 μL control in 99 μL of prepared solution). (13) Gently add an appropriate amount of PrP to the reaction cocktail / master mix. (14) Mix by inversion. Do not vortex. (15) Gently inject 10 μL of the master mix into the microfluidic channel. (16) Seal the PTFE tube and place the microfluidic chip on a hot plate (45°C). (17) Connect the piezoelectric converter to the AC function generator. (18) Operate the function generator in a cycle of 30 seconds on and 30 seconds off for 4 hours. (19) Remove the gloves and discard them in the yellow barrel. (20) Clean the hood and the RT-QuIC bench. If the hood or anything that has come into contact with 50% bleach, wipe it with 70% EtOH. • All potentially infectious items, including disposable lab coats and gloves, should be placed in the yellow waste bin (if in doubt, place in the yellow waste bin). (Once the vibration protocol is complete) (21) Remove the microfluidic chip from the hot plate (22) Place the microfluidic chip on a fluorescence microscope (excitation wavelength and emission wavelength = 427 nm / 450 nm). (23) Measure the fluorescence intensity 20 times. Master Mix [Table 1]

[0058] As described herein, CWD is one exemplary disease that can be amplified within a sound-fluidic apparatus. Compared with conventional RT-QuIC, the method described here accelerates amyloid aggregation dynamics by utilizing microfluidic channels to limit shear-induced secondary prion amplification on a micrometer scale. This method is also compatible with a wide range of other sound actuators, such as thin finger-like membranes (sharp-edge structures) and sound cavities, instead of the immediate amplification using liquid and gas cavity structures for sound operation. This method is also compatible with various detection methods, such as fluorescence intensity measurement, aggregation analysis based on gold nanoparticles, and capillary action measurement.

[0059] The described examples include a functional prototype of a microfluidic device that utilizes sound to effectively amplify misfolded proteins. The microfluidic device can detect misfolded CWD prions within hours. This device and technology would also be useful for detecting misfolded α-synuclein associated with Parkinson's disease. Therefore, sound-based protein amplification would have broad utility for detecting an array of human and animal protein misfolding disorders. The microfluidic device, a method of mixing recombinant proteins with misfolded seeds, can amplify and detect protein misfolding disorders. Rapid detection of prion and protein misfolding disorders, including CWD, BSE, scrapie, Creutzfeldt-Jakob disease, Parkinson's disease, Alzheimer's disease, and PPID, is possible. Furthermore, the device is highly portable, enabling rapid diagnosis in a wide variety of environments.

[0060] The following examples are described below.

[0061] Example 1 In a system, the system comprises a housing configured to contain a main channel containing a fluid solution comprising a sample comprising a plurality of proteins; one or more side channels fluidly communicating with the main channel, each of which side channels is configured to establish a liquid-gas interface between the fluid solution in the main channel and the air contained within the one or more side channels; and at least one sound-generating element configured to generate sound waves in at least one of the gas in the one or more side channels or the fluid solution in the main channel, thereby causing vibration of the liquid-gas interface and mixing of the fluid solution in the main channel.

[0062] Example 2 The system in Example 1, wherein the main channel has a width of 100 micrometers to 500 micrometers.

[0063] Example 3 The system in Example 2 has a width of approximately 300 micrometers.

[0064] Example 4 A system according to any one of Examples 1 to 3, wherein each of the one or more side channels has a width of 50 micrometers to 500 micrometers.

[0065] Example 5 The system in Example 4 has a width of approximately 100 micrometers.

[0066] Example 6 A system according to any one of Examples 1 to 5, wherein the width of each of the one or more side channels is smaller than the width of the main channel.

[0067] Example 7 The system according to any one of Examples 1 to 6, wherein the sound generating element comprises a piezoelectric converter or a speaker.

[0068] Example 8 A system according to any one of Examples 1 to 7, wherein the sound generating element is in contact with a part of the housing.

[0069] Example 9 The housing comprises a glass slide and a first layer disposed on the glass slide and at least partially defining the main flow channel between the glass slide and the first layer, wherein the system further comprises a first pipe fluid-connected to the inlet of the main flow channel and a second pipe fluid-connected to the outlet of the main flow channel, the system according to any one of Examples 1 to 8.

[0070] Example 10 The system according to Embodiment 9, wherein the housing comprises a second layer made of PDMS, PMMA, or glass, which is disposed around at least a portion of the first layer and defines a space between the first layer and the second layer, and a hydrophobic fluid disposed within the space between the first layer and the second layer.

[0071] Example 11 The system according to any one of Embodiments 1 to 10 further comprises a control device configured to energize a sound-generating element for one or more periods of time.

[0072] Example 12 A system in any one of Examples 1 to 11, wherein the at least one sound-generating element is configured to generate sound waves in the gas or thin film of one or more side channels to bring the misfolded protein into contact with other proteins in the plurality of proteins, thereby amplifying a certain amount of the misfolded protein to a detectable level.

[0073] Example 13 A system in any one of Examples 1 to 12, wherein at least a portion of the housing is transparent and allows for the measurement of the fluorescence intensity of at least some of the multiple proteins in the sample in the main channel after mixing.

[0074] Example 14 The system in any one of Examples 1 to 13 further comprises one or more thin films protruding into the main channel, wherein each of the one or more thin films is configured to establish direct contact with the fluid solution in the main channel.

