Method for detecting calcium ions inside and outside cells at high sensitivity

Abstract

The invention discloses a high-sensitivity method for detecting calcium ions inside and outside cells, and belongs to the field of laser technology and photonics. An optical microcavity doped with a fluorescent dye is used as a micro laser, the surface of the optical microcavity is functionalized, the optical microcavity is connected with a calcium ion probe to form a sensing system, the sensing system is placed in solutions containing different free calcium concentrations and different types of cells, the change of a microcavity emission spectrum is monitored, and the calcium level is rapidly analyzed on the basis of the displacement difference of a central wavelength. The detection limit of calcium ions reaches pM order of magnitude, the dynamic range at least reaches 6 order of magnitude, and non-toxic, rapid, real-time and accurate quantification of calcium concentrations in different cells is realized.

Description

Technical Field

[0001] The invention belongs to the field of laser technology and photonics, and specifically relates to a method for using an optical microcavity to perform high-sensitivity detection of intracellular and extracellular calcium ions. Background Art

[0002] Since electromagnetic waves will be reflected when they propagate from optically dense media to optically sparse media, in a rotationally symmetric geometric structure, continuous total reflection will occur when light propagates along the inner wall of the geometric structure boundary, and the light beam will be constrained on the annular boundary, thus producing the whispering gallery phenomenon. When the optical path of the light beam propagating around the geometric structure boundary satisfies an integer multiple of the wavelength, interference enhancement, i.e., resonance, will occur. We call this annular structure used to constrain the light field a whispering gallery mode optical microcavity. Since the whispering gallery mode optical microcavity has an ultra-high quality factor and a small mode volume, it can greatly enhance the interaction between light and matter. Therefore, more and more studies are using it to study high-sensitivity optical sensing. In order to further improve the sensitivity of the whispering gallery mode optical microcavity, the optical microcavity is generally combined with other technologies, such as local surface plasmon enhancement, energy resonance transfer technology, self-heterodyne laser beat frequency measurement, etc. These methods can effectively improve the detection sensitivity or reduce the detection limit. There have been some reports on optical microcavities in the field of cells, such as labeling and tracking cell activities, detecting intracellular small molecules, monitoring the heart contraction process, and quantitative analysis of cell secretions.

[0003] Calcium ions are one of the most important signaling molecules in cells, regulating a variety of cellular functions. Usually, there is a significant difference in the concentration of intracellular and extracellular calcium ions. Usually, the extracellular calcium concentration is 1.2mM, and the cytoplasmic free calcium concentration is about 100nM at rest. The concentration gradient of 10,000 times makes the intracellular calcium ion level change significantly during information transmission. Intracellular calcium homeostasis is necessary for normal cell function. Studies have found that calcium imbalance is considered to be one of the important factors in the occurrence of many diseases, such as cancer, atrial arrhythmia and Alzheimer's disease. Exploring ion dynamics in the cellular environment is very important for the diagnosis and treatment of early diseases. At present, many methods for detecting intracellular calcium ion concentration have been developed, such as genetically encoded calcium indicators (GECIs), fluorescent probe indicators, nanobiosensors, nuclear magnetic resonance spectroscopy, etc. However, these methods have certain limitations for the detection of calcium ions, such as the photobleaching phenomenon of indicators, inaccurate measurements, weak signals, expensive imaging equipment, and challenges encountered in large-scale quantitative analysis, which limit the detection and analysis of intracellular calcium ions.

[0004] In view of the above problems, it is particularly important to develop a method for rapid and accurate quantitative analysis of intracellular calcium ions, which is the key to the early diagnosis and prevention of diseases related to calcium imbalance. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides an optical microsphere laser doped with green fluorescent material inside the resonant cavity, and uses chemical reaction to connect the red calcium ion probe to the surface of the microcavity through covalent bonds to form a sensing system. By pumping the optical microcavity, the different surrounding calcium concentrations cause the emission spectrum to shift, and the wavelength shift distance is detected to perform quantitative analysis of the calcium level.

[0006] The present invention is achieved through the following technical contents:

[0007] 1. The whispering gallery mode microsphere cavity is selected as the microlaser for emitting laser light. This mode of microcavity has the advantages of ultra-high quality factor, small mode volume and label-free sensing. The polystyrene microspheres doped with green fluorescent material used are in the micrometer range in diameter and are non-toxic, easy to enter cells, and can also be used as donors for energy transfer.

