CN114878438B - Intracellular and extracellular photoelectric integrated detection platform and construction method and application thereof - Google Patents
Intracellular and extracellular photoelectric integrated detection platform and construction method and application thereof Download PDFInfo
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- 230000003834 intracellular effect Effects 0.000 title claims abstract description 65
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- 239000000126 substance Substances 0.000 claims abstract description 10
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- 238000000338 in vitro Methods 0.000 claims abstract description 7
- 238000001727 in vivo Methods 0.000 claims abstract description 6
- 230000003287 optical effect Effects 0.000 claims abstract description 4
- 239000013307 optical fiber Substances 0.000 claims description 53
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- 239000000835 fiber Substances 0.000 claims description 17
- 108091008102 DNA aptamers Proteins 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 13
- 230000005693 optoelectronics Effects 0.000 claims description 9
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- KTWOOEGAPBSYNW-UHFFFAOYSA-N ferrocene Chemical compound [Fe+2].C=1C=C[CH-]C=1.C=1C=C[CH-]C=1 KTWOOEGAPBSYNW-UHFFFAOYSA-N 0.000 claims description 4
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 3
- 229910021607 Silver chloride Inorganic materials 0.000 claims description 2
- 229910010413 TiO 2 Inorganic materials 0.000 claims description 2
- 238000005253 cladding Methods 0.000 claims description 2
- 230000008878 coupling Effects 0.000 claims description 2
- 238000010168 coupling process Methods 0.000 claims description 2
- 238000005859 coupling reaction Methods 0.000 claims description 2
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- 229910052697 platinum Inorganic materials 0.000 claims description 2
- 229910052709 silver Inorganic materials 0.000 claims description 2
- 239000004332 silver Substances 0.000 claims description 2
- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 claims description 2
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- 229910052946 acanthite Inorganic materials 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1434—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its optical arrangement
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G01N15/01—
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N2015/1006—Investigating individual particles for cytology
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1434—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its optical arrangement
- G01N2015/144—Imaging characterised by its optical setup
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Abstract
The invention discloses an intracellular and extracellular photoelectric integrated detection platform, which comprises: an intracellular fluorescence detection system and an extracellular photoelectric detection system; the intracellular fluorescence detection system comprises a light source part, a scanning system, a first detection system and a first signal collection detection system; the extracellular photoelectric detection system comprises a light source part, a second detection system and a second signal collection detection system. The invention also provides a construction method of the detection platform and application of the detection platform in-vivo and/or in-vitro collection of intracellular fluorescent signals and/or extracellular photocurrent signals respectively or simultaneously. The built intracellular and extracellular photoelectric integrated detection platform can realize simultaneous collection of intracellular and extracellular signals, is suitable for quantitative analysis of detection substances, and has simple instrument and equipment assembly and excellent performance. And the fluorescent probe can be widely applied to fluorescent probes with other wavelengths by replacing the optical filter, and has low cost and high benefit.
Description
Technical Field
The invention belongs to the technical field of spectrum imaging and biosensing, and relates to an intracellular and extracellular photoelectric integrated detection platform, and a construction method and application thereof.
Background
The brain is the highest part of the central nervous system, and many diseases are closely related to the concentration imbalance of related substances in the brain environment, so that the research on the related substances of different brain areas has important significance. Conventional electrophysiological techniques when used in brain imaging analysis often have limitations of insufficient spatial resolution and it is difficult to distinguish between chemical signals of different substances. Fluorescent imaging technology has evolved over the years to provide powerful support for detecting signal changes from different chemicals. However, in the conventional fluorescence apparatus, it is difficult to perform simultaneous detection of a plurality of substances both inside and outside the cell, and therefore, there is an urgent need to develop an apparatus for simultaneously collecting an intracellular fluorescence signal and an extracellular photocurrent signal in a brain region of a living body.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide a method for constructing an intracellular and extracellular photoelectric integrated detection platform and application thereof, wherein the intracellular and extracellular photoelectric integrated detection platform can be used for respectively and simultaneously collecting intracellular fluorescence signals and/or extracellular photocurrent signals in vivo and/or in vitro.
The invention provides an intracellular and extracellular photoelectric integrated detection platform, which is shown in the figure 1.
The intracellular and extracellular photoelectric integrated detection platform comprises the following components:
1. an intracellular fluorescence detection system;
a light source section: the light source part is a fiber laser. The excitation center wavelength is 488nm plus or minus 0.5nm; the setting range of the output power is 0-500mW; line width is less than 0.1nm; the output interface is SMA905 or FC/PC; the operating voltage is 220V.
