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Method for producing internal reference nano PH sensor and using for in-cell PH non-trauma monitoring

A pH sensor and internal reference technology, applied in the field of nanomaterials, can solve the problems of complex preparation process of PEBBLES sensor, sensitive dye measurement error, interference pH measurement, etc., and achieve biocompatibility, improved reliability, and stable physical and chemical properties. Effect

Inactive Publication Date: 2005-04-27
HUNAN UNIV
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Problems solved by technology

[0002] Many important life activities such as cell growth and differentiation, calcium regulation, cell apoptosis, etc. are closely related to intracellular pH. Since the size of cells ranges from a few microns to tens of microns, it is necessary to develop Small sensors, nano- and micro-level optical fibers and photoelectrodes developed earlier need to penetrate cells to detect intracellular pH, but at the same time they can also cause damage to cells, causing physiological changes in cells and interfering with pH measurement
The development of non-invasive, high-sensitivity, and rapid-response real-time detection methods is a new requirement for the development of sensors. The preparation process of the PEBBLES sensor that arises at the historic moment is complicated, and the sensitive dyes embedded are limited to lipophilic dyes. , The method of gene gun delivery into cells requires expensive instruments and requires professional operation
Although the newly developed liposome sensor is easy to enter the cell, after the fusion of the support material and the cell membrane, the release of the encapsulated sensitive dye into the cytoplasm will cause damage to the cell, and the sensitive dye is easily interfered by the background fluorescence signal, resulting in measurement error
Fluctuations of the excitation light source, photobleaching, transmission changes of the optical fiber, and light scattering of the sample are all likely to cause changes in the measurement signal, so the sensor embedded with a single sensitive dye cannot accurately reflect the change of pH through the change of the fluorescence signal, so The above sensors are difficult to achieve non-invasive, highly sensitive, accurate and real-time dynamic monitoring of intracellular pH changes

Method used

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  • Method for producing internal reference nano PH sensor and using for in-cell PH non-trauma monitoring
  • Method for producing internal reference nano PH sensor and using for in-cell PH non-trauma monitoring
  • Method for producing internal reference nano PH sensor and using for in-cell PH non-trauma monitoring

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1, preparing a silicon-shell fluorescent nanometer pH sensor with an internal reference:

[0043] The first step is to configure the solutions of sensitive dye and reference dye: equimolar 0.1mol / L saturated FITC solution of fluorescein isothiocyanate and aminosilylating reagent γ-aminopropyltriethoxysilane (APTES) Mix and react overnight at room temperature in the dark, then mix the obtained product FITC-APTES (80 μL) with 0.1 mol / L biruthenium pyridine solution (10 μL) and set aside.

[0044] In the second step, cyclohexane (7.5mL), surfactant Triton X-100 (TritonX-100, 1.8mL) and co-surfactant n-hexanol (1.8mL) were mixed evenly, and 310 μL of water was added as the dispersed phase , stir until the solution is a clear and translucent water-in-oil microemulsion, add the sensitive dye and reference dye mixture prepared in the first step, stir evenly, add 200 μL of silylating reagent orthoethyl silicate (TEOS) and 200 μL of catalytic ammonia water, The reaction l...

Embodiment 2

[0045] Example 2, a silicon-shell fluorescent nanopH sensor with an internal reference is used for non-invasive monitoring of pH in macrophages

[0046] The first step is to draw the standard curve of the nanosensor’s response to pH. The specific process is: add 5 μL of nanoparticles to 200 μL of buffer solution with different pH, react for 5 minutes, and drop the solution onto the laser confocal microscope stage On the glass slide, record the respective relative fluorescence intensity of sensitive dye fluorescein isothiocyanate FITC and reference dye biruthenium pyridine Rubpy in the nanoparticle, calculate the fluorescence ratio of the two (I FITC / I Rubpy ), and the experiment was repeated three times. Taking pH as the abscissa, I FITC / I Rubpy Draw the response curve of nanoparticles to pH for the ordinate, such as image 3 , the linear range of the nanosensor to pH response is 4-7.5, since the pH change in the cytoplasm and organelles is just in the range of pH5-7 und...

Embodiment 3

[0050] Example 3, a silicon-shell fluorescent nanopH sensor with an internal reference is used for non-invasive monitoring of pH in liver cancer cells

[0051] In the first step, the liver cancer cells were pre-cultured to 70% coverage 24 hours before observing the intracellular pH, and the culture medium was discarded. Dilute and disperse the pre-sonicated 8μL nanosensor suspension with 1mL serum-free medium, then add it to the culture dish with liver cancer cells, and incubate at 37°C for 4 hours. Since the size of the nanosensor is about 40nm, liver cancer The cells can endocytose the sensor into the cell, realizing the non-invasive introduction of the nanometer pH sensor into the liver cancer cells. Wash the cells three times with cold phosphate buffered saline (PBS, pH7.4) before observation, remove excess nanosensors in the medium that have not been phagocytosed by cells and nanosensors non-specifically adsorbed on the surface of liver cancer cells, and then add 1 mL pH6...

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Abstract

The present invention aims at synthesizing one kind of nanometer pH sensor with embedded reference dye and sensitive dye and stable performance for non-invasive monitoring of dynamic pH variation inside cell. The preparation process includes mixing oil phase cyclohexane, surfactant Triton X-100 and co-surfactant n-hexanol and water as dispersive phase to obtain water-in-oil micro emulsion; adding mixed sensitive and reference dye liquid via stirring; adding ethyl acetate as sylylation reagent, TEOS and catalyst ammonia water to react, demulsifying with acetone, washing with absolute ethanol and super-purified water to obtain inner reference nanometer sensor with silicon shell. The inner reference nanometer sensor is used in non-invasive dynamic intracellular pH monitoring.

Description

Technical field: [0001] The invention relates to the field of nanomaterials, in particular to a preparation method of a nanometer pH sensor with an internal reference and its application to real-time monitoring of intracellular pH changes. Background technique: [0002] Many important life activities such as cell growth and differentiation, calcium regulation, cell apoptosis, etc. are closely related to intracellular pH. Since the size of cells is between several microns to tens of microns, measuring intracellular pH changes requires the development of Small sensors, nano- and micro-level optical fibers and photoelectrodes developed earlier need to penetrate cells to detect intracellular pH, but at the same time they can also cause damage to cells, causing physiological changes in cells and interfering with pH measurement. The development of non-invasive, high-sensitivity, and rapid-response real-time detection methods is a new requirement for the development of sensors. The...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61B5/145G01N21/01G01N21/76G01N21/80
Inventor 王柯敏谭蔚泓彭姣凤王燕何晓晓邢新丽
Owner HUNAN UNIV
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