CN114062334B - Fluorescent probe for measuring oxygen and hydrogen ions, preparation method and application - Google Patents

Abstract

The object of the present invention is to provide fluorescent probes, preparation methods and uses for measuring oxygen and hydrogen ions, including substance one: tris(4,7-phenyl-1,10-phenanthroline)ruthenium(II) di( Hexafluorophosphate), substance two: polyfluorescein isothiocyanate allylamine salt; prepare substance one and substance two in a container into an aqueous solution with a concentration of 20 mg/mL and an ethanol solution with a concentration of 10 mg/mL respectively, and then add the substance two Mix together with 5% Nafion membrane solution at a volume ratio of 1:2:4. Compared with traditional macroscopic pH electrochemistry and optical fiber sensors, which cannot accurately monitor pH changes in microenvironments such as cells, among commonly used pH detection methods, the probe of the present invention can effectively realize microenvironment detection. The detection method based on fluorescence signal changes has High sensitivity, good selectivity and non-invasiveness.

Description

Fluorescent probe for measuring oxygen and hydrogen ions, preparation method and use

Technical field

The invention belongs to the technical field of cellular aerobic respiration and anaerobic glycolysis detection, and in particular relates to fluorescent probes for measuring oxygen and hydrogen ions, preparation methods and uses.

Background technique

Oxygen is a ubiquitous reactant and product that also plays an important role in many chemical and biochemical processes. The quantification of oxygen concentration has always been an important issue in many application fields such as environmental quality monitoring, transportation, medicine, and agriculture. Therefore, it is of great significance to develop efficient sensors that can accurately measure oxygen concentration, and researchers have devoted a lot of efforts to it.

For the wide applicability of such sensors, several solutions have been proposed, such as solid electrolyte-based potentiometric, galvanic and metal oxide-based semiconductor resistance-type sensors for measurements at high temperatures, as well as Clark electrode-based sensors for Sensor that measures oxygen. Common disadvantages of these sensors are relatively long response times, high oxygen consumption, and poisoning of the sensor surface by organic compounds. The above problems can be circumvented by using optical oxygen sensors, which are mainly based on the quenching of luminescence by a series of chemical substances. The most common type of optical oxygen sensing material is based on the dynamic quenching of the luminescence of dye molecules, and its light emission depends on ambient oxygen concentration. Fluorescent technologies entered the field because they offer non-invasive, high-sensitivity, rapid and reversible measurements, and low toxicity. This sensing consumes no oxygen, resulting in more reliable measurements and is free from electrical interference.

Luminescence is a well-known analytical method that is widely used for the specific detection of molecular compounds. In short, it is a spontaneous relaxation process in which a luminous body absorbs light to elevate electrons from the ground state energy level to the excited state, and then returns the electrons to the ground state. In the subsequent transition, a photon is emitted with energy equal to the difference between the excited state and the ground state.

Fluorescence-based oxygen sensors work on the quenching of this fluorescence. During this process, the fluorescence of the dye weakens due to interactions with oxygen molecules. To obtain feasible measurements, it is important to use long-lived fluorophores. Most luminescent indicators used for oxygen sensing suffer from the disadvantages of short-wavelength excitation and small Stokes shifts, thereby increasing the complexity of the required measurement instrumentation.

Hydrogen ions play an important role in regulating the life activities of organisms. The occurrence of some life phenomena is often accompanied by changes in hydrogen ion concentration. Therefore, it is of great significance to detect pH changes in real time in microenvironments such as the cellular level. Traditional macroscopic pH electrochemistry and optical fiber sensors cannot accurately monitor pH changes in microenvironments such as cells. The development of molecular structure or nanoscale pH sensors can effectively realize microenvironment detection. Among commonly used pH detection methods, detection based on fluorescence signal changes The method has the advantages of high sensitivity, good selectivity and non-invasiveness, and has received widespread attention from researchers.

The energy sources for life activities are mainly cellular aerobic respiration and anaerobic glycolysis. These two major biochemical reaction channels undergo complex changes in different states of cells, resulting in changes in cellular energy metabolism patterns. Conducting research on the intrinsic molecular mechanisms of "reprogramming" of cellular energy metabolism can provide important hints for uncovering the mysteries of life activities (such as exploring tumor treatment targets). This has been a hot topic in life science research in recent years.

Contents of the invention

The purpose of the present invention is to provide a fluorescent probe, a preparation method and a use for measuring oxygen and hydrogen ions, and to solve the existing problems of relatively long response time for fluorescence detection of oxygen and hydrogen ions, large oxygen consumption and poisoning of the sensor surface caused by organic compounds. .

