Device for observing protein aggregation behavior and method for observing protein aggregation behavior using the same

Abstract

The present invention relates to a device for observing the aggregation behavior of proteins capable of observing the initial aggregation behavior of proteins, and a method for observing the aggregation behavior of proteins using the same.

Description

Technology Field

[0001] The present invention relates to an apparatus for observing protein aggregation behavior and a method for observing protein aggregation behavior using the same. More specifically, the invention relates to an apparatus for observing protein aggregation behavior capable of observing the initial aggregation behavior of proteins and a method for observing protein aggregation behavior using the same. Background Technology

[0003] Protein aggregation refers to the phenomenon in which proteins accumulate abnormally to form aggregates, and these abnormal aggregates can be toxic. Toxic aggregates adversely affect surrounding cells and can cause degenerative diseases such as neurological disorders, type 2 diabetes, and cancer. In particular, amyloidosis is a group of diseases caused by the abnormal aggregation and accumulation of a protein called amyloid; representative neurological diseases such as Alzheimer's disease and Parkinson's disease fall under this category. Furthermore, there exist various types of amyloidosis that exhibit diverse manifestations depending on the causative protein and the affected organ.

[0004] One of the important pathological features of amyloid diseases is the formation of protein-peptide aggregates called flakes. For example, amyloid-beta (amyloid beta 1-42), known as the primary causative protein of Alzheimer's disease, abnormally aggregates and accumulates around synapses and neurons to form plaques. This is known to cause synaptic dysfunction and neuronal death, ultimately leading to degenerative brain diseases. Recent studies have revealed that intermediate aggregates generated during the plaque formation process—such as oligomers, protofibrils, and fibrils—possess neurotoxicity and are associated with the onset of the disease.

[0005] For this reason, in order to understand the pathogenesis of protein aggregation-inducing diseases and develop treatments, mechanisms and methods capable of screening protein aggregation stages are required.

[0006] Currently, the main methods used to screen protein aggregation stages are to quantitatively measure the degree of aggregation using reagents such as Thioflavin T (ThT) or Congo red that selectively bind to protein peptide ends, or to track the formation and stages of aggregation structures using circular dichroism (CD).

[0007] However, this method has limitations in that the signal appears weak because sufficient substances reacting with the reagent are not formed during the initial agglutination reaction. As a result, it is difficult to effectively screen the initial agglutination reaction.

[0008] Although there are attempts to track protein aggregation behavior and stages using microscopes, the spatial resolution of conventional microscopes is insufficient to observe small aggregates, such as peptide monomers or oligomers, which are several nanometers in size. In addition, additional pretreatment steps such as fixation, staining, and tagging are required for observation, which can make the experimental process complex and reduce the accuracy of the results.

[0009] Therefore, there is a need for observation instruments and quantification methods capable of effectively screening the early aggregation reactions of protein aggregation-inducing diseases. Such technology can be utilized not only for the early diagnosis of diseases based on initial aggregation behavior but also for screening drugs such as protein aggregation inhibitors or anti-aggregation agents. This invention was developed to address these technical requirements. Prior art literature

[0011] Korean Patent Publication No. 10-2017-0133622 The problem to be solved

[0012] One objective of the present invention is to provide a device for observing the aggregation behavior of proteins that can observe the initial aggregation behavior of proteins without the use of reagents or pretreatment processes.

[0013] One objective of the present invention is to provide a method for observing the aggregation behavior of a protein using the above-described protein aggregation behavior observation device. means of solving the problem

[0015] One embodiment of the present invention provides an apparatus for observing the aggregation behavior of proteins, comprising: an upper chip and a lower chip comprising a two-dimensional material; a flow path formed between the upper chip and the lower chip; a pump for supplying proteins to the flow path; and a transmission electron microscope for observing the proteins.

[0016] The upper chip and lower chip may be formed by stacking one to five layers of a two-dimensional material.

[0017] The above two-dimensional material may be at least one of the group consisting of graphene, borophen, transition metal dichalcogenide (TMDC), and hexagonal boron nitride (hexagonal-BN).

[0018] The above two-dimensional material may have its surface modified with hydrogen or oxygen.

[0019] Another embodiment of the present invention provides a method for observing the aggregation behavior of proteins, comprising the steps of: supplying proteins to a flow channel formed by stacking two-dimensional materials; irradiating the proteins supplied to the flow channel with an electron beam to take an image; and analyzing the image taken.

