Apparatus and method for isoelectric aggregation with pH measurement

Abstract

Embodiments described herein relate to a device or apparatus for isoelectric aggregation precipitation of proteins on a horizontal plane, an apparatus and method for separating and precipitating an analyte of interest from a sample by sequentially adjusting the pH of the solution and pooling and separating the analyte of interest according to corresponding isoelectric points of the analyte of interest. The apparatus and method include subjecting a sample to fluid kinematics resting on a horizontal plane, applying an electric field parallel to the horizontal plane through an anode and a cathode on both sides of the sample, and then pooling and precipitating an analyte fraction of interest according to their respective isoelectric point movement.

Description

Technical Field

[0001] This invention belongs to the field of protein isoelectric aggregation precipitation technology. Some embodiments described herein relate to apparatus and methods for collecting and depositing protein samples. Background Technology

[0002] Isoelectric aggregation precipitation of proteins is a commonly used method for protein separation.

[0003] Existing technologies, such as the "Method for Preparing Albumin" (Authorization Announcement No.: CN103012581B).

[0004] Current technologies primarily adjust the pH of a solution by adding acids or bases. However, without pH measurement, substances with similar isoelectric points to the target analyte can easily precipitate along with it. Furthermore, the pH adjustment precision of current technologies is limited to one decimal place.

[0005] Electrophoresis, including isoelectric focusing (IEF), is a common technique for protein separation. IEF is an electrophoretic technique that separates proteins and other amphoteric solutes based on their isoelectric points (pI) along a pH gradient. The synthetic carrier amphoteric electrolyte is a small amphoteric molecule that rapidly establishes a pH gradient after an electric field is applied. Once the pH gradient is established, slower-moving proteins and other amphoteric molecules concentrate at their pI. In IEF, the electric field drives protein movement to separate proteins. The process is as follows: when the pH of the protein's environment is not equal to its isoelectric point (pI), the protein becomes charged in the pH gradient and moves towards the opposite electrode until it reaches an environment where the pH equals its isoelectric point (pI), where it remains. Finally, all proteins remain at positions in the pH gradient where the pH equals their respective isoelectric points (pI).

[0006] Isoelectric focusing (IEF) technology utilizes the properties of synthetic carrier amphoteric electrolytes to rapidly establish a pH gradient after applying an electric field. With continuous application of the electric field, slower-moving proteins and other amphoteric molecules concentrate at their pI (particle size index). One drawback of existing IEF technology is that the electrolyte used to establish the pH gradient is incompatible with common downstream analyses and mass spectrometry methods.

[0007] In isoelectric focusing (IEF) technology, the synthetic carrier amphoteric electrolyte is replaced with electrolyzed water. Although no pH gradient is formed after an electric field is applied, redox reactions occur at the anode and cathode, leading to changes in the solution's pH. The cathode reaction for acidic water electrolysis is: The anode reaction of acidic water electrolysis is: The cathode reaction of alkaline water electrolysis is: Anode reaction in alkaline water electrolysis: .

[0008] Most electrophoresis techniques, including isoelectric focusing (IEF), involve anodes and cathodes that are conductive to the sample, electrolyte buffer, electrolyte solution, sample buffer, or sample diluent. Applying a voltage to the anode and cathode creates a closed-loop circuit with a continuous current flow. However, in some cases, such as when the sample volume is very small, the anode or cathode is insulated from the sample (but must be close), or both the anode and cathode are insulated from the sample (but must be close), initially, a certain current flows across the anode and cathode, and a certain degree of chemical reaction occurs at the electrode conductive to the sample. However, this current stops within a short time, and the chemical reaction at the electrode also ceases. The voltage across the two electrodes then becomes independent of the protons in the sample. and / or hydroxide ions The concentration will reach equilibrium.

[0009] One of the improved techniques of isoelectric focusing (IEF) technology, the invention patent "Method for Single-Channel Free-Flow Electrophoresis with Sequential pH Adjustment" (application publication number CN114728213A), also strives to achieve the elimination of the use of synthetic carrier amphoteric electrolytes. This patent is mainly for the preparative stage; the apparatus or equipment described in this patent has inherent limitations for the analytical stage, necessitating the development of new technologies.

[0010] Medical testing often requires measuring the content of a single analyte, but it also requires measuring the total content of several analytes at once, such as the total content of several proteins.

[0011] Analytical-grade electrophoresis apparatus or equipment that is convenient for microscopic examination is an important requirement in industries such as pharmaceuticals.

[0012] In existing technologies, including the aforementioned patented technologies, the electric field driving electrophoresis is unidirectional, and within the effective region, the electric field lines are essentially parallel. For analytical-grade electrophoresis operations with small sample volumes, a convergent or divergent electric field can be particularly significant.

[0013] The prior art, including the aforementioned patent, adjusts the pH of the electrolyte by modifying the ratio of MES and BisTris in the electrolyte solution. Specifically, it also uses metering pumps or valves and electrolyte temperature to adjust the pH. This prior art method of adjusting pH does not involve pH measurement; it relies entirely on empirical knowledge of the relationship between MES, BisTris, temperature, and electrolyte pH. For preparative-grade electrolytes with large sample volumes and in motion, this might be adequate for pH measurement. However, for analytical-grade electrolytes with small sample volumes and in a static state, the accuracy is insufficient, or should be improved, to ensure accurate precipitation of the analyte of interest when the sample solution contains other substances with similar isoelectric points.

[0014] Existing technologies, such as the methods for single-channel free-flow electrophoresis with sequential pH adjustment (CN114728213B), methods for removing proteins from protein-rich wastewater (CN102659233A), and methods for preparing albumin (CN103012581B), describe pH values ​​with only one decimal place in their specifications and / or claims. This indicates that existing electrophoresis and isoelectric point precipitation techniques can only adjust the pH of buffer solutions or sample solutions to one decimal place. In some practical applications, such as medical testing, the difference between the isoelectric point (pI) of other substances in the sample solution and the analyte of interest, as well as the difference between the isoelectric points of analytes, is less than 0.1. Even if the isoelectric point (pI) difference is greater than 0.1, in many cases, the adjustment precision of only one decimal place can lead to the precipitated analyte being contaminated with other analytes or substances, reducing the purity of the analyte of interest after precipitation. In conclusion, the existing methods for adjusting pH values ​​that have been disclosed clearly cannot meet practical needs.

[0015] Furthermore, existing methods for adjusting pH, namely, utilizing the differences in kPa among different buffer solutions to adjust the pH of the sample solution or buffer solution by adjusting the ratio of different buffer solutions, employ pumps and other methods to apply fluid dynamics forces to generate convection, in order to facilitate the movement of hydroxide ions... and protons While a uniform distribution is desirable, thermal diffusion remains the primary thermodynamic force driving the sample solution to achieve a uniform and accurate pH, in order to ensure the sample solution pH accurately meets requirements, such as reaching the isoelectric point (pI) of the analyte of interest. Generally, reaching thermodynamic equilibrium of the sample solution pH accurately through thermal diffusion takes a relatively long time.

[0016] In conclusion, the existing methods for adjusting pH values ​​that have been disclosed are clearly insufficient to meet practical needs, and a completely new technology in terms of principle and mechanism is required.

[0017] In view of the shortcomings and limitations of current technologies and the practical needs of industries such as pharmaceuticals, this disclosure describes an apparatus and method for free electrophoresis without fluid dynamics barriers for the collection and precipitation of analytes of interest in fluid kinetics-stationary samples. This disclosure also describes the principle and operation of adjusting the pH of the sample solution through electrodynamic equilibrium. The apparatus described herein is operable to collect and separate analytes with specific pIs. The apparatus described herein can also be operated to collect a series of analytes with specific pIs and precipitate these analytes for measuring the total content of these analytes. Compared with prior art, the apparatus described herein keeps the sample in a stationary state, replacing the prior art where the sample must be in a flowing state, reducing sample consumption and facilitating real-time microscopic observation of the sample. The apparatus described herein also employs real-time pH measurement, and precisely adjusts the sample pH by adjusting the cathode voltage and adjusting the anode voltage, so as to accurately precipitate analytes with specific pIs and reject the erroneous precipitation of analytes with similar pIs. The device described herein employs a unique design and separate operation specifically for collecting analytes of interest, allowing trace amounts of analytes in a sample to be concentrated in a small area for convenient further manipulation. Some embodiments described herein do not require a synthetic carrier amphoteric electrolyte; instead, electrolysis of water containing dilute sulfuric acid or sodium hydroxide solution is used.