In order to solve the above technical problems, the present invention is implemented through the following technical solutions:

Fluorescent probe complex for measuring oxygen and hydrogen ions, including substance one: tris(4,7-phenyl-1,10-phenanthroline)ruthenium(II) bis(hexafluorophosphate), CAS number: The specific structure of 123148-15-2 is:

Among the dyes and compounds that exhibit oxygen fluorescence quenching and have been used as oxygen sensors, we can find transition metal complexes of palladium, platinum and ruthenium, the most commonly used of which are ruthenium complexes. These fluorophores possess the following characteristics: high photochemical stability, high molar absorptivity, long lifetime derived from metal-to-ligand charge transfer (MLCT) excited states, and large Stokes shift, making them useful in fluorescence-based oxygen sensors. Attractive fluorophores.

The photochemistry and photophysics of Ru(II) complexes have attracted the interest of chemists because of their bright luminescence in room temperature solutions, moderate quantum yields, long excited state lifetimes, and spectrally distinguishable metal redox. states, tunable electronic properties, undergo energy and electron transfer processes, and chemical and photostability.

Although there are several types of ruthenium(II) polypyridine complexes, one of the most frequently explored types is the trichelate complexes, which are coordination saturated and kinetically inert, exhibiting excellent chemical stability, e.g. Following intraperitoneal injection of radiolabeled [ ^106Ru (phen) ₃ ][ _ClO4 ] ₂ in rats and mice, they appeared to be metabolically stable when intact cations were detected in excreted urine within approximately 12 hours. Other examples of this exceptional stability include survival in boiling concentrated acids or bases, or the use of the ruthenium(II) polypyridine complex as a digestive marker in ruminants since it is not absorbed by the animal's intestines. This chemical stability is one of the reasons why ruthenium(II) polypyridine complexes are so extensively studied as biological probes or therapeutics. In general, the complex should be considered as robust as the free ligand unless irradiation causes loss of the ligand. Importantly, trichelate ruthenium(II) polypyridine complexes, which are chiral (propeller molecules with D3 point group symmetry), are typically isolated as racemic mixtures of Λ and Δ enantiomers.

Among various ruthenium(II) polypyridine trichelate complexes, it is known that tris(4,7-phenyl-1,10-phenanthroline)ruthenium(II) complex has better properties than other polypyridine complexes. Longer excited state lifetime and therefore expected to exhibit higher sensitivity. The long lifetime of the excited state also facilitates the design of relatively inexpensive sensing systems based on decay time measurements.

Substance 2: Polyfluorescein isothiocyanate allylamine salt; CAS N/A MFCD05665548

Fluorescein isothiocyanate (FITC) is a common pH-sensitive fluorescent dye molecule that can respond quickly to changes in pH value and has high sensitivity. Poly(fluorescein isothiocyanate allylamine hydrochloride) (PFIAH) is a type of fluorescein isothiocyanate derivative. In alkaline solution, the isothiocyanate group of fluorescein isothiocyanate (FITC) can be covalently bonded to the free amino group of poly(allylamine) to form a stable PFIAH compound without changing the spectral characteristics of fluorescein. And the chemical properties of the bound substances (such as antigens and antibodies), its high molar absorption coefficient, large fluorescence quantum yield and strong photostability make it widely used in the biomedical field as a sensitive fluorescent labeling substance. Such as fluorescent tracing of antigens and antibodies and rapid diagnosis of diseases. FITC is an organic weak acid. Its fluorescence intensity is very sensitive to the concentration of hydrogen ions in the solution. The fluorescence intensity gradually increases with the increase of pH. The change of fluorescence signal based on the nanosensor can quickly respond to the change of pH value, so it can be used as hydrogen ion. Sensitive indicators are used in the research of hydrogen ion sensors. The excitation and emission wavelengths used by FITC as fluorescent probes and hydrogen ion indicators are usually around 490nm and 520nm, where FITC has a high fluorescence quantum yield.

The cellular metabolic process is regulated by intracellular pH. The detection of intracellular pH can provide important information for studying the physiological and pathological processes of individual cells. The biocompatibility and cell phagocytosis efficiency of pH nanosensors are important factors affecting the detection of intracellular pH. . Modification of FITC by poly(allylamine) can improve the biocompatibility and cell phagocytosis efficiency of nanosensors, which will promote the application of nanosensors in intracellular pH detection.