[0075] Example 15 A method comprising: a fluid solution containing a sample comprising a plurality of proteins in a main channel of a housing, wherein the housing comprises at least one of one or more side channels or one or more thin films that are in fluid communication with the main channel, and each of the one or more side channels or multiple thin films is configured to establish a liquid-gas interface between the fluid solution or direct contact in the main channel and the gas contained in the one or more side channels; and generating sound waves in the gas of the one or more side channels or at least one gas of the fluid solution in the main channel via at least one sound generating element, thereby causing vibration of the liquid-gas interface and mixing of the fluid solution in the main channel.

[0076] Example 16 The method in Example 15, in which generating sound waves also causes amplification of any misfolded proteins within multiple proteins.

[0077] Example 17 The method in Example 16, comprising generating sound waves to cause a detectable amount of the misfolded protein present in the sample to be released in less than two hours.

[0078] Example 18 The method in any one of Examples 15 to 17, wherein the plurality of proteins comprises one or more misfolded proteins associated with a disorder of incorrect protein folding.

[0079] Example 19 A method in Example 18 for a disorder of incorrect protein folding, including chronic wasting disease.

[0080] Example 20 The method described above further comprises detecting the properties of the plurality of proteins using an aggregate analysis sample based on gold nanoparticles, as in any one of Examples 15 to 19.

[0081] Example 21 The method in any one of Examples 15 to 20 further comprises detecting the properties of the plurality of proteins using at least one externally inserted or integrated capillary.

[0082] Various examples of the present disclosure have been described. Any combination of the described systems, operations, or functions is possible. These examples and other examples are within the scope of the following claims.

Claims

1. In the system, The aforementioned system, It is a housing, A main channel configured to contain a fluid solution containing a sample containing multiple proteins, A housing that defines one or more side passages that are in fluid communication with the main passage, each of which is configured to establish a liquid-gas interface between the fluid solution in the main passage and the gas contained in the one or more side passages, A system comprising at least one sound-generating element configured to generate sound waves in at least one of the gas in one or more side channels or the fluid solution in the main channel, the sound-generating element causing vibration at the interface between the liquid and the gas and mixing of the fluid solution in the main channel.

2. The system according to claim 1, wherein the main channel has a width of 100 micrometers to 500 micrometers.

3. The system according to claim 2, wherein the width is approximately 300 micrometers.

4. The system according to any one of claims 1 to 3, wherein each of the one or more side channels has a width of 50 micrometers to 500 micrometers.

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

6. The system according to any one of claims 1 to 5, wherein the width of each of the one or more side channels is smaller than the width of the main channel.

7. The system according to any one of claims 1 to 6, wherein the sound generating element comprises a piezoelectric converter or a speaker.

8. The system according to any one of claims 1 to 7, wherein the sound generating element is in contact with a part of the housing.

9. The aforementioned housing is Glass slide and, The apparatus comprises a first layer, which is disposed on the glass slide and at least partially defines the main flow channel between the glass slide and the first layer, and which comprises one of polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), or glass. The aforementioned system, The inlet of the main flow path and the first pipe connected to the fluid, The system according to any one of claims 1 to 8, further comprising a second pipe connected to the outlet of the main flow path.

10. The aforementioned housing is A second layer comprising PDMS, PMMA, or glass, positioned around at least a portion of the first layer and defining the space between the first layer and the second layer, The system according to claim 9, further comprising a hydrophobic fluid disposed in the space between the first layer and the second layer.

11. The system according to any one of claims 1 to 10, further comprising a control device configured to energize the sound generating element for one or more periods of time.

12. The system according to any one of claims 1 to 11, wherein the at least one sound-generating element is configured to generate sound waves in the gas or thin film of one or more side channels to bring the misfolded protein into contact with other proteins in the plurality of proteins and amplify a certain amount of the misfolded protein to a detectable level.

13. The system according to any one of claims 1 to 12, wherein at least a portion of the housing is transparent and allows for the measurement of the fluorescence intensity of at least some of the plurality of proteins in the sample in the main channel after mixing.

14. The system according to any one of claims 1 to 13, further comprising one or more thin films protruding into the main channel, wherein each of the one or more thin films is configured to establish direct contact with the fluid solution in the main channel.

15. In the method, The aforementioned method, The housing contains a fluid solution containing a sample comprising multiple proteins within its main channel, the housing comprises at least one of one or more side channels or one or more thin films that are in fluid communication with the main channel, and each of the one or more side channels or thin films is configured to establish a liquid-gas interface between the fluid solution or direct contact in the main channel and the gas contained in the one or more side channels. A method comprising generating sound waves in at least one gas in one or more side channels or in at least one gas in the fluid solution in the main channel via at least one sound-generating element, thereby causing vibration at the liquid-gas interface and mixing of the fluid solution in the main channel.

16. The method according to claim 15, wherein generating the sound waves also causes amplification of any misfolded proteins within the plurality of proteins.

17. The method according to claim 16, wherein generating the sound waves is performed to cause a detectable amount of the misfolded protein present in the sample to be released in less than two hours.

18. The method according to any one of claims 15 to 17, wherein the plurality of proteins comprises one or more misfolded proteins associated with a disease of incorrect protein folding.

19. The method according to claim 18, wherein the disorder of incorrect protein folding includes chronic wasting disease.

20. The method according to any one of claims 15 to 19, further comprising detecting the properties of the plurality of proteins using an aggregate analysis sample based on gold nanoparticles.

21. The method according to any one of claims 15 to 20, further comprising detecting the properties of the plurality of proteins using at least one externally inserted or integrated capillary.

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