[0018] As described above, the device disclosed herein employs pH measurement technology to improve the accuracy of pH adjustment and generate information feedback for adjusting the pH of the sample solution.

[0019] Existing technologies for measuring the pH of solutions mainly rely on pH test strips, which depend on visual observation and color comparison to determine the pH of the solution. This method is highly subjective, and accurate pH results vary from person to person.

[0020] Existing technologies for measuring solution pH include pH electrode probe technology. However, pH electrode probe technology has the drawback of requiring frequent calibration. Ordinary pH electrodes are relatively large and cannot measure the pH of minute amounts of solution (e.g., a drop of aqueous solution, approximately 0.05 ml). Miniature pH electrode probes are available on the market; however, they require sophisticated manufacturing processes and are expensive. If funds allow, they can be considered as one option for pH measurement or as a supplement to other pH measurement technologies.

[0021] The technique of measuring solution pH using fluorescent substances has undergone decades of use and improvement. Among them, pyranine (HPTS, Solvent Green 7) is widely used due to its unique and excellent properties, such as excited-state proton transfer (ESPT) properties. Although ESPT properties have been utilized for more than half a century, the use of pyranine as a fluorescent probe to detect the surrounding hydration layer is only a recent development.

[0022] The completed research and development work has proven that the fluorescent substance HPTS (pyranine) is successful in pH measurement in liposomes.

[0023] Measuring solution pH using the excitation and emission spectra of fluorescent substances presents a significant challenge: the selection of the fluorescent substance support and minimizing its influence on the fluorescent substance, including minimizing or eliminating covalent bonds between the support and the fluorescent substance. The development of silica (silica) as a fluorescent substance support technology has yielded promising results. This article discloses the latest advancements in fluorescent probe technology in this area. Summary of the Invention

[0024] Some of the embodiments described herein relate to apparatus and methods for collecting, precipitating, and separating samples containing biological materials or analytes such as proteins.

[0025] Some embodiments described herein relate to an instrument configured to allow a sample, placed at hydrodynamic rest on a horizontal plane without any hydrodynamic force other than gravity, to electrophoretically move and accumulate a mixture of analytes contained therein, and to sequentially precipitate at its isoelectric point (pI). An anode and cathode are positioned on either side of the sample. When energized (i.e., when a potential is applied to the anode and cathode), the anode and cathode can jointly induce an electric field parallel to the horizontal plane and oriented through the sample. As discussed further in detail below, analytes having an isoelectric point (pI) different from the pH of the sample solution can migrate along the direction of the electric field (parallel to the horizontal plane and pointing towards an electrode, such as the anode) to the edge of the sample solution near the electrode (e.g., the anode). The pH of the sample solution is then sequentially adjusted such that the analyte mixture precipitates at its isoelectric point.

[0026] In some embodiments, the anode in the device is configured to be insulated from the sample solution, and the cathode in the device is configured to be conductive to the sample solution. When energized (i.e., when a potential is applied to the anode and cathode), the sample solution is in an alkaline state. Within appropriate ranges, the lower the cathode voltage (more negative), and / or the higher the anode voltage (more positive), the higher the pH of the sample solution. As discussed further in detail herein, the pH of the sample solution can be controlled by adjusting the anode and / or cathode voltage in conjunction with the addition of acid or base.

[0027] In some embodiments, the solvent of the sample solution is water, which is the main component of the sample solution. The pH of the sample solution can be controlled by controlling the anode voltage and / or cathode voltage. As described above, through specific settings, for example, the anode in the device is set to be insulated from the sample solution, and the cathode in the device is set to be conductive to the sample solution, the anodic half-reaction is inhibited; however, the cathodic half-reaction proceeds smoothly, with the protons... The reduction and hydroxide ions The increase of ions and their diffusion to other regions of the solution through electric field force and thermal diffusion makes the voltage applied to the electrode more sensitive to the protons in the solution. and hydroxide ions The concentration reaches electrochemical dynamics equilibrium.

[0028] In some embodiments, after energization (i.e., when a potential is applied to the anode and cathode), no chemical reaction occurs on the anode surface because the anode is insulated from the sample solution. However, the edge region of the sample solution near the anode will repel cations, for example... It attracts anions, such as hydroxide ions. This causes the pH in this area to rise. After energizing, because the cathode is conductive to the sample solution, a chemical reaction occurs on the cathode surface—the cathode reaction described above for acidic and alkaline water electrolysis. Both reactions result in an increase in the pH near the cathode. As the reaction on the cathode surface continues, the hydroxide ions produced by the reaction increase... Leaving the cathode (which requires a slightly higher voltage difference to be applied between the anode and cathode), hydroxide ions in the sample solution near the cathode... More and more hydroxide ions are appearing in the sample solution. The movement of ions under the influence of an electric field and their spontaneous diffusion cause hydroxide ions in the sample solution to... The pH of the sample solution gradually becomes uniformly distributed, reaching a uniform alkaline state. After gradually increasing to a certain value, the pH of the sample solution stops increasing, reaching electrodynamic equilibrium with the cathode potential and anode potential.

[0029] In some embodiments, after energizing, changing the cathode and anode potentials allows the pH of the sample solution to reach a new electrodynamic equilibrium with respect to these potentials, thus altering the pH of the sample solution. Since there are no hydrodynamic obstacles in the sample solution, hydroxide ions... and protons The rapid fluid kinematics of the proton, combined with the small volume of the sample solution, results in a short time to reach a new electrodynamic equilibrium. This allows for quick and precise pH adjustment of the sample, enabling proteins of interest to be maintained at the correct pH for the precise duration, accurately precipitating at their isoelectric points and rejecting the erroneous precipitation of proteins with similar isoelectric points. In some embodiments, a new electrodynamic equilibrium can be reached from the previous one within 2-3 seconds after changing the cathode and / or anodic potentials.

[0030] In some embodiments, the initial pH of the sample solution is less than 7, and the initial state of the sample solution is acidic. This is because the cathode reactions in acidic and alkaline water electrolysis consume protons. (proton) and the generation of hydroxide ions Both of these processes increase the pH in the region near the cathode. Therefore, after a period of time following the application of electricity, the pH of the sample solution will gradually increase. The specific pH value that the sample solution can reach depends on the potential difference between the anode and the cathode.

[0031] In some embodiments described herein, the thermodynamic voltage of the electrode reaction at the anode and cathode surfaces is correlated with temperature, meaning the voltage required for the electrode reaction can be altered by changing the temperature. However, in actual cathode reactions, a voltage lower than the theoretical reaction voltage (e.g., -1.23V at 25°C) must be applied for the electrode reaction to proceed normally. This lower voltage is primarily used to overcome the inherent activation energy barrier present on the cathode, as well as other resistances, such as solution resistance and contact resistance.

[0032] In some embodiments, in addition to selecting suitable electrode materials, the effective active area of ​​the electrode is also an important factor affecting the voltage required for the electrode reaction. By optimizing the electrode preparation method, for example, by introducing nanostructures, the electrochemical active surface area of ​​the electrode can be increased.

[0033] In some embodiments, bubbles are generated on the electrode surface during the electrode reaction. These bubbles should be removed from the electrode surface as much as possible to reduce the time required to reach electrodynamic equilibrium between voltage and pH, reduce the time required to adjust pH, and improve pH accuracy.