[0008] 2. Select fluorescent substance 2 with multiple excitation wavelengths as a probe for identifying calcium ions. This fluorescent probe has the function of specific recognition of calcium ions. Its absorption spectrum has a good overlap with the emission spectrum of fluorescent substance 1 inside the microcavity, which meets the conditions for energy transfer. It is covalently coupled to the surface of the optical microcavity through chemical reaction to form a sensing system.

[0009] 3. Prepare the sensing probe for monitoring the dual resonance peak. After drying the microsphere cavity doped with fluorescent substance 1, put it into a plasma cleaning machine and introduce oxygen. The oxygen is excited by the electric field energy in the electric field, dissociating into oxygen plasma, which contacts the surface of the optical microcavity to react chemically, and modifies the surface with carboxyl groups. After 10 minutes of treatment, it is redispersed in the buffer solution, and EDC / NHS crosslinking agent is added to activate the carboxyl groups on the surface of the microspheres. Subsequently, calcium ion probes are added and placed on a shaker to allow the microspheres to evenly contact and react with the calcium ion probes. After 2 hours, the microsphere solution with the calcium probe coupled to the surface is taken out, washed with pure water, and resuspended in PBS buffer solution for storage.

[0010] The prepared sample is placed in the microscopic excitation light path, and a 473nm pump source with a wavelength suitable for the inside of the microsphere is used to irradiate the sample. When the fluorescent substance 1 is excited, the light beam will be confined in the microsphere, and through multiple total internal reflections at the interface of the microsphere, when the light reaches the origin of the same phase after one cycle, the resonance condition of the cyclic light is met, and the whispering gallery mode spectrum is observed at this time. When calcium ions exist in the solution, due to the existence of energy transfer, the microsphere cavity will transfer part of the energy to the calcium ion probe on the surface, achieving synchronous luminescence, so that the fluorescent substance 1 and the calcium ion probe (fluorescent substance 2) in the microsphere emit dual-resonance wavelengths at the same time.

[0011] As the level of free calcium in the surrounding environment changes, the position and wavelength difference of the dual resonance wavelengths emitted by the microsphere cavity and the calcium probe also vary. When the calcium ion concentration in the solution is high, almost all calcium probes on the surface of the microsphere cavity will recognize calcium, and more energy will be transferred from the microsphere cavity to the calcium probe, resulting in increased loss of the microsphere cavity during the excitation process, while the gain obtained by the calcium probe increases, resulting in a blue shift in the central resonance wavelength emitted by the microsphere cavity, a red shift in the central resonance wavelength emitted by the calcium probe, and an increase in the wavelength difference between the dual resonance wavelengths. Conversely, when the calcium ion concentration in the solution is low, the central resonance wavelength emitted by the microsphere cavity is red-shifted, the central resonance wavelength emitted by the calcium probe is blue-shifted, and the wavelength difference between the dual resonance wavelengths is reduced. The relationship between calcium ion concentration and the dual resonance wavelength difference is used to draw a standard curve, which is used as the basis for quantifying calcium levels.

[0012] The prepared sensing system can not only detect the calcium level in the solution, but also detect the free calcium concentration in the cell by incubating it with different cells. The microsphere cavity is internalized into the cell through endocytosis, and the microsphere cavity in the cell is irradiated after sample preparation. The wavelength difference between the dual resonance wavelengths emitted by the microsphere cavity in the cell is monitored, and the free calcium level in different cells can be calculated using the above standard curve as a reference.

[0013] Compared with the prior art, the present invention has the following advantages:

[0014] 1. Optical microcavities are particularly suitable for the field of cell sensing due to their advantages such as ultra-high quality factor and small mode volume. The changes in the laser spectrum emitted by the microcavity can monitor tiny disturbances in the surrounding environment. Combined with energy transfer technology, it greatly increases the detection range and sensitivity of the target object.

[0015] 2. Using wavelength difference as the basis for detection is more accurate and stable. When the microsphere cavity is irradiated for a period of time, the wavelength of the spectrum obtained remains stable, getting rid of the influence of photobleaching.

[0016] 3. The selected polystyrene microsphere cavity has high biocompatibility, is non-toxic to cells, is in the micrometer range in size, and can enter cells by itself through endocytosis, making it particularly suitable for cell sensing.

[0017] 4. It can dynamically monitor calcium levels in real time. By laser pumping the microsphere cavity, the emitted laser spectrum and the double resonance wavelength difference can be quickly displayed in real time in the computer software, and the calcium concentration in the solution and in the cell can be immediately calculated through the standard curve. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Double resonance spectra of DG and Rhod-4 in solutions with different calcium concentrations

[0019] Figure 2 Laser emission spectrum of microsphere sensor probe in Hela DETAILED DESCRIPTION

[0020] The present invention is further described below in conjunction with experimental cases, but the present invention is not limited to the following experimental cases.