[0020] The step of capturing the above image may involve synchronizing the electron beam irradiation and the image capture, and then recording the synchronized time at regular intervals.

[0021] The total amount of electron beam irradiated during the step of capturing the above image is 800 e - / A 2 It may be less than.

[0022] The step of analyzing the captured image may include: selecting protein particles in a single image; classifying the selected protein particles into similar forms; and quantifying the degree of aggregation of the classified protein particles.

[0023] It may further include a step of calculating the aggregation speed according to the above-mentioned quantified aggregation degree. Effects of the invention

[0025] The protein aggregation behavior observation device according to the embodiment of the present invention is designed to enable the quantification of the degree of aggregation and the calculation of the aggregation speed, and thus has the advantage of being able to observe the initial aggregation behavior of proteins without the need for reagents or pretreatment processes.

[0026] Furthermore, the method for observing protein aggregation behavior using a protein aggregation behavior observation device according to an embodiment of the present invention can quantify the degree of aggregation and calculate the aggregation rate, and thus can be utilized as a new medical diagnostic method for diagnosing the severity of disease. In addition, it can provide a rapid criterion for judging the efficacy of drugs developed to inhibit and mitigate protein aggregation, and thus can be utilized as an efficient drug screening method from a pharmaceutical perspective. Brief explanation of the drawing

[0028] FIG. 1 is a cross-sectional view of an upper chip (10), a lower chip (20), and a flow path (30) formed between the upper chip (10) and the lower chip (20) applied to a protein aggregation behavior observation device (1) according to the present invention. FIG. 2 is a flowchart of a method for observing the aggregation behavior of proteins according to one embodiment of the present invention. Figure 3 is a flowchart of the step (S300) of analyzing a captured image. FIG. 4 is a schematic diagram of a device for observing the aggregation behavior of proteins according to one embodiment of the present invention. Figure 5 illustrates the effect of the electron beam when observing proteins using an electron microscope. Figure 6 is a real-time observation of the initial aggregation behavior of amyloid beta protein. Figure 7 shows the process of quantifying protein aggregation. Figure 8 shows the degree of aggregation of proteins classified based on morphological similarity. Figure 9 is a graph showing the total number of aggregated particles classified according to the time frame of the real-time observation video. Figure 10 is an example of time-series observation, showing the results of tracking the aggregation behavior of amyloid proteins at intervals of about 5-10 minutes. Figure 11 is a graph showing the change in cohesion over time. Specific details for implementing the invention

[0029] Expressions such as "comprising" as used in this specification should be understood as open-ended terms implying the possibility of including other technical features.

[0030] As used herein, "as an example," "as an embodiment," and "preferably" refer to embodiments of the invention that may provide certain advantages under certain conditions, and are not intended to exclude other embodiments from the scope of the invention.

[0031] In this specification, "aggregation" refers to a phenomenon in which molecules or particles combine or clump together to form a larger structure, and "protein aggregation" refers to a phenomenon in which protein molecules interact abnormally due to denaturation or partial structural changes to form an aggregate.

[0032] In this specification, "aggregate" means an assembly formed by proteins, particles, or molecules combining or clumping together through interaction.

[0033] In this specification, "two-dimensional material" means a material that is thin at the atomic level and consists of one or more atomic layers.

[0034] An apparatus for observing the aggregation behavior of proteins according to one embodiment of the present invention comprises an upper chip and a lower chip comprising a two-dimensional material, a flow path formed between the upper chip and the lower chip, a pump for supplying proteins to the flow path, and a transmission electron microscope for observing proteins.

[0035] FIG. 1 is a cross-sectional view of an upper chip (10), a lower chip (20), and a flow path (30) formed between the upper chip (10) and the lower chip (20) applied to a protein aggregation behavior observation device (1) according to the present invention.

[0036] Referring to FIG. 1, an upper chip (10) is stacked on top of a lower chip (20) to form a channel (30) through which protein can be supplied. The protein can be continuously circulated and supplied through the channel (30).

[0037] The upper chip (10) and lower chip (20) above contain a two-dimensional material.

[0038] The upper chip (10) and lower chip (20) may each independently further include one or more of a substrate (110, 210), a support (120, 220), and a spacer (40).