[0034] In some embodiments described herein, the anode of the DC power supply that generates the voltage (i.e., the potential generator that applies a potential to the anode and cathode) should be robustly grounded so that the pH of the sample solution can be stably increased to the desired value by reducing the cathode voltage.

[0035] In some embodiments, a series of uniformly distributed pH probes are fixed on a glass slide in the device. These probes can measure the pH of the sample solution on the glass slide, providing data for precise adjustment of the sample solution pH. In some embodiments, the pH probes are pH electrode probes.

[0036] In some embodiments, one side of a glass slide in the apparatus is covered with a uniform, transparent film containing a fluorescent substance, and the covered side of the slide is horizontally fixed to the stage of a fluorescence microscope with the covered side facing upwards. In some embodiments, the sample solution is subjected only to the hydrodynamic forces of gravity and is held at rest on the covered slide by hydrodynamic kinematics, allowing the pH of the sample solution to be measured.

[0037] In some embodiments, the glass slide is made of quartz.

[0038] In some embodiments, the main component of the transparent film on the glass slide is silica sol, wherein examples of silica sol include aqueous solutions of silicates ("water glass") and silanols, wherein examples of silanols include tetramethoxysilane (TMOS), tetraethoxysilane (TEOS), and γ-aminopropyltriethoxysilane (APTES, silane coupling agent KH-550).

[0039] Because the covalent bonds between the carrier and the fluorescent substance can alter the absorption and emission spectra of the fluorescent substance, in some embodiments, fluorescent substances and processes that do not chemically react with the carrier are selected. In some embodiments, the fluorescent substance is trisodium 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS). The attached figures show the excitation and emission spectra of HPTS, as well as the logarithm of the HPTS excitation light intensity ratio versus pH value.

[0040] In some embodiments, the relevant technical solution for measuring the pH of the sample solution is as follows: Step 1, Preparation of the curing coating on the glass slide: a) Dissolve HPTS in 28% ammonia water at a weight ratio of 210 g / kg; b) Coat APTES onto a quartz glass slide; c) Drop the ammonia solution obtained in step 1a) onto the APTES coating on the glass slide obtained in step 1b), covering the APTES coating; d) Dry the glass slide with the coating obtained in step 1c) at 10-20 degrees Celsius (24 hours); e) Store the glass slide with the dried and cured coating in a light-proof environment at -15°C (shelf life 24 months). Step 2, Calibration: a) Drop a solution with a known pH value onto the glass slide coating obtained in step 1; b) Measure the excitation light intensity of HPTS molecules at wavelengths of 400 nm and 450 nm on the coating, and calculate the intensity ratio logarithm; c) Use mathematical statistics to find the relationship curve between the pH value and the intensity ratio logarithm. Step 3, Measurement: a) Add the liquid to be tested to the coated side of the glass slide; b) Repeat step 2b); c) Use the logarithm of the intensity ratio obtained in step 3b) to compare with the relationship curve obtained in step 2c) to determine the pH value of the liquid being tested.

[0041] In some embodiments, examples of mathematical statistical methods used in the calibration step of a technical solution for measuring the pH of a sample solution include univariate linear regression, multiple linear regression, and nonlinear regression. In some embodiments, examples of mathematical statistical methods used in the calibration step of a technical solution for measuring the pH of a sample solution include the least squares method.

[0042] In some embodiments, the ambient temperature of the sample solution is maintained between 5 and 25 degrees Celsius. In some embodiments, the ambient temperature of the sample solution is adjusted so that the relationship between the cathode voltage and the pH of the sample solution is within a range that is favorable for controlling the pH of the sample solution by the cathode voltage; for example, the relationship between the cathode voltage and the pH of the sample solution is approximately linear.

[0043] In some embodiments, a transparent film containing fluorescent material on a glass slide dissolves upon contact with an aqueous solution, releasing the fluorescent material. This allows the fluorescent material to directly contact the sample solution, enabling accurate measurement of the sample solution's pH.

[0044] In some embodiments, the liquid addition device in the apparatus can add precise amounts of acid or alkali to the sample solution to adjust the pH of the sample solution, assisting other methods for adjusting the pH of the sample solution.

[0045] In some embodiments, the liquid addition device in the apparatus can precisely add liquid to the sample solution, examples of which include hydrophilic organic solvents and / or various salts, to promote the precipitation of specific analytes in the sample.

[0046] In some embodiments, the operation process is as follows: 1) A glass slide with a transparent film containing fluorescent material on one side is fixed on the stage of a fluorescence microscope with the film facing upwards; 2) The sample is added to the glass slide; 3) The sample volume and sample pH are estimated, and the amount of acid or base (e.g., dilute sulfuric acid or sodium hydroxide) needed to make the pH of the sample solution greater than the isoelectric point of the analyte of interest with the highest isoelectric point is estimated. Based on the estimation, acid or base is added to the sample; 4) The fluorescence microscope is started, and the pH of the sample solution is measured. The pH of the sample solution is adjusted according to the measurement results so that the pH of the sample solution is greater than the isoelectric point of the analyte of interest with the highest isoelectric point. If necessary, the above measurement and adjustment process can be repeated multiple times; 5) A potential is applied to the anode and cathode so that all analytes of interest and other analytes with isoelectric points lower than the pH of the sample solution move to a small region in the sample solution near the anode; 6) The pH of the sample solution is lowered to the isoelectric point of the analyte of interest with the highest isoelectric point (referred to as "first analyte") and maintained, so that the first analyte precipitates; 7) Operation 6) above is repeated so that all analytes of interest precipitate sequentially.

[0047] In some embodiments, protein isoelectric point precipitation, involving proton diffusion and the reorganization of the protein molecule's outer hydration shell, requires a certain amount of time, thus necessitating the maintenance of a specific ambient pH for a given period. Conversely, non-target analytes will not precipitate after a short period when the ambient pH equals their isoelectric point.

[0048] In some embodiments, as the analyte of interest precipitates sequentially, the pH of the sample solution gradually decreases. During this pH decrease, the sample solution pH remains at the isoelectric point of the non-target analyte for a very short time, preventing precipitation. When the sample solution pH falls below the isoelectric point of the non-target analyte, the non-target analyte becomes positively charged and begins to migrate towards the cathode, leaving the small region.

[0049] As described herein, in some embodiments, to improve the purity and percentage of the precipitated analyte of interest in the total sample, the operation is performed in two steps, forming two small regions. In the first region, a non-target analyte present in a higher concentration, such as albumin in blood, is precipitated. In some embodiments, to further improve the purity of the precipitated analyte of interest, the operation can be performed in three or more steps. The preceding steps precipitate various non-target analytes from the sample, and the final step precipitates all analytes of interest. Attached Figure Description

[0050] Instruction manual attached Figure 1The diagram illustrates the assembly of an apparatus according to an embodiment, in which a sample, placed on a horizontal plane, contains an analyte mixture that is electrophoretically moved and aggregated, and precipitates sequentially at its isoelectric point (pI). It shows five components: a fluorescence microscope, a coated slide, typically made of quartz, fixed to the stage of the fluorescence microscope, anodes and cathodes on either side of the slide, typically made of metal, and a handheld liquid addition device.

[0051] Instruction manual attached Figure 2 The following is shown from a top-down perspective: a glass slide, a sample solution on a glass slide coating, a small region in the sample solution where the analyte of interest is concentrated, an anode, a cathode, a conductor that conducts electricity with the sample solution, and electric field lines, according to an embodiment.

[0052] Instruction manual attached Figure 3 It consists of a glass slide and a transparent thin film containing fluorescent material covering the slide.

[0053] Instruction manual attached Figure 4 The HPTS excitation and emission spectra are shown. (See attached image.) Figure 4 The excitation and emission spectra of HPTS at ambient pH values ​​of 4, 7, and 10 are shown.

[0054] Instruction manual attached Figure 5 The figure shows the relationship between the logarithm of the ratio of excitation light intensity of 450 nm and 400 nm HPTS and pH value.

