Method for adjusting pH of solution with measured pH

Abstract

Embodiments described herein relate to devices and methods for measuring the pH of an adjusted solution. The apparatus and method include subjecting a sample to fluid kinematics resting on a horizontal plane and applying an electric field parallel to the horizontal plane through an anode and a cathode on both sides of the sample. Embodiments described herein relate to devices or apparatuses without other hydrodynamic powers in the horizontal plane except gravity, and apparatuses and methods for measuring the pH of an adjusted solution. The apparatus and method include subjecting a sample to fluid kinematics resting on a horizontal plane and applying an electric field parallel to the horizontal plane through an anode and a cathode on both sides of the sample.

Description

Technical Field

[0001] Some of the embodiments described herein relate to apparatus and methods for measuring and adjusting the pH of a solution. Background Technology

[0002] Techniques such as electrophoresis require adjustment of the solution pH. Protein isoelectric point precipitation techniques also require precise pH adjustment.

[0003] In electrophoresis, the electrolyte is replaced with electrolyzed water. When an electric field is applied, redox reactions occur at the anode and cathode, causing a change 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: .

[0004] In most electrophoresis techniques, the anode and cathode 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 will flow across the anode and cathode, and a certain degree of chemical reaction will occur at the electrode conductive to the sample. However, the current will stop within a short time, and the chemical reaction at the electrode will also cease. The voltage across the two electrodes will then be related to the protons in the sample. and / or hydroxide ions The concentration will reach equilibrium.

[0005] Electrophoresis technology and its improved techniques, such as the invention patent "Method for Single-Channel Free-Flow Electrophoresis with Sequential pH Adjustment" (application publication number CN114728213A), all require adjustment of the sample solution pH.

[0006] Devices or equipment for measuring and adjusting the pH of solutions that facilitate microscopic examination are an important requirement in industries such as pharmaceuticals.

[0007] 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.

[0008] 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.

[0009] 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.

[0010] 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.

[0011] In view of the shortcomings and limitations of current technology and the practical needs of industries such as pharmaceuticals, this disclosure describes the principle and operation of adjusting the pH of a sample solution through electrodynamic equilibrium, by adjusting the cathode voltage and the anode voltage to precisely adjust the sample pH. Some embodiments described herein do not require an electrolyte, but instead use electrolyzed water containing dilute sulfuric acid or sodium hydroxide solution.

[0012] To accurately adjust the pH of a solution, a feedback mechanism must be introduced, meaning that the pH of the solution needs to be measured in real time.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] The research and development work that has now been completed has proven that the fluorescent substance HPTS (pyranine) is successful in pH measurement in liposomes.

[0017] 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

[0018] 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), an electrode reaction occurs at the cathode surface, and the pH of the sample solution increases. 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 an acid or base.

[0019] 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 the diffusion of hydroxide ions through electric field force and thermal diffusion Diffusion to other regions of the solution, and causing protons to... The protons move to the cathode surface, participate in the electrode reaction, become hydrogen gas, and leave the cathode, thus affecting the voltage applied to the electrode and the solution protons. and hydroxide ions The concentration reaches electrochemical dynamics equilibrium.

[0020] 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 sample solution gradually becomes uniformly distributed, and its pH reaches a uniform alkaline state. The pH of the sample solution gradually increases to a certain value and then stops increasing, reaching electrodynamic or electrochemical dynamic equilibrium with the cathode and anode potentials.

[0021] 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.

[0022] 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 cathodic reactions of 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] In some embodiments described herein, 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 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.

[0027] In some embodiments, such as those described in the instruction manual Figure 1 The relevant technical solution for measuring the pH of a sample solution is as follows: Step 1, Calibration: a) Add HPTS to the solution and measure the pH value using a method for measuring pH, such as a pH electrode probe; b) Measure the excitation light intensity of HPTS molecules at wavelengths of 400 nm and 450 nm in the solution, 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 2, Measurement: a) Measure the excitation light intensity of HPTS molecules at wavelengths of 400 nm and 450 nm in the solution, and calculate the intensity ratio logarithm; b) Compare the intensity ratio logarithm obtained in a) with the relationship curve obtained in step 1c) to determine the pH value of the solution being tested.

[0028] 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.

[0029] In some embodiments, such as those described in the instruction manual Figure 2 and 3 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.

[0030] 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.

[0031] In some embodiments, the glass slide is made of quartz.

[0032] 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).

[0033] Since 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] In some embodiments, the liquid addition device in the apparatus can precisely add liquid to the sample solution. Attached Figure Description

[0039] Instruction manual attached Figure 1 The sample solution, anode, cathode, and power supply are shown.

[0040] Instruction manual attached Figure 2 The slide, sample solution on a slide coating, anode, cathode, conductor conducting electricity between the cathode and the sample solution, and electric field lines are shown from a top-down perspective, according to an embodiment.

[0041] Instruction manual attached Figure 3 It consists of a glass slide and a transparent thin film containing fluorescent material covering the slide.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] Instruction manual attached Figure 7 The Forster loop is shown.

[0046] manual Figure 8 The molecular structure of the fluorescent substance HPTS (pyranine) is shown.

[0047] manual Figure 9 The fluorescent substance HPTS (pyranine) is shown to be used for pH measurement in liposomes. Detailed Implementation

[0048] This disclosure includes providing an apparatus and method for real-time measurement and adjustment of solution pH. 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 movable device.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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).

[0053] 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)).

[0054] As described herein, the small region is a small region in the sample solution near the edge of the electrode (e.g., the anode). As described herein, features (e.g., electric field and horizontal plane) are “parallel” when they are substantially parallel. As used herein, substantially parallel means features oriented toward each other at 0 degrees (plus or minus 5 degrees).

[0055] 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.