[0039] The material of the above substrate may be silicon (Si).

[0040] The above support can be placed on one side of the substrate.

[0041] The material of the above support may be silicon nitride (SiNx) or silicon oxide (SiOx), etc.

[0042] The above two-dimensional material (130, 230) can be placed on one surface of the support.

[0043] The above two-dimensional material (130, 230) may be laminated in 1 to 5 layers on one surface of the support.

[0044] The above two-dimensional material (130, 230) may be laminated by transferring it in a dry or wet manner.

[0045] The above two-dimensional material (130, 230) can be formed on the innermost part of the upper chip (10) and lower chip (20) so as to interact with proteins.

[0046] The above two-dimensional material (130, 230) may be at least one of the group consisting of graphene, borophen, transition metal dichalcogenide (TMDC), and hexagonal boron nitride (hexagonal-BN) to prevent loss of resolution and clarity.

[0047] The above two-dimensional material (130, 230) may have its surface modified with hydrogen or oxygen to improve affinity with proteins.

[0048] The above surface modification can be performed by plasma treatment, chemical vapor deposition (CVD), ultraviolet (UV) irradiation, oxygen plasma process, wet chemical treatment, or heat treatment.

[0049] The above two-dimensional material (130, 230) can be applied to evaluate the interaction between the cell membrane and the protein by directly culturing cells on the two-dimensional material (130, 230) or placing cells on it for selective interaction with the protein.

[0050] The spacer (40) can be positioned at both ends between the upper chip (10) and the lower chip (20). Due to the spacer (40), a channel (30) is formed, which is a space through which proteins can pass when the lower chip (20) and the upper chip (10) are stacked. Therefore, the height of the channel (30) space is determined according to the height of the spacer (40).

[0051] The above spacer (40) can be formed of a metal, ceramic, or polymer material.

[0052] Next, I will explain the pump.

[0053] The pump supplies protein to the flow path formed between the upper chip and the lower chip.

[0054] The above pump may be a syringe pump.

[0055] The above syringe pump may supply the solution in the syringe (capacity of 0.5 mL to 5 mL) at a constant rate (supply rate per constant hour).

[0056] The above syringe pump may include a storage container (syringe), housing, motor, rotor, shaft, bearing, motor controller, temperature control device, etc.

[0057] The above storage container (syringe) has a multi-channel that can connect one or two, and may store a protein solution, a buffer solution, or a drug in each syringe. The solution may be supplied by a rotor connected to the motor of the syringe pump pushing the thumb pusher of the syringe to discharge the solution from the syringe to the tip. At this time, the supply speed is determined by adjusting the speed of the motor, and preferably, the speed may be 0.5 ul / min to 5 ul / min.

[0058] A Luer lock connector can be connected to the tip of the above syringe, and a PEEK tube can be used to directly supply the protein solution to the flow path of the chip.

[0059] The above storage tank may store protein and supply it to the Euro.

[0060] The above storage tank can store additional solutions other than protein and supply them to the flow path.

[0061] The solution other than the above protein may be a solution that affects protein aggregation, and non-limiting examples may include drugs that can inhibit or alleviate protein aggregation, buffer solutions that can affect the aggregation phenomenon, etc.

[0062] The above pump can control the protein supply rate by adjusting the slope.

[0063] The above pump may be tilted at an angle of 10° or less, 9° or less, 8° or less, or 7° or less from the ground. If the above range is satisfied, protein aggregation can be inhibited while the protein is supplied to the two-dimensional material of the upper chip or lower chip.

[0064] The following describes the transmission electron microscope.

[0065] The above transmission electron microscope may observe protein aggregation phenomena in real time by irradiating an electron beam onto a protein passing through a channel.

[0066] The above real-time observation results can be output to the detector as a video or image.

[0067] The electron beam of the above transmission electron microscope may penetrate two-dimensional materials and flow paths.

[0068] In the above transmission electron microscope, the moment the electron beam is irradiated and the time when it is output as a video or image can be synchronized.

[0069] The aforementioned synchronization refers to the process of aligning the moment an electron beam is irradiated onto a sample in a transmission electron microscope with the time when the corresponding image data is recorded and output by the detector. This enables the generation of real-time images based on changes in the electron beam or the accurate tracking of dynamic changes in a sample under specific conditions. The synchronization process is achieved through timing adjustments between the electron signal and the detection system, contributing to improved image precision and temporal resolution.