[0055] Instruction manual attached Figure 6 The diagram illustrates the photoprotolytic cycle of the fluorescent material HPTS when irradiated with incident light of a specific wavelength. This means that the fluorescent material undergoes a process of fragmentation under illumination, including the release of protons, entering an excited state and emitting fluorescence, and then returning to the ground state.

[0056] Instruction manual attached Figure 7 The Forster loop is shown.

[0057] manual Figure 8 The molecular structure of the fluorescent substance HPTS (pyranine) is shown.

[0058] manual Figure 9 The fluorescent substance HPTS (pyranine) is shown to be used for pH measurement in liposomes.

[0059] manual Figure 10 The slide, the probe (pH probe) fixed on the slide, the positive electrode (anode), the negative electrode (cathode), the sample solution, and the electric field lines are shown from a top-down perspective, according to an embodiment.

[0060] Instruction manual attached Figure 11 This is a flowchart of a method, according to an embodiment, for pooling a mixture of one or more proteins and analytes of interest into a small region and sequentially precipitating them. Detailed Implementation

[0061] This disclosure includes an apparatus and method for collecting and separating proteins from a protein solution. The apparatus includes a fluorescence microscope (or a conventional biological microscope), a slide fixed to the microscope stage, an anode and a cathode fixed to both sides of the slide, a transparent film containing a fluorescent substance covering the slide, and a liquid addition device held or mechanically fixed above the slide by an adjustable movement mechanism.

[0062] While various embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, substitutions, and alternatives will occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives may be adopted to the embodiments of the present disclosure described herein.

[0063] As used in this specification, the singular forms “a,” “an,” and “the” include plural indicators unless the context clearly specifies otherwise. Thus, for example, the term “member” is intended to mean a single member or a combination of members, and “material” is intended to mean one or more materials or a combination thereof.

[0064] As used herein, the term "protein" or "protein analogue" refers to proteins, oligopeptides, peptides, and analogues, including proteins and amino acid analogues containing non-naturally occurring amino acids and their structures. The term "protein" or "protein analogue" also refers to various proteins, oligopeptides, peptides, and analogues having different isoelectric points.

[0065] As used herein, the term "analyte" means any molecule or compound that is to be detected or moved, aggregated, precipitated, and / or precipitated as described herein. Suitable analytes may include, but are not limited to, small chemical molecules, such as, for example, environmental molecules, clinical molecules, chemicals, contaminants, and / or biomolecules. More specifically, such chemical molecules may include, but are not limited to, pesticides, insecticides, toxins, therapeutic and / or abused drugs, hormones, antibiotics, antibodies, organic materials, proteins (e.g., enzymes, immunoglobulins, and / or glycoproteins), nucleic acids (e.g., DNA and / or RNA), lipids, lectins, carbohydrates, whole cells (e.g., prokaryotic cells such as pathogenic bacteria and / or eukaryotic cells such as mammalian tumor cells), viruses, spores, polysaccharides, glycoproteins, metabolites, cofactors, nucleotides, polynucleotides, transition state analogs, inhibitors, nutrient solutions, electrolytes, growth factors, and other biomolecules and / or non-biomolecules, as well as fragments and combinations thereof. Some of the analytes described herein may be proteins, such as enzymes, drugs, cells, antibodies, antigens, cell membrane antigens and / or receptors or ligands thereof (e.g., neural receptors or ligands thereof, hormone receptors or ligands thereof, nutrient receptors or ligands thereof, and / or cell surface receptors or ligands thereof).

[0066] As used herein, the term "sample" refers to a composition containing one or more analytes to be detected, separated, moved, pooled, precipitated, and / or precipitated. Samples can be heterogeneous, containing various components (e.g., different proteins) or homogeneous, containing one component. In some cases, samples can be naturally occurring biological materials and / or artificial materials. Furthermore, samples can be in natural or denatured forms. In some cases, samples can be single cells (or single cell contents) or multiple cells (or multiple cell contents), blood samples, tissue samples, skin samples, urine samples, water samples, and / or soil samples. In some cases, samples can be derived from living organisms, such as eukaryotes, prokaryotes, mammals, humans, yeast, and / or bacteria, or samples can be derived from viruses. In some cases, samples can be one or more stem cells (e.g., any cell that can be isolated for an unlimited time period and generate specific cells). Suitable examples of stem cells can include, but are not limited to, embryonic stem cells (e.g., human embryonic stem cells (hES)) and non-embryonic stem cells (e.g., mesenchymal, hematopoietic, induced pluripotent stem cells (iPS cells), or adult stem cells (MSCs)).

[0067] The instruments and methods disclosed herein generally relate to the collection, separation, precipitation, and / or sedimentation of analytes of interest contained in a sample based on their isoelectric points (pI). In some embodiments, various analytes of interest may be sequentially separated and / or precipitated. The instruments and methods described herein relate to a free electrophoresis method in which a sample, held at hydrodynamic rest on a horizontal plane without any hydrodynamic force other than gravity, moves and collects a mixture of analytes contained in the sample under the influence of an electric field parallel to the horizontal plane, precipitating them at their isoelectric points (pI). These small regions are small areas in the sample solution near the edge of the sample solution close to an electrode (e.g., an anode). As described herein, features (e.g., the electric field and the horizontal plane) are “parallel” when they are substantially parallel. As used herein, substantially parallel means features oriented relative to each other at 0 degrees (plus or less than 5 degrees).

[0068] In some embodiments, the anode is configured to be insulated from the sample solution, and the cathode is configured to be conductive to the sample solution. Upon energization (i.e., when a potential is applied to the anode and cathode), the pH of the sample solution gradually increases and becomes higher than the isoelectric point (pI) of all analytes of interest. All analytes of interest in the sample solution are negatively charged and driven to a small region near the anode by the electric field applied to the apparatus or device as described herein. In some embodiments, by changing the pH of the sample solution in descending order of the isoelectric point (pI) of the analytes of interest, precipitates of different pI values, such as proteins, can be formed one at a time in the small region near the anode, resulting in the separation of the analytes, depending on their charge, from the sample solution.

[0069] Before describing the apparatus and methods of this disclosure in more detail, it should be understood that this disclosure is not limited to the specific embodiments described, and therefore changes are naturally possible. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to limit the scope of the claims.

[0070] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0071] It will be apparent to those skilled in the art upon reading this disclosure that each individual, separate component and feature described and illustrated herein can be readily separated from or combined with any other plurality of features without departing from the scope or spirit of the apparatus and methods of this disclosure. Any described method may be performed in the order of the described events or in any other logically possible order.

[0072] The aspects of this disclosure include electrophoretic migration and / or separation devices and pH measurement devices. The electrophoretic migration and / or separation devices and pH measurement devices can be adapted to perform a variety of migration and / or separations of interest, and combinations thereof. In some embodiments, the migration and / or separation devices are automated, meaning that the devices are capable of performing electrophoretic migration and / or separation without user intervention.

[0073] In some embodiments, electrophoretic migration and separation devices are designed to perform the migration, collection, and / or separation of proteins in blood or blood-diluent mixtures for further staining and / or testing. The devices can be scalable and can process specific proteins in whole blood and other body fluid samples.

[0074] The device disclosed herein can be used as a standalone protein isolation device or as part of an integrated device.

[0075] Figure 1 A free electrophoresis apparatus or device is described according to an embodiment, in which a sample is placed at rest on a horizontal plane by hydrodynamics without being driven by hydrodynamic forces other than gravity, and the analyte mixture contained therein is electrophoretically moved and precipitated sequentially by isoelectric point (pI). The apparatus includes: (1) a fluorescence microscope (including, a main switch 101, a sample holder (also, a cathode) 104, a stage Z-axis knob 105, a stage X-axis and Y-axis knobs 106, a fluorescence assembly chamber 107, an ND filter 110, a focusing lens knob 109, a field diaphragm knob 111, an aperture diaphragm knob 112, etc.); (2) a film-upright slide 103; (3) an anode 102; (4) a cathode (also, a sample holder) 104; and (5) a handheld burette 113. Figure 1 As shown, the anode 102 is a flattened cylinder perpendicular to the horizontal, and the cathode 104 is an annular shape parallel to the horizontal plane. As used herein, parallel or substantially parallel means a characteristic offset of less than 5 degrees, less than 2 degrees, or less than 0 degrees, including all ranges and subranges within the range.