[0021] Implementation Case 1: Calcium chloride solution of different concentrations was added to the solution, ethylene glycol bis-α-aminoethyl ether tetraacetic acid solution was added to chelate calcium ions, the free calcium ions in the solution were adjusted to the required concentration, the prepared microsphere sensor probe was added to the solution, and placed in a vortex oscillator for uniform dispersion, and then the microsphere cavity was excited by a laser, and the emission spectrum was observed and collected on a computer.

[0022] The specific experimental steps are as follows:

[0023] (1) A polystyrene optical microcavity doped with dragon green dye was selected as a microlaser, with a doping mass fraction of 2% and a diameter of 15.25 μm. The red calcium ion probe was Rhod-4. The emission spectrum of DG and the absorption spectrum of Rhod-4 overlapped by more than 60%, ensuring the occurrence of energy transfer. 5 μl of the polystyrene optical microcavity was centrifuged and dried, and placed in a plasma cleaner for carboxyl modification. The treatment time was 10-15 min, and oxygen was introduced. The oxygen content introduced each time was 0.1 MPa. The power of the plasma cleaner was set to 100 W and the vacuum pressure was 40 Pa. The oxygen was affected by the electric field energy in the electric field, dissociated into oxygen plasma, and chemically reacted with the surface of the optical microcavity to modify the carboxyl group on its surface, and then redispersed in 10 μl of MES buffer.

[0024] (2) Weigh EDC powder and NHS powder, add MES buffer to prepare 5 mg / ml solution, add 5 μl EDC solution and 5 μl NHS solution to the optical microcavity dispersed in 10 μl MES buffer, respectively, mix well and place in a shaker to activate the carboxyl groups on the microcavity surface. The shaker speed is set to 200 r / min and the temperature is 22°C. After reacting for 15 minutes, take out and place in a low-temperature high-speed centrifuge at a speed of 3000 rpm and a temperature of 20°C. After 5 minutes, remove the supernatant, add 10 μl PBS buffer to resuspend the micro-optical microcavity, and then add 5 μl Rhod-4 solution with a concentration of 50 μM. After mixing and dispersing well, place in a shaker to allow the microspheres to evenly contact with the calcium ion probe to react. The shaker speed is set to 200 r / min and the temperature is 22°C. After 2 hours, take out the optical microcavity solution with the calcium probe coupled on the surface, wash it and resuspend it in PBS buffer for storage.

[0025] (3) Generally speaking, water contains trace amounts of calcium ions. In order to ensure the accuracy of the calcium concentration in the detection solution, we used 10mM ethylene glycol bis-α-aminoethyl ether tetraacetic acid solution to chelate the calcium ions in the buffer solution, and mixed 30mM 3-(N-morpholino)propanesulfonic acid solution and 100mM potassium chloride solution as the buffer solution, wherein the pH of the buffer solution was 7.2. At 22°C and pH = 7.2, adding different concentrations of calcium chloride solution to the buffer solution can obtain different concentrations of free calcium, as shown in Table 1. We added different concentrations of calcium chloride to the buffer solution containing 10mM ethylene glycol bis-α-aminoethyl ether tetraacetic acid solution, 9mM, 6mM, 4mM, 2mM, 1mM, 100uM, 100nM, 25nM, 10nM, 1nM, 0nM, and placed the mixed solution in an oscillator to disperse it evenly, then dripped it into a cover glass, took another cover glass to cover it from one side, and sealed it with nail polish. After the sample was prepared, it was placed in the excitation system.

[0026] (4) Turn on the 473 nm laser to excite the sample, observe and collect the changes in the excitation spectrum emitted by the microsphere cavity, measure 30 sets of data for each concentration, and take the average value of the central resonance wavelength, such as Figure 1 As shown, two groups of laser peaks are obtained, namely the microsphere cavity resonance peak and the Rhod-4 resonance peak. The position and wavelength difference (Δλ) of the double peaks are different with different free calcium concentrations in the solution. When the free calcium concentration in the solution is 1350nm, the double peak wavelength difference is 19.01nm. At this time, the DG resonance peak is located at 523.53nm and the Rhod-4 resonance peak is located at 542.54nm. As the free calcium concentration decreases, the double peak wavelength difference gradually decreases, the DG resonance peak moves to the long wave, and the Rhod-4 resonance peak moves to the short wave. When the free calcium concentration is small to a certain extent, when the free calcium concentration is 380fM, only the DG single peak output is observed.