[0070] After the above synchronization, the electron beam can be irradiated at regular intervals, and the total irradiation dose is 1000 e - / A 2 below, 900 e - / A 2 Less than or equal to 800 e - / A 2 The incident dose and irradiation interval can be adjusted so as to be less than or equal to the above.

[0071] FIG. 2 is a flowchart of a method for observing the aggregation behavior of proteins according to one embodiment of the present invention.

[0072] Referring to FIG. 2, a method for observing the aggregation behavior of proteins includes the step of supplying proteins to a flow path (S100), the step of irradiating the proteins with an electron beam to take an image (S200), and the step of analyzing the image (S300).

[0073] The step of supplying protein to the above-mentioned flow path (S100) may be a step of supplying protein to a flow path formed between an upper chip and a lower chip containing a two-dimensional material.

[0074] The above protein supply can be performed on a pump.

[0075] The above pump can control the protein concentration or flow rate inside the fluid path.

[0076] The above pump can supply a solution that affects protein aggregation, in addition to protein, to the flow path.

[0077] The solution affecting the protein aggregation mentioned above may be a drug capable of inhibiting or mitigating protein aggregation, a buffer solution capable of affecting the aggregation phenomenon, etc.

[0078] The solution affecting the protein aggregation described above can simulate the natural state or aggregation conditions of proteins by controlling the fluid environment, such as pH and ionic strength, and such control enables precise observation and analysis of protein aggregation behavior.

[0079] The step of irradiating the protein with an electron beam to capture an image (S200) is a step of using a transmission electron microscope (TEM) to irradiate the protein passing through the fluid channel with an electron beam and recording structural changes and aggregation phenomena of the protein in real time.

[0080] The step (S200) of irradiating the above protein with an electron beam to take an image allows the electron beam to pass through the protein and two-dimensional material inside the fluid channel, and can generate a high-resolution image capable of analyzing the size, shape, and distribution of protein aggregates.

[0081] The total amount of electron beam irradiated in the step (S200) of capturing the above image is 1000 e - / A 2 below, 900 e - / A 2 Less than or equal to 800 e - / A 2 It may be less than or equal to the above range, and if the above range is satisfied, protein damage can be minimized and the accuracy of observation can be increased.

[0082] The step of capturing the above image may involve synchronizing the electron beam irradiation and the image capture, and then recording the synchronized time at regular intervals.

[0083] The step of capturing the above images can track temporal changes in protein aggregation behavior by recording images at regular time intervals based on a synchronized point in time. Through this, the initial state, aggregate growth, and final form during the protein aggregation process can be precisely analyzed.

[0084] In addition, image data can be processed and analyzed in real time to derive quantitative data regarding protein aggregation phenomena (e.g., aggregate size distribution, concentration changes, etc.). This process can be utilized to evaluate the efficacy of protein aggregation inhibitors or promoters, or to understand the kinetic characteristics of protein aggregation under specific environmental conditions.

[0085] The image captured in the step of capturing the image (S200) can be stored in a digital format and used as data for subsequent analysis.

[0086] Figure 3 is a flowchart of the step (S300) of analyzing a captured image.

[0087] The step of analyzing the captured image (S300) includes the step of selecting protein particles in a single image (S310), the step of classifying the selected protein particles into similar forms (S320), and the step of quantifying the degree of aggregation of the classified protein particles (S330).

[0088] The step of selecting protein particles in the single image (S310) is a step of selecting protein particles in a single image or a single frame of the captured protein. At this time, the protein particles may be structures exhibiting a specific aggregation state, such as single molecules, dimers, or trimers, and may be selected based on characteristics of size, shape, or electron density. Additionally, the selected protein particles may be separated from background noise or other artifacts of the image and purified into a form suitable for subsequent analysis.

[0089] The step of classifying the selected protein particles into similar forms (S320) is a step of grouping the selected protein particles according to size, shape, electron density distribution, or structural features. In this step, protein particles of similar forms can be classified into the same class.

[0090] The step of quantifying the degree of aggregation of the classified protein particles (S330) is a step of quantitatively evaluating the relative concentration, size, and structural characteristics of aggregates by analyzing the number and distribution of protein particles within each class. In this process, the density, size distribution, and aggregate formation ratio between particles derived from 2D or 3D data are calculated, and the formation kinetics and stability of protein aggregates can be numerically expressed. The quantified degree of aggregation can be used to compare the difference between the natural state of the protein and the aggregation conditions, or to evaluate the effect of an aggregation inhibitor or promoter.