[0076] Figure 2 A top-down magnified schematic diagram is shown of a glass slide 201, a sample solution 205 on the glass slide, an anode 207, a cathode 203, a small region 206 on one side of the sample solution, and electric field lines 202, according to an embodiment.

[0077] Figure 1 Slide 103 is... Figure 2 Slide 201.

[0078] Figure 10 The diagram shows a top-down enlarged schematic diagram of a glass slide, a sample solution on the glass slide, a probe (pH probe), a positive electrode (anode), a negative electrode (cathode), and electric field lines, according to an embodiment.

[0079] In some embodiments, the glass slides involved in this disclosure are made of hydrophobic and non-conductive materials, such as quartz and transparent plastic, but may be made of any suitable material.

[0080] In some embodiments, the flat cylindrical anode involved in this disclosure is close to the edge of the sample solution on the glass slide coating, but is insulated from the sample edge.

[0081] although Figure 1 The liquid addition device in the embodiments is described as a handheld burette, but it should be understood that any object or structure suitable for adding liquid can be used to add liquid to a sample solution, such as a handheld burette, a handheld microsyringe, an automatic burette, a microfluidic chip, and a surface acoustic wave microdroplet on-demand spraying device.

[0082] Figure 1 The cathode of the embodiment is depicted as a ring-shaped glass slide clip. It should be understood that any suitable metallic object or structure can also be used as a cathode or anode while fulfilling other functions.

[0083] When an electric current is applied to the cathode and anode, an electric field is induced. In some embodiments, the apparatus or device may include an anode and a cathode. In some embodiments, the electrodes (i.e., the cathode and / or anode) may be made of platinum. In some embodiments, the electrodes may be made of copper. In some embodiments, the electrodes may be made of graphite. In some embodiments, the electrodes may be made of titanium. In some embodiments, the electrodes may be made of brass. In some embodiments, the electrodes may be made of silver. In some embodiments, the electrodes may be made of carbon fiber material. In some embodiments, the electrodes may be made of gold. In some embodiments, the electrodes may be made of stainless steel. In some embodiments, the electrodes may be made of any material suitable for the electrophoresis process.

[0084] In some embodiments, the annular cathode and the edge of the sample solution on the glass slide coating can be electrically connected by a conductor. The conductor can be a strip or block of metal (e.g., copper, platinum, and silver, and any suitable material) spanning the edge of the sample solution and the annular cathode.

[0085] In some embodiments, the pH of the sample solution is adjusted to be greater than 7, and all analytes of interest in the sample solution carry a negative charge, experiencing an electric force directed from the cathode to the anode. Therefore, Figure 2 The direction of the electric field lines is depicted as pointing from the cathode to the anode.

[0086] In some embodiments, the cathode and the sample solution are electrically connected through a conductor, wherein the term "cathode" also refers to the conductor that is electrically connected to the cathode, and the term "cathode reaction" also refers to a reaction that occurs on the surface of the conductor portion that is physically in contact with the sample solution and is electrically connected to the cathode.

[0087] In some embodiments, the sample solution on the slide coating can be added to the slide coating by means of a liquid addition device or other device after the slide coating is fixed upward on the stage of a fluorescence microscope.

[0088] In some embodiments, the sample solution on the slide coating can be added to the slide coating by means of a liquid addition device or other device before the slide coating is fixed upward on the stage of the fluorescence microscope.

[0089] In some embodiments, the liquid addition device may be located directly above or to the side of the slide. A liquid addition device positioned to the side of the slide can be advantageous because the liquid outlet of such a device is located precisely above a region of the sample solution, for example, a small area where the analyte of interest accumulates in the sample solution.

[0090] As disclosed herein, “a small region of analyte of interest in a sample solution” means, on an area basis, a reduction of at least 62.5%, at least 75%, at least 90%, at least 95%, or at least 99% of the area occupied by the sample solution when it is placed on a glass slide, including all ranges and subranges thereof, relative to the area occupied by the sample solution.

[0091] In some embodiments, the potentials applied to the anode and cathode are sufficiently large that the electric fields induced at the anode and cathode not only prevent the diffusion of analytes of interest in the sample solution but also drive these analytes to converge along the electric field lines until the negatively charged analytes settle in a small region, which is the "small region of convergence of analytes of interest in the sample solution" disclosed herein. In some embodiments, the small region contains all the analytes of interest in the sample solution. As disclosed herein, "small region of convergence of analytes of interest in the sample solution" means that, relative to the various analytes of interest contained in the sample solution, the content of each analyte of interest in the small region is increased to at least 50%, at least 62.5%, at least 75%, at least 90%, at least 95%, at least 99%, at least 100%, including all ranges and subranges therein, based on the number of analyte molecules, relative to the total number of all molecules of all analytes of interest contained in the sample solution.

[0092] In some embodiments, the slide is 76 mm long, 26 mm wide, and 1 mm thick. A uniform film is applied to the slide, with a film thickness of 0.1-0.5 mm.

[0093] In some embodiments, the transparent film containing fluorescent material on the glass slide can slowly dissolve when it comes into contact with an aqueous solution, allowing the fluorescent material molecules originally encapsulated by the film to directly and physically contact the aqueous solution. This makes the pH of the fluorescent material molecules' environment the pH of the sample solution, enabling accurate measurement of the sample solution's pH.

[0094] In some embodiments, as described herein, one or more analytes of interest and other non-target analytes in the sample solution can be driven by an electric field to converge into a small region. Then, using the difference in isoelectric point (pI) between the analyte of interest and the other non-target analytes, the pH is measured and adjusted so that one or more analytes of interest precipitate at their respective isoelectric points within (and outside) the small region, separating them from the sample solution. This allows the other non-target analytes to remain in the remaining sample solution, which are then washed away by a laminar flow of purified water off the slide. The analytes of interest that have converged and precipitated on the slide (in some embodiments, also comprising mixtures) remain on the slide for further processing.

[0095] As discussed in this article, laminar flow is the opposite of turbulence, which is a liquid flow state with a relatively low fluid velocity.

[0096] In some embodiments, the pH of the sample solution is adjusted or assisted in by adjusting the temperature of the sample solution.

[0097] In some embodiments, electrolyte buffers, such as MES buffers or BisTris buffers, are avoided because they typically contain polymers that may affect the electrophoretic aggregation and isoelectric point precipitation of analytes. Due to the use of fluorescent pH measurement and probe-based pH measurement techniques, real-time pH feedback is available during pH adjustment. Based on this feedback, the pH of the sample solution can be accurately and effectively adjusted by adjusting the cathode (and anode) voltages. In some embodiments, the accuracy and precision of adjusting the pH of the sample solution by adjusting the cathode (and anode) voltages in conjunction with real-time measurement of the sample solution pH is at least an order of magnitude greater than the accuracy and precision of adjusting the pH of the sample solution or buffer solution by adjusting the buffer ratio (e.g., using a pump).

[0098] Instruction manual attached Figure 11 The diagram shown is a flowchart of a method according to an embodiment for pooling a mixture of one or more proteins and analytes of interest into a small region and sequentially precipitating a precipitate.

[0099] Instruction manual attached Figure 4The excitation and emission spectra of the fluorescent material HPTS, according to an embodiment, are shown. In some embodiments, only the 400 nm and 450 nm excitation spectra of HPTS are used for different sample solution pH values. Figure 4 As shown, the excitation wavelengths are 400 nm and 450 nm, but the emission wavelength is 510 nm. This not only improves measurement accuracy but also simplifies operation and equipment. The principle is that HPTS exhibits excited-state proton transfer (ESPT) performance. That is, the logarithm of the ratio of excitation light intensities at different wavelengths of HPTS to the pH value of the sample solution is a monotonic function over a large pH range, and also a linear function over a large pH range.