[0056] 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.

[0057] 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.

[0058] This disclosure includes a pH measuring device. The pH measuring device can be adapted to perform a variety of operations of interest. In some embodiments, the device operation is automatic, meaning that the device is capable of performing electrophoretic migration and / or separation without user intervention.

[0059] In some embodiments, the device may be scalable and may be able to process specific proteins in whole blood and other body fluid samples.

[0060] The device disclosed herein can be used as a standalone pH measuring device or as part of an integrated device.

[0061] Figure 1 An apparatus or device for adjusting the pH of a solution according to an embodiment is described. The apparatus includes: (1) a power source 101; (2) a wire 102; (3) a cathode (immersed in the solution) 103; (4) a solution 104; and (5) an anode (insulated from the solution from the outside) 105.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] In some embodiments, the cathode and the edge of the sample solution on the glass slide coating can be electrically connected via 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] In some embodiments, the pH of the sample solution is adjusted or assisted in by adjusting the temperature of the sample solution.

[0074] In some embodiments, the accuracy and precision of controlling the pH of a sample solution by adjusting the cathode (and anode) voltage in conjunction with real-time measurement of the sample solution pH is adjusted by at least an order of magnitude more than the accuracy and precision of adjusting the pH of a sample solution or buffer solution by adjusting the buffer ratio (e.g., using a pump).

[0075] Instruction manual attached Figure 4 The 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.

[0076] 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.

[0077] HPTS is a type of fluorescent probe called photoacids, one of the characteristics of which is that it can release protons under light conditions.

[0078] 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.

[0079] 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.

[0080] 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 .

[0081] 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 a wavelength of 450 nm, both being 510 nm.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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).

[0089] 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.

[0090] 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.

[0091] 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.

[0092] In some embodiments, the pH of the sample solution is altered by changing the concentrations of various ions in the sample solution relative to the electrodynamic equilibrium achieved by the anode and / or cathode voltages, instead of adjusting the pH by adding electrolyte buffers, such as MES and BisTris, and modifying the buffer ratio. While it is not desirable to be bound by any single theory, 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), for example, a cathode (or anode) voltage versus sample solution pH curve that is closer to a linear relationship.

[0093] 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.

[0094] 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 above-described physicochemical processes are used to rapidly and accurately adjust the sample pH. In some embodiments, a new electrodynamic equilibrium can be reached within 2-3 seconds after changing the cathode and / or anodic potentials, allowing the sample solution to promptly reach and exit a stable and accurate pH.

[0095] In some embodiments, such as those described in the instruction manual Figure 1 Before using fluorescent substances to measure the pH of a solution, other methods of pH measurement must be used, such as pH electrode probes, to calibrate and obtain the relationship curve between pH and fluorescence intensity. The purpose is that measuring the pH of a solution by fluorescence intensity is much faster than directly using a pH electrode probe, which can meet the requirements of real-time pH measurement and sensitive adjustment of the solution pH.

[0096] In some embodiments, such as those described in the instruction manual Figure 2 and 3 By measuring the pH of a solution through fluorescence intensity, it not only meets the requirements for real-time pH measurement and sensitive adjustment of solution pH, but also solves the technical obstacle of pH electrode probes being too large to measure the pH of minute amounts of solution (such as a drop on a plane).

[0097] 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.

[0098] 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.

[0099] 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, enabling the pH of the sample solution to be measured in real time by means of the fluorescent substance in the sample solution.

[0100] In some embodiments disclosed herein, for example, the solution pH is greater than 7, the anode is insulated from the sample solution and the cathode is conductive to the sample solution; however, in other embodiments, for example, the setup where the solution pH is less than 7, the anode is conductive to the sample solution and the cathode is insulated from the sample solution is more effective.

[0101] 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.

[0102] 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.

[0103] 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 and method for adjusting the pH of a sample, comprising: A DC power supply and its anode and cathode, wherein, The anode and the cathode are configured such that: the anode is insulated from the sample and the cathode is conductive to the sample, or the anode is conductive to the sample and the cathode is insulated from the sample, wherein the positions of the anode and the cathode are both close to the sample or are wholly or partially in the sample; A voltage is applied to the anode and the cathode, inducing an electric field through the sample, causing an electrode reaction to occur on the electrodes that are conductive to the sample; and The pH value of the sample is adjusted by adjusting the voltage applied to the anode and the cathode by the DC power supply.

2. The method of claim 1, further comprising mixing the sample with a sample diluent to form a sample solution before voltage is applied to the anode and the cathode.

3. According to claims 1 and 2, it further comprises that after the sample is mixed with the sample diluent to form the sample solution, the sample solution is placed on a horizontally placed glass slide, wherein, pH probes are fixed to the surface of the glass slide.

4. According to claims 1 and 2, it further comprises adding a fluorescent substance to the sample diluent before mixing the sample with the sample diluent to form the sample solution, or adding a fluorescent substance to the sample solution afterward.

5. According to claims 1 and 2, it further comprises that after the sample is mixed with the sample diluent to form the sample solution, the sample solution is placed on a horizontally placed glass slide, wherein, The surface of the glass slide is covered with a transparent film containing fluorescent material.

6. According to claims 1, 2, and 5, it further comprises that after the sample solution is placed on a horizontally positioned slide, the slide is fixed on the stage of a fluorescence microscope.

7. The claim 1, further comprising a liquid adding device for adding liquid to the sample, wherein the liquid adding device is an adjustable moving device held manually above the sample or mechanically fixed above the sample, examples of the liquid adding device including a handheld dropper, a handheld microsyringe, an automatic burette, a microfluidic chip, and a surface acoustic wave microdroplet on-demand spraying device, and examples of the liquid added to the sample by the liquid adding device including acids or bases.

Images