Substance three: 5% Nafion membrane solution.

Nafion membrane is a perfluorosulfonic acid-type proton exchange membrane developed and produced by DuPont in the United States. This proton exchange membrane has excellent proton (H ⁺ ) conductivity. This is produced by copolymerizing a perfluorinated vinyl ether comonomer with tetrafluoroethylene (TFE), resulting in the following chemical structure:

DuPont offers a wide range of polymer ingredients and dispersant combinations. Typical uses include the manufacture of thin films and coating formulations for fuel cell membranes, catalyst coatings, sensors and various electrochemical applications.

The preparation method of fluorescent probes for measuring oxygen and hydrogen ions includes the following steps:

Prepare substance one and substance two into an aqueous solution with a concentration of 20 mg/mL and an ethanol solution with a concentration of 10 mg/mL respectively in a container; the container is an Erlenmeyer flask.

Then mix the two with 5% Nafion membrane solution at a volume ratio of 1:2:4, stir for 10 minutes to ensure uniform mixing, and then apply the resulting mixed solution dropwise onto an acrylic plate, and dry in a vacuum oven at 25°C for 3 hours. , you can get the required probe.

The use of fluorescent probes to measure oxygen and hydrogen ions requires the use of the XF series extracellular energy analyzer, whose core components include a 96-well or 24-well probe plate and cell plate;

Inoculate cell culture medium with 10,000-300,000 cells per well in the cell plate;

Apply the fluorescent probe to the tip of the probe tube corresponding to the probe board, install the probe board and the cell plate overlappingly, and then put them into the XF series extracellular energy analyzer. The corresponding probe tube of the XF series extracellular energy analyzer has several bundles of optical fibers that emit and collect excitation light;

Fluorescent probes mainly detect real-time changes in oxygen and hydrogen ions in cell culture fluid. The change rate of oxygen represents the intensity of aerobic respiration, and the change rate of hydrogen ions, the acidification rate, represents the rate of anaerobic glycolysis.

For the XF96 probe card, apply 2ul to each probe tube tip, and for the XF24 probe card, apply 5ul and dry at 50°C.

The invention has the following beneficial effects:

The use of tris(4,7-phenyl-1,10-phenanthroline)ruthenium(II) complex has a longer excited state lifetime than other polypyridine complexes and is therefore expected to exhibit higher sensitivity. The long lifetime of the excited state also facilitates the design of relatively inexpensive sensing systems based on decay time measurements.

Poly(fluorescein isothiocyanateallylamine hydrochloride), PFIAH, is used, which is a type of fluorescein isothiocyanate derivative. Modification of FITC by poly(allylamine) can improve the biocompatibility and cell phagocytosis efficiency of nanosensors, which will promote the application of nanosensors in intracellular pH detection.

Compared with traditional macroscopic pH electrochemistry and optical fiber sensors that cannot accurately monitor pH changes in microenvironments such as cells, the probe of the present invention can effectively realize microenvironment detection. Among commonly used pH detection methods, detection methods based on changes in fluorescence signals have high Sensitivity, good selectivity and non-invasiveness.

Of course, any product implementing the present invention does not necessarily need to achieve all the above-mentioned advantages at the same time.

Description of the drawings

In order to explain the technical solutions of the embodiments of the present invention more clearly, the drawings needed to describe the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without exerting creative efforts.

Figure 1: Standard proportion diagram of the Nafion membrane solution of the present invention.

Figure 2: 96-well probe plate and cell plate of the present invention.

Figure 3: Schematic diagram of the use of the 96-well probe plate and cell plate of the present invention.

Figure 4: A graph produced by detecting the change rate of oxygen and the change rate of hydrogen ions according to the present invention.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, rather than all the embodiments.

A fluorescent probe complex for measuring oxygen and hydrogen ions, including substance one: tris(4,7-phenyl-1,10-phenanthroline)ruthenium(II) bis(hexafluorophosphate), with a specific structure: :

Substance 2: Polyfluorescein isothiocyanate allylamine salt;

Substance three: 5% Nafion membrane solution.

What we use is 5% Nafion membrane solution, the composition is shown in Figure 1:

The preparation method of fluorescent probes for measuring oxygen and hydrogen ions includes the following steps:

Prepare substance one and substance two into an aqueous solution with a concentration of 20 mg/mL and an ethanol solution with a concentration of 10 mg/mL respectively in a container; the container is an Erlenmeyer flask.