[0100] Instruction manual attached Figure 5 The diagram shows the logarithm of the ratio of the intensity of the emitted light (510 nm) produced by the 450 nm and 400 nm excitation light of the HPTS as a function of the pH value of the sample solution. This function shows that it is a monotonic function over a large pH range, and also a linear function over a large pH range. In some embodiments of this disclosure, the excellent properties of the HPTS ensure that the pH of the sample solution can be measured accurately and rapidly.

[0101] HPTS is a type of fluorescent probe called photoacids, one of the characteristics of which is that it can release protons under light conditions.

[0102] Instruction manual attached Figure 4 As shown, the fluorescent substance HPTS molecule has at least two excitation light spectral peaks, but HPTS has only one emission light spectral peak. Figure 6 The diagram shows that the excitation spectral peak of HPTS at a wavelength of 450 (or 460) nm corresponds to the emission spectral peak at a wavelength of 510 nm, and the excitation spectral peak of HPTS at a wavelength of 400 nm corresponds to the emission spectral peak at a wavelength of 445 nm. However, Figure 4 The HPTS did not show an emission spectrum peak at a wavelength of 445 nm. This is because the HPTS entered a state of flux after receiving excitation light at a wavelength of 400 nm. Figure 7 The Forster cycle shown emits fluorescence at 510 nm but not at 445 nm.

[0103] Instruction manual attached Figure 5 The curve showing the relationship between the logarithm of the excitation light intensity ratio of the fluorescent substance HPTS and pH value is shown.

[0104] In some embodiments, the method for measuring the excitation light intensity of the fluorescent material HPTS at 400 nm and 450 (460) nm is as follows: the intensity of the 450 nm or 400 nm incident light incident on the sample solution (which has been dissolved or partially dissolved and contains sufficient fluorescent material HPTS) on the glass slide film is gradually increased, and the intensity of the 510 nm emission light emitted by the fluorescent material HPTS in the excitation light incident area is received and measured. When the increase in the intensity of the 450 nm or 400 nm incident light no longer causes an increase in the intensity of the 510 nm emission light, the intensity of this maximum intensity of the 510 nm emission light is the excitation light intensity at 450 nm or 400 nm, respectively. and .

[0105] Figure 8 This shows that HPTS molecules contain a complete [molecule] at lower pH. HPTS minutes State, when pH is high, the outside of HPTS Will lose Only HPTS molecules are in Therefore, HPTS molecules have different molecular structures in acidic and alkaline environments, resulting in completely different excitation and emission spectra. Furthermore, the excited states of HPTS molecules... It can transform into an excited state. This transformation is called ESPT (excited-state proton transfer). The fluorescent substance HPTS molecule has ESPT properties, so that the fluorescence wavelength produced by excitation light at a wavelength of 400 nm is the same as that of excitation light at 450 nm, which is 510 nm.

[0106] Figure 6 The left side shows The transition between the ground and excited states of an HPTS molecule requires one proton from the environment for each 400 nm excitation light received and 445 nm emission light emitted. Participation, because if there is no [participation] in the environment HPTS will It exists in form. Figure 6 The right side shows The transition between the ground and excited states of the HPTS molecule requires one hydroxyl group in the environment for each 450 nm excitation light received and 500 nm emission light emitted. Participation, because if there is no [participation] in the environment HPTS will The form exists. Therefore, each detected 510 nm emission light generated by 400 nm excitation light represents one HPTS molecule and one proton surrounding it in the solution. Similarly, each detected fraction of 510 nm emission light generated by 450 nm excitation light represents one HPTS molecule and one hydroxyl group surrounding it in the solution. The maximum intensity of the detected 510 nm emission light represents the protons present around the HTPS molecule. Or hydroxide ions The number of protons (assuming there are enough HPTS molecules in the solution) determines the ratio of the excitation light intensity to the number of protons in the solution. Content and hydroxide ions The ratio of content. The logarithm of the ratio of excitation light intensity. It exhibits a monotonic or even linear functional relationship with the solution pH value.

[0107] As described in this article, in acidic solutions, HPTS molecules are in a state of... The state, 400 nm excitation light causes HPTS molecules to... ground state transition to Excited state, An excited state loses a proton during the ESPT process and becomes Excited state, then, Excited state transition to In its ground state, it emits light at a wavelength of 510 nanometers. Because the environment is an acidic solution, ground state transforms into Ground state. Therefore, the 400 nm excitation light caused the HPTS molecule to undergo a cycle of states.

[0108] As described in this article, in alkaline solutions, HPTS molecules are in a state of... The state, 450 nm excitation light causes HPTS molecules to... ground state transition to Excited state, then, Excited state transition to In its ground state, it emits light at a wavelength of 510 nanometers. Because the environment is an alkaline solution, The ground state remains unchanged. The ground state will not transform into the ground state. Ground state. Therefore, the 450 nm excitation light simply causes the HPTS molecule to undergo a process of being excited by light and emitting fluorescence, returning the HPTS molecule to its ground state.

[0109] Although the above description assumes acidic and alkaline solution conditions to clearly illustrate the changes in the molecular state of the fluorescent substance HPTS, in some embodiments, the specific changes in the molecular state of HPTS do not require the overall solution to be acidic or alkaline; only that the HPTS molecules are surrounded by protons. Or hydroxide ions Therefore, the intensity of the emitted light at 510 nanometers represents the number of protons surrounding the HPTS molecule. or hydroxide ions The probability of occurrence (or near occurrence) is linearly related to the pH of the solution, if other factors are not considered.

[0110] To understand the technique disclosed herein for measuring the pH of a sample solution using the properties of fluorescent molecules, it should be understood that the 510 nm emission intensity of the HPTS molecule is actually due to incident light at 400 nm or 450 nm. or An indicator of the probability of a state change from the ground state to an excited state. If there are protons in the sample solution... More The probability of transitioning from the ground state to an excited state is high, especially if there are hydroxide ions in the sample solution. More The probability of transitioning from the ground state to an excited state is high. However, considering the properties of the sample solution solvent and protons... or hydroxide ions The intensity of the 510 nm emitted by the HPTS molecule is actually determined by the distance from the HPTS molecule and the respective locations where protons or hydroxide ions approach the HPTS molecule. or The probability of HPTS molecules changing state from the ground state to the excited state. This probability depends on the concentrations of protons and hydroxide ions in the sample solution; the higher the concentrations of protons and hydroxide ions, the greater the probability. Under statistically significant conditions (i.e., when there are sufficient HPTS fluorescent molecules), the emission intensity at 510 nm is related to the proton concentration. Concentration and hydroxide ions The concentrations are directly proportional.

[0111] As described in this article, the intensity of emitted light at 510 nm is related to protons. Concentration and hydroxide ions The concentrations are directly proportional, but the fluorescence intensity is also related to the solvent properties and temperature. Here, the fluorescence intensity ratio eliminates the influence of solvent and other factors. This is the physical basis for measuring the pH of a sample solution by measuring the intensity of light emitted by HPTS molecules.

[0112] As described in this article, HPTS has the following advantages: 1. The wavelengths of incident light and emitted fluorescence that HPTS can receive are both within the visible light range; 2. HPTS has a larger Stokes shift than other fluorescent substances, which is beneficial for detection and resolution; 3. HPTS has high solubility in water, and because HPTS molecules contain sulfonates, HPTS carries a negative charge over a wide pH range; and 4. The pKa value of HPTS is near the physiological pH of the human body (e.g., 7.35-7.45).

[0113] This article discloses Figure 5 The illustrated curve showing the relationship between emitted light intensity and solution pH is highly sensitive to solvent properties, including ionic strength. In some embodiments, a calibration test must be performed with the same solvent and the corresponding curve plotted before each measurement of solution pH.