[0091] In the present invention, the degree of aggregation is an indicator that quantitatively represents the extent to which protein particles form aggregates. This includes states ranging from single molecules (monomers) to aggregates formed by the combination of multiple molecules, and can be calculated based on the size, shape, distribution, and concentration of aggregates of the protein particles. The degree of aggregation is calculated by quantifying changes in morphological similarity, inter-particle distance, size distribution, or particle count based on image data of observed protein particles. In particular, the present invention precisely evaluates the degree of aggregation by classifying it from primary aggregates to higher-order aggregates, which can be utilized as an important criterion for analyzing the aggregation rate and the effect of drugs.

[0092] The step of analyzing the captured image (S300) may further include the step of calculating the aggregation speed according to the quantified aggregation degree (S340).

[0093] The step of calculating the aggregation speed according to the quantified aggregation degree (S340) is a process of calculating the rate at which protein aggregation proceeds by analyzing the change in the quantified aggregation degree over time. In this step, the rate of increase in aggregation degree for each time interval is measured from the data arranged in chronological order, and the average or maximum value of the aggregation speed is derived through this. The aggregation speed may vary depending on the protein concentration, environmental conditions (pH, temperature, ionic strength, etc.), and whether drug treatment is applied, thereby allowing for a clear understanding of the kinetic characteristics of protein aggregation. The calculated aggregation speed can be utilized to evaluate the effects of aggregation inhibitors or promoters, or to provide basic data on aggregation phenomena associated with diseases.

[0095] The present invention will be described in more detail below through examples. These examples are merely illustrative of the present invention, and therefore the scope of the present invention should not be interpreted as being limited by these examples.

[0097] Test example

[0098] FIG. 4 is a schematic diagram of a device for observing the aggregation behavior of proteins according to one embodiment of the present invention. Using the device of FIG. 4, the aggregation behavior of amyloid beta 1-42 protein, which is believed to cause Alzheimer's disease, was observed, and a method for quantifying the degree of aggregation and an aggregation rate constant for judging the drug screening effect were calculated.

[0099] Referring to Fig. 4, a graphene film is placed on the surface of two chip-shaped devices, and the device is placed in a holder capable of supplying liquid. Then, unaggregated monomers of aggregating proteins are supplied through an external pump, and the phenomenon of proteins aggregating from monomers can be observed through a transmission electron microscope.

[0100] Figure 5 illustrates the effect of an electron beam when observing proteins using an electron microscope. Since protein degradation and damage occur when a high-energy electron beam is irradiated onto a protein, it is important to minimize electron beam exposure. As shown in Figure 5, excessive exposure to the electron beam oxidizes and damages proteins; therefore, the ideally accumulated electron beam irradiation dose (incident electron, e) - / A 2 ) to 800 e - / A 2 It is important to maintain it at 800 e under limited observation conditions. - / A 2 The electron beam must be efficiently split to analyze time-series changes. For example, 10 e - / A 2 When continuously observed with an incident dose of s, real-time observation is possible for about 80 seconds, but when observed at intervals of 10 minutes, aggregation behavior can be observed for more than 1 hour.

[0101] Figure 6 is a real-time observation of the initial aggregation behavior of amyloid beta proteins. Referring to Figure 6, the observed small, black dot-shaped particles are proteins, and dynamic changes in shape and size were observed during an observation period of one minute. This demonstrates the dynamic equilibrium state, a common characteristic of proteins, showing that they maintain a state of morphological and chemical equilibrium while changing over a short period of time.

[0102] Figure 7 illustrates the process of quantifying protein aggregation. According to Figure 7, the degree of aggregation of protein aggregates observed in video frame images and time-series images was quantified based on morphology. To this end, protein particles were classified according to morphological similarity and divided into first-order aggregates, fifth-order aggregates, and high-order aggregates. Additionally, in this process, the classification criteria can be subdivided into three or more stages.

[0103] Figure 8 shows the degree of protein aggregation classified based on morphological similarity. Referring to Figure 8, images of representative particles classified by degree of aggregation based on morphological similarity show that as the degree of aggregation increases, the clumping of the particles becomes more pronounced. At this time, it can be seen that various previously known shapes, such as curved, spherical, and disc shapes, coexist in terms of the size and shape of the amyloid particles.