Then mix the two with 5% Nafion membrane solution at a volume ratio of 1:2:4, stir for 10 minutes to ensure uniform mixing, and then apply the resulting mixed solution dropwise onto an acrylic plate, and dry in a vacuum oven at 25°C for 3 hours. , you can get the required probe.

The use of fluorescent probes for measuring oxygen and hydrogen ions requires the use of the XF series extracellular energy analyzer, whose core components include a 96-well or 24-well probe plate and a cell plate; the XF series extracellular energy analyzer invented by Agilent is The most authoritative instrument for studying such issues. Its core components (consumables) are a set of 96-well (or 24-well) probe plate and cell plate. XF9 type consumables are shown in Figure 2.

A probe card has 96 hollow, transparent bottom probe tubes (sensor probes) at the lower end, which go deep into the corresponding cell wells (well). The core part is the white fluorescent probe compound (solid statesensor) at the tip of the probe tube. ), as well as several bundles of fiber optics in the probe tube that emit and collect excitation light. The fluorescent probe complex mainly detects real-time changes in oxygen and hydrogen ions in cell culture fluid. The oxygen change rate (OCR) represents the intensity of aerobic respiration (mitochondrial respiration), and the hydrogen ion change rate is the acidification rate (ECAR). Rate of anaerobic glycolysis. The working diagram is shown in Figure 3.

When the sensor probe penetrates into the well of the cell plate, the fluorescent probe complex (solid statesensor) forms a photosensitive intermediate substance with oxygen and hydrogen ions in the cell culture medium respectively, and receives the characteristic excitation provided by optical fibers (fiberoptics). Light, produce characteristic emitted light, and receive the emitted light, convert it into electrical signals, and transmit them to the data analysis system. In this way, the real-time energy metabolism changes of cells can be accurately described by this indirect test.

Test the suitable pH range for glycolysis, which is within the physiological range of cells, generally between PH7.2-7.6, and its ECAR value is between 5-300mph/min. Test the suitable oxygen partial pressure range for aerobic respiration, which is generally between 50-160mmHg. Its OCR value is 5-200pmol/min. The change curve is shown in Figure 4.

The preferred embodiments of the invention disclosed above are only intended to help illustrate the invention. The preferred embodiments do not describe all details, nor do they limit the invention to the specific implementations described. Obviously, many modifications and variations are possible in light of the contents of this specification. These embodiments are selected and described in detail in this specification to better explain the principles and practical applications of the present invention, so that those skilled in the art can better understand and utilize the present invention. The invention is limited only by the claims and their full scope and equivalents.

Claims (3)

1. A fluorescent probe complex for measuring oxygen and hydrogen ions, characterized by: Including substance one: tris(4,7-phenyl-1,10-phenanthroline)ruthenium(II) bis(hexafluorophosphate), the specific structure is: ; Substance 2: Polyfluorescein isothiocyanate allylamine salt; Substance three: 5% Nafion membrane solution; The preparation method of fluorescent probes for measuring oxygen and hydrogen ions includes the following steps: Prepare substance one and substance two in a container into an aqueous solution with a concentration of 20 mg/mL and an ethanol solution with a concentration of 10 mg/mL respectively; Then mix the two with 5% Nafion membrane solution at a volume ratio of 1:2:4, stir for 10 minutes to ensure uniform mixing, and then apply the resulting mixed solution dropwise onto an acrylic plate, and dry in a vacuum oven at 50°C for 3 hours. , you can get the required probe. 2. The use of a fluorescent probe complex for measuring oxygen and hydrogen ions according to claim 1, characterized in that: an XF series extracellular energy analyzer needs to be used, the core components of which include a 96-well or 24-well probe plate and cell plate; Inoculate cell culture medium with 10,000-300,000 cells per well in the cell plate; Apply the fluorescent probe compound to the tip of the probe tube corresponding to the probe board, install the probe board and the cell plate overlappingly, and then place them into the XF series extracellular energy analyzer. The XF series extracellular energy analyzer should be placed in the corresponding probe tube. Having several bundles of optical fibers that emit and collect excitation light; Fluorescent probes mainly detect real-time changes in oxygen and hydrogen ions in cell culture fluid. The change rate of oxygen represents the intensity of aerobic respiration, and the change rate of hydrogen ions, the acidification rate, represents the rate of anaerobic glycolysis. 3. The use of a fluorescent probe complex for measuring oxygen and hydrogen ions according to claim 2, characterized in that: for the XF96 probe card, the tip of each detection tube is coated with 2ul, and for the XF24 probe card, Apply 5ul and dry at 50℃.