[0114] As described in this disclosure, the ESPT process must be carried out in solution. In some embodiments, HPTS molecules are dissolved in the solution to avoid contact with solid surfaces. In some embodiments, to prevent HPTS molecules from contacting solid surfaces, a transparent film containing HPTS molecules on a glass slide can be dissolved in an aqueous solution, so that when the sample solution is added to the glass slide film, it quickly dissolves the glass slide film, and at the same time, the HPTS molecules also dissolve, thus achieving a state where the HPTS molecules are dissolved in water.

[0115] In this disclosure, one of the HPTS excitation wavelengths is 400 nm, and another is 450 or 460 nm. The difference between 450 and 460 nm is due to variations in wording by different researchers; the actual wavelength of the emitted light from HPTS should be between 450 and 460 nm. Other HPTS excitation wavelengths disclosed in this disclosure, such as the 400 nm incident wavelength and the emitted wavelength, such as 510 nm, have an accuracy of only ten decimal places. In some embodiments, it is necessary to determine the specific operating value of the HPTS excitation wavelength before actual operation.

[0116] This disclosure provides an apparatus and method for sample separation in which a sample solution is placed at rest on a horizontal plane by hydrodynamic kinematics without any other hydrodynamic driving force other than gravity, and the analyte mixture contained in the sample is electrophoretically moved, aggregated, and precipitated sequentially at its isoelectric point (pI). Figure 11 This is a flowchart of a method for collecting and separating analytes of interest according to an embodiment.

[0117] In some embodiments, as described herein, the analyte mixture in the sample solution may include peptides having different isoelectric points of 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, including all ranges and subranges therein. In some embodiments, the pH of the sample solution is changed by altering the concentrations of various ions in the sample solution relative to the electrokinetic equilibrium achieved by the anode and / or cathode voltages, instead of using methods such as adding electrolyte buffers, e.g., MES and BisTris, and modifying the buffer ratio to adjust the pH of the sample solution. It is not intended to be bound by any theory that the pKa value of the sample solution will change in response to temperature changes; however, in some embodiments, changes in ambient temperature are primarily used to select a cathode (or anode) voltage versus sample solution pH curve that is more conducive to adjusting the pH of the sample solution by adjusting the electrodes (anode and / or cathode), e.g., a cathode (or anode) voltage versus sample solution pH curve that is closer to a linear relationship.

[0118] In some embodiments, the anode in the device is configured to be insulated from the sample solution, and the cathode in the device is configured to be conductive to the sample solution. When energized (i.e., when a potential is applied to the anode and cathode), a chemical reaction occurs at the cathode surface. The reactions at the cathode surface differ for acidic and alkaline solutions; however, the result is always an alkaline sample solution near the cathode. Within appropriate ranges, the lower the cathode voltage (more negative), and / or the higher the anode voltage (more positive), the higher the pH of the sample solution. As discussed further in detail herein, the pH of the sample solution can be controlled by adjusting the anode and / or cathode voltage in conjunction with the addition of acid or base.

[0119] In some embodiments, the solvent of the sample solution is water, which is the main component of the sample solution, and the pH of the sample solution can be controlled by controlling the anode voltage and / or cathode voltage. As described above, the anode in the device is configured to be insulated from the sample solution, and the cathode in the device is configured to be conductive to the sample solution. After energization (i.e., when a potential is applied to the anode and cathode), no chemical reaction occurs on the anode surface because the anode is insulated from the sample solution. However, due to the electric field force, the edge region of the sample solution near the anode will repel cations, such as protons. Aggregates anions, such as hydroxide ions. This causes the pH in this area to rise. After energizing, because the cathode and sample solution are conductive, a chemical reaction occurs on the cathode surface—the cathode reaction described above for acidic and alkaline water electrolysis. Both reactions result in an increase in the pH of the area near the cathode. As the reaction on the cathode surface continues, the hydroxide ions in the sample solution near the cathode increase. More and more hydroxide ions are appearing in the sample solution. The motion of particles under the influence of an electric field and their spontaneous diffusion cause hydroxide ions in the sample solution to... As the pH of the sample solution gradually increases and becomes more uniformly distributed, it eventually reaches a certain value and then plateaus, achieving electrodynamic equilibrium with the cathode and anodic potentials. Changing the cathode and anodic potentials will also alter the pH of the sample solution, leading to a new electrodynamic equilibrium. Because there are no hydrodynamic obstacles in the sample solution, hydroxide ions... and protons Driven by an electric field, the fluid kinematics moves at high speeds, and given the small volume of the sample solution, the time to reach a new electrodynamic equilibrium is very short. In some embodiments, the aforementioned physicochemical processes are used to rapidly and precisely adjust the sample pH, allowing proteins of interest to remain at the accurate pH for an accurate time, precipitating at their isoelectric points and rejecting the erroneous precipitation of proteins with similar isoelectric points. In some embodiments, a new electrodynamic equilibrium can be reached within 2-3 seconds after changing the cathode and / or anodic potentials, enabling the sample solution to promptly reach and exit a stable and accurate pH.

[0120] In some embodiments, the anode in the device is configured to be insulated from the sample solution, and the cathode in the device is configured to be conductive to the sample solution. The pH of the sample solution can be controlled by adjusting the anode voltage and the cathode voltage. The anode of the DC power supply that generates the voltage (i.e., the potential generator that applies a potential to the anode and cathode) should be robustly grounded.

[0121] In some embodiments, the analytes of interest in the sample solution are pooled into a small region, rather than being achieved by applying voltage to the anode and cathode in a single step. In some embodiments, the sample solution can be added to the slide film in two stages. The first addition of sample solution leaves the edge of the solution at a distance from the anode. After measuring and adjusting the pH of the sample solution, voltage is applied, causing the analytes to pool in a small region at a distance from the anode. The pH is then adjusted again, causing analytes other than the analytes of interest, such as albumin (which is present in larger quantities), to precipitate first. The second addition of a diluent without the analytes leaves the edge of the sample solution on the slide film close to (but not in contact with) the anode. The pH is again measured and adjusted, and voltage is applied again, causing the remaining analytes in the sample solution to pool in a small region near the anode. The pH is then measured and adjusted again, causing the analytes of interest (e.g., proteins) to precipitate at their respective isoelectric points. After rinsing the slide with a laminar flow of pure water, two protein precipitation regions remain on the slide. The protein in the region closer to the anode is the analyte of interest. In some embodiments, by adding the solution twice as described above—first adding the sample solution, then adding the diluent, and performing related operations—the purity of the analyte of interest in a small region near the anode on the slide after the operation is completed, and the ratio of the analyte of interest in the small region to all the analytes of interest contained in the sample, can be significantly improved.

[0122] In some embodiments, the above-mentioned process of adjusting the pH of the sample solution by two measurements and then using electricity to collect the analytes in the sample solution to form two small regions where the analytes are concentrated can be changed to the process of adjusting the pH of the sample solution by three or more measurements and then using electricity to collect the analytes in the sample solution to form three or more small regions where the analytes are concentrated.

[0123] In some embodiments, if it is necessary to repeatedly precipitate several or a dozen analytes of interest, the anode and cathode can be connected to an automatic control system. The automatic system controls the voltage of the anode and cathode and maintains the voltage stable for a certain period of time to control the migration and aggregation of analytes in the sample solution and the precipitation of analytes at their respective isoelectric points.

[0124] In some embodiments, isoelectric point precipitation (adjusting the solution pH to the isoelectric point of the analyte) is the primary method for analyte separation, while other methods include salting out (using a high-concentration salt solution to change the ionic strength), organic solvent precipitation (reducing the dielectric constant of the solution by means of ethanol, etc.), and / or alkaloid reagent precipitation.

[0125] In some embodiments, salting out (using a high-concentration salt solution to change the ionic strength), organic solvent precipitation (reducing the dielectric constant of the solution by means of ethanol, etc.), and / or alkaloid reagent precipitation requires the addition of a specific liquid to the sample solution, which is done by means of the liquid addition device disclosed herein.