[0104] Figure 9 graphs the total number of aggregated particles classified according to the time frame of the real-time observation video. Referring to Figure 9, it shows that during real-time observation of about 1 minute, even if the particles exhibit dynamic movement, the overall total amount does not change significantly and remains constant. This demonstrates the dynamic equilibrium state of the protein, confirming that proteins with varying degrees of aggregation maintain a constant ratio of shape during short moments.

[0105] The aggregation behavior of proteins of actual pathological interest occurs over time, days, months, and years, requiring a method to track and observe it. To this end, a system capable of tracking changes in aggregation over a set period was developed. An important point here is the need to synchronize time-interval observation with electron beam irradiation to prevent protein damage caused by the electron beam. For example, during the aging period when observation is not taking place, the electron beam must be blocked or diverted to prevent it from reaching the observation area.

[0106] Figure 10 is an example of time-series observation, showing the results of tracking the aggregation behavior of amyloid proteins at intervals of approximately 5 to 10 minutes. Referring to Figure 10, it can be seen that the number of particles with high aggregation increases over time. Although observation was conducted at the minute level in the example, long-period aggregation analysis at the hourly and daily levels is also possible through the optimization of observation conditions.

[0107] Figure 11 shows the graph of the change in cohesion over time. Referring to Figure 11, it can be seen that the aggregation of high-cohesion aggregates occurs rapidly over time. At this time, the cohesion levels increase exponentially over time ( It follows ), and the aggregation rate constant, k = 1 / t 1 The aggregation rate constant for each degree of aggregation can be calculated through the following method. The aggregation pattern under the given conditions can be determined by comparing the calculated aggregation rate constants for each case. In this case, it can be seen that the environment is one where aggregation is dominant, as the aggregation rate of aggregates with a high degree of aggregation of 3rd grade or higher is higher than in other cases.

[0108] By using these methods to analyze the degree of aggregation and aggregation rate under mixed conditions with protein aggregation inhibitory / mitigating drugs, it can be utilized for initial screening of drug efficacy. Explanation of the symbols

[0110] 10: Upper chip 20: Bottom chip 30: Euro 40: Spacer 110: Upper substrate 120: Upper support 130: Upper two-dimensional material 210: Lower substrate 220: Lower support 230: Lower two-dimensional material

Claims

Claim 1 An apparatus for observing the aggregation behavior of proteins, comprising: an upper chip and a lower chip containing a two-dimensional material; a flow path formed between the upper chip and the lower chip; a pump for supplying protein to the flow path; and a transmission electron microscope for observing the protein. Claim 2 A device for observing protein aggregation behavior according to claim 1, wherein the upper chip and the lower chip are formed by stacking 1 to 5 layers of a two-dimensional material. Claim 3 A device for observing protein aggregation behavior according to claim 1, wherein the two-dimensional material is at least one of the group consisting of graphene, borophen, transition metal dichalcogenide (TMDC), and hexagonal boron nitride (hexagonal-BN). Claim 4 A device for observing the aggregation behavior of proteins according to claim 1, wherein the two-dimensional material has a surface modified with hydrogen or oxygen. Claim 5 A method for observing the aggregation behavior of proteins, comprising: a step of supplying proteins to a flow channel formed by stacking two-dimensional materials; a step of irradiating the proteins supplied to the flow channel with an electron beam to take an image; and a step of analyzing the image taken. Claim 6 A method for observing the aggregation behavior of proteins according to claim 5, wherein the step of capturing the image is to synchronize the electron beam irradiation and the image capture, and then record the synchronized time points at regular intervals. Claim 7 In claim 5, the total amount of electron beam irradiated in the step of capturing the image is 800 e - / A 2 Lee Ha-in, Method for observing protein aggregation behavior. Claim 8 A method for observing the aggregation behavior of proteins according to claim 5, wherein the step of analyzing the captured image comprises: selecting protein particles in a single image; classifying the selected protein particles into similar forms; and quantifying the degree of aggregation of the classified protein particles. Claim 9 A method for observing the aggregation behavior of a protein, further comprising the step of calculating the aggregation speed according to the quantified aggregation degree in claim 8.

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