[0027] (5) Using the different dual excitation wavelength differences obtained at different calcium levels, a concentration-frequency shift curve is plotted as a standard for quantifying calcium levels.

[0028] This experiment shows that the microsphere sensor probe prepared by the invention can quantify the free calcium level in the solution. The detection limit obtained by the method of the present invention reaches the pM level, and the highest calcium level that can be detected is 1 μM, realizing a wide range of calcium concentration detection from pM-μM, which is 6 orders of magnitude higher in sensitivity than conventional detection methods.

[0029] Implementation Case 2: The prepared microsphere sensor probes were incubated with Hela cells respectively. After the microspheres were internalized into the cells, the cell samples were prepared and placed in the excitation system. The changes in the double resonance peaks emitted by the microsphere cavity and Rhod-4 in different cells were observed, and the standard curve was used to quantitatively analyze the intracellular free calcium concentration.

[0030] The specific experimental steps are as follows:

[0031] (1) 300 μl of Hela cells were added to a culture dish with a coverslip, placed in a cell culture incubator, set the temperature to 37°C, and maintain the carbon dioxide content at 5%. The cells were incubated for 24 hours. After 24 hours, the culture dish was taken out and the cell growth state was observed under a microscope to be in the logarithmic growth phase. Then 5 μl of the prepared sensor system was added, and the culture dish was gently shaken to make it evenly dispersed. Then, it was placed in a cell culture incubator and incubated for 3-4 hours. Due to the small size and non-toxic characteristics of the microsphere cavity, most of the microspheres will enter the cells through endocytosis and will not destroy the cell morphology.

[0032] (2) After the cells are internalized into the microsphere cavity, wash the cell pick with PBS buffer, take another pick to cover it from one side, seal it with nail polish and place it in the excitation system, turn on the 473nm laser, excite the sample, and observe the laser spectrum emitted by the microcavity sensor system in different cells. Measure 30 sets of data for each concentration and take the average value of the central resonance wavelength. Figure 2 As shown, the Δλ of Hela cells is 16.06 nm, corresponding to an intracellular calcium concentration of 54.60 nM.

[0033] This experiment shows that the microsphere sensor probe prepared by the invention can quantify the level of free calcium in cells, and proposes a new intracellular calcium measurement method with single-cell resolution, which can quantify the intracellular free calcium concentration more quickly and accurately.

[0034] Table 1 Free calcium concentration corresponding to different calcium chloride solutions

[0035]

Claims

1. A method for highly sensitive detection of intracellular and extracellular calcium ions, characterized in that: An optical microcavity doped with fluorescent material is selected as a microlaser, and a calcium ion fluorescent probe is modified on the surface of the optical microcavity through a chemical reaction to form a sensing system. The system is placed in a solution containing calcium ions. The calcium ion probes connected around the microcavity will recognize free calcium and bind to it. After being excited by a 473nm laser pump source that can excite the optical microcavity, the microcavity will transfer part of the energy to the calcium probe. Finally, a double laser resonance peak emitted by the fluorescent material and the calcium ion fluorescent probe is obtained at the same time. The change in the wavelength of the double resonance peak caused by the different calcium levels in the solution is used as the detection target.

2. The method for highly sensitive detection of intracellular and extracellular calcium ions according to claim 1, characterized in that: The absorption spectrum range of the calcium ion probe used overlaps with the emission spectrum range of the fluorescent material doped inside the microcavity by more than 60%, satisfying the conditions for energy transfer to occur.

3. The method for highly sensitive detection of intracellular and extracellular calcium ions according to claim 1, characterized in that: Under different calcium levels, the frequency shift difference between the dual resonance peaks will change. A 473nm laser is used to continuously pump the optical microcavity for more than 10 minutes, and the frequency shift difference is used as the detection target and as a basis for quantifying the calcium concentration.

4. The method for highly sensitive detection of intracellular and extracellular calcium ions according to claim 1, characterized in that: The optical microcavity doped with fluorescent material is an optical microcavity doped with dragon green dye with a doping mass fraction of 2% as a microlaser, and Rhod-4 is selected as a calcium ion fluorescent probe.

5. The method for highly sensitive detection of intracellular and extracellular calcium ions according to claim 1, characterized in that: The preparation of the sensing system is specifically as follows: oxygen is filled into a plasma cleaner, the treatment time is 10-15 minutes, the oxygen pressure introduced each time is 0.1 MPa, the power of the plasma cleaner is set to 100 W, and then the carboxyl groups on the surface of the microspheres are activated by a cross-linking agent, and Rhod-4 with an amino structure is added, and the reaction is placed in a shaker for 2 hours at a temperature of 22°C to form a sensing probe.

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