[0126] In some embodiments, the sample solution is a mixture of human blood and a sample diluent. In some embodiments, the human blood is fresh whole blood from the fingertip. In some embodiments, the human blood is anticoagulated whole blood from a vein. In some embodiments, the diluent is dilute sulfuric acid. In some embodiments, the diluent is sodium hydroxide (NaOH) solution. In some embodiments, the diluent is any solution, buffer, electrolyte, and / or other liquid suitable for diluting human blood and facilitating the movement, precipitation, sedimentation, and / or separation of proteins therein.

[0127] In some embodiments, a fluorescent substance, such as HPTS, is dissolved in a sample diluent and mixed with the sample to form a sample solution containing the fluorescent substance, allowing the pH of the sample solution to be measured in real time by the fluorescent substance in the sample solution. While this avoids the cumbersome process of laminating the glass slide, it adds the step of ensuring the fluorescent substance is evenly distributed in the diluent and / or sample solution.

[0128] In some embodiments, the apparatus or methods disclosed herein are used for staining and / or detection of proteins in various human samples, wherein examples of proteins in the human samples include: hepatitis B virus macroprotein, anti-human globulin, α-thalassemia gene / protein, β-thalassemia gene / protein, hemoglobin A2 / F / A1c, protein C, protein S, free protein S, ceruloplasmin, PD-L1 protein, p53 protein, Golgi protein 73 (GP73), alpha-fetoprotein (AFP), alpha-fetoprotein isoform (AFP-L3) ratio, nuclear matrix protein 22, heat shock protein 90α, abnormal glycan glycoprotein (TAP), type IV gel Original protein, C-reactive protein, high-sensitivity C-reactive protein, full-range C-reactive protein, S100-β protein, α1-acid glycoprotein, α1-microglobulin, α2-macroglobulin, β2-microglobulin, β-amyloid 1-40 (Aβ1-40), β-amyloid 1-42 (Aβ1-42), β-amyloid protein, Alzheimer's-associated neurofilament protein (AD7C-NTP), cancer antigen 125 (carbohydrate antigen 125, CA125), cancer antigen 15-3 (carbohydrate antigen 15-3, CA15-3), cancer antigen 72-4 (carbohydrate antigen 72-4, CA72-4), carcinoembryonic antigen (CEA) content, laminin (laminoid protein) White), haptoglobin (heparin-binding protein), heparin-binding protein (HBP), Golgi protein 73 (GP73), myoglobin, glial fibrillary acidic protein (GFAP), sperm SP10 protein, chitosanase 3-like protein 1, soluble transferrin receptor (sTfR), citrullinated protein, squamous cell carcinoma antigen (SCC), immunoglobulin A (IgA), immunoglobulin G (IgG), immunoglobulin G subtypes (such as IgG1, IgG2, IgG3, IgG4), immunoglobulin M (IgM), B-type natriuretic peptide (BNP), atrial natriuretic peptide (ANP), albumin, prostatic small effusive protein (PSEP), ischemic repair Albumin, α-synuclein (SNCA), epididymal protein 4 (HE4), pregnancy-associated plasma protein A (PAPP-A), lactoferrin, retinol-binding protein, glycated hemoglobin, glycated serum albumin, glycated serum protein (fructosamine), carbohydrate antigen 19-9 (CA19-9), carbohydrate antigen 242 (CA242), carbohydrate antigen 50 (CA50), glucose-deficient transferrin (CDT), ferritin content, fibronectin (fibronectin, FN), cardiac myosin-binding protein C, heart-type fatty acid-binding protein, thyroglobulin (TG), sex hormone-binding globulin (SHBG), thyroid-binding globulin, α1-antitrypsin (α1-protease inhibitor), and / or lipoproteins.

[0129] The electrophoretic separation device disclosed herein is suitable for the aggregation and separation of specific proteins in blood and body fluid samples.

[0130] In some embodiments disclosed herein, the anode is insulated from the sample solution and the cathode is conductive to the sample solution; however, in other embodiments, the arrangement of the anode being conductive to the sample solution and the cathode being insulated from the sample solution provides better operational performance.

[0131] The illustrative examples and detailed descriptions in this disclosure, including those in the claims and specification, are intended solely to demonstrate the feasibility of this disclosure, embody the spirit of the claims, and facilitate a clear understanding of this disclosure by the reader. They are not intended to limit the scope of any claim. Any changes and / or modifications to this disclosure that are obvious to those skilled in the art shall still fall within the spirit and / or scope of the appended claims.

[0132] Furthermore, all examples, embodiments, and conditional language in this disclosure, including those in the appended claims and specification, are intended to help the reader understand the principles of this disclosure and the concepts contributed by the inventors to advance the prior art, and are to be construed as not being limited to these specific descriptive examples and / or embodiments.

[0133] Furthermore, all statements regarding the principles, aspects, and implementation methods of this disclosure, as well as specific examples thereof, are intended to cover equivalents of its structure and function. Such equivalents include both currently known equivalents and future equivalents not currently known, i.e., any element performing the same function or its derivatives, regardless of its structure. Therefore, the scope of this disclosure is not limited to the examples and / or embodiments shown and described herein. Rather, the scope and spirit of this disclosure are embodied solely by the appended claims.

Claims

1. An apparatus for isoelectric focusing precipitation of a sample, comprising: a horizontal plane to hold the sample in free standing; an anode and a cathode disposed on both sides of the sample; The anode and cathode are arranged with the sample so that: one, the anode is electrically conductive with the sample and the cathode is electrically conductive with the sample, two, the anode is electrically insulating with the sample and the cathode is electrically conductive with the sample, three, the anode is electrically conductive with the sample and the cathode is electrically insulating with the sample, or four, the anode is electrically insulating with the sample and the cathode is electrically insulating with the sample, wherein, the anode is in close proximity or electrically conductive to the sample with the cathode; the anode and the cathode are applied with a voltage to induce an electric field parallel to the horizontal plane, such that the electric field passes through the sample, such that an analyte of interest in the sample is moved by isoelectric focusing to concentrate in a small area in the sample; and, the analyte of interest is precipitated by isoelectric focusing.

2. The apparatus of claim 1, further comprising measuring and adjusting the pH of the sample before the anode and the cathode are applied with a voltage.

3. The apparatus of claims 1 and 3, further comprising mixing the sample with a sample diluent before measuring and adjusting the pH of the sample.

4. The apparatus of claims 1 and 2, further comprising measuring and adjusting the pH of the sample solution before the analyte of interest is precipitated by isoelectric focusing.

5. The apparatus of claims 1 and 2, wherein, The isoelectric focusing precipitation method examples also include salting out, organic solvent precipitation, addition of non-ionic polymer precipitation, addition of polyelectrolyte precipitation, and / or any suitable method to precipitate the analyte of interest.

6. The apparatus of claim 1, wherein, The horizontal plane is a glass slide plane, wherein the glass slide surface is covered with a transparent film containing fluorescent substances, or, the glass slide surface is fixed with some pH probes.

7. The apparatus of claims 1, 2 and 7, further comprising separating the glass slide from the rest of the apparatus after the analyte of interest is precipitated by isoelectric focusing, to flush the solution on the glass slide with a layer of pure water, and / or, to let the glass slide stand to dry.

8. The apparatus of claim 1, further comprising a liquid adding device to add liquid to the sample, wherein the liquid adding device is manually held above the sample or mechanically fixed above the sample with an adjustable moving device, wherein examples of the liquid adding device include a hand-held dropper, a hand-held microsyringe, an automated burette, a microfluidic chip, and a surface acoustic wave microdroplet on-demand ejection device, wherein examples of the liquid added to the sample by the liquid adding device include acids or bases, hydrophilic organic solvents and various inorganic salts, to adjust the pH of the sample and / or to facilitate the precipitation of the analyte of interest.

9. The apparatus of claim 1, wherein, The anode is a flat cylinder and the cathode is a horizontal ring, or, the cathode is a flat cylinder and the anode is a horizontal ring.

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