Scanning system: the scanning system core is based on a laser confocal unit and comprises a first filter, a second filter, a first reflecting mirror, a second reflecting mirror, a scanner, an objective lens and the like. When excitation light is generated from the fiber laser, the excitation light sequentially passes through the first filter, the first reflecting mirror, the second filter and the second reflecting mirror to enter the scanner, and then passes through the objective lens to be focused; the first filter is a band-pass filter, and can transmit 488nm light; the second filter is a notch filter for filtering 488nm incident light; the first reflecting mirror, the second reflecting mirror and the third reflecting mirror are all total reflecting mirrors, the scanner is a laser confocal scanning unit, the magnification of the objective lens is 10×, and the numerical aperture NA is 0.25. As shown in fig. 2.
A first detection system: the first detection system is mainly based on multimode optical fiber signal transmission and collection. The tail end of the optical fiber is drawn and tapered to form an optical fiber with a tapered head, incident light enters the optical fiber after being focused by the objective lens, excitation light is emitted from the tapered end of the optical fiber, and the excitation probe generates a fluorescent signal and is collected through the same optical fiber. The fiber was a multimode fiber with a core diameter of 200 microns and a cladding thickness of 12.5 microns. The Numerical Aperture (NA) is 0.22 and the transmission range is 400-1100nm. The taper length of the fiber end was 500 μm and the diameter of the fiber tip was 500nm. As shown in fig. 4.
The first signal collecting system is partially overlapped with the scanning system and comprises an objective lens, a scanner, a second reflecting mirror, a second filter, a third filter, a fourth filter, a third reflecting mirror and a fluorescence detector; after the fluorescent signals collected by the optical fibers return to the scanner, the fluorescent signals with corresponding wavelengths are returned to the fluorescent spectrometer through the second reflector, the second filter, the third filter, the fourth filter and the third reflector, and the fluorescent signals with corresponding wavelengths are read out by the fluorescent detector. The scanner is a confocal laser scanning unit. The objective magnification was 10×, and the numerical aperture NA was 0.25. The second reflecting mirror is a total reflecting mirror; the second filter is a notch filter for filtering 488nm incident light; the third filter plate and the fourth filter plate are both band-pass filter plates, and the third reflector is a total reflector and is used for separating fluorescent signals and guiding the fluorescent signals into a fluorescent detector. As shown in fig. 3. The third filter (6) is used for separating out a 700nm fluorescent signal, the fourth filter (7) is used for separating out a 600nm fluorescent signal, and the third reflector (8) is used for reflecting a 515nm fluorescent signal.
2. Extracellular photodetection system:
the light source part in the extracellular photoelectric detection system is shared with the intracellular fluorescence detection system;
a second detection system: the second detection system is partially overlapped with the first detection system, is a photoelectrochemical sensor based on an optical fiber with a conical head part, and sequentially modifies ITO and TiO on the surface of the optical fiber 2 、Ag 2 S, DNA aptamer; the ITO modified on the surface of the optical fiber in the second detection system is used for making the optical fiber conductive, and TiO 2 And Ag 2 S is the electron transmission part in the photoelectrochemical sensor, and the DNA aptamer is used for specifically identifying the substance to be detected. As shown in fig. 5;
a second signal collection system: the second signal collecting system is a loop formed by a three-electrode system consisting of a working electrode, a reference electrode and an auxiliary electrode and an electrochemical workstation through a lead, and photocurrent signals are output from the electrochemical workstation; the working electrode is an electrode based on optical fibers, the reference electrode is a silver/silver chloride electrode, the auxiliary electrode is a platinum electrode, and the electrochemical workstation is a Chenhachi 660 electrochemical workstation.
The invention provides a method for constructing an intracellular and extracellular photoelectric integrated detection platform, which comprises the following steps:
step 1: assembling a confocal scanning unit, comprising the following sub-steps:
step 1-1: a first filter plate, a first reflecting mirror, a second filter plate and a second reflecting mirror are sequentially arranged;
step 1-2: installing a scanner and an objective lens;
step 2: setting up a scanning system, which specifically comprises the following substeps:
step 2-1: connecting an optical fiber laser with a confocal scanning unit through an optical fiber;
step 2-2: the optical path is debugged, and the test excitation light can pass through the confocal scanning unit and is transmitted out by the objective lens;
step 3: building a first detection system and a second detection system, which concretely comprises the following substeps:
step 3-1: tapering the optical fiber to obtain the optical fiber with the tapered head, and improving the optical fiber signal collection efficiency;
step 3-2: sequentially modifying ITO and TiO on the surface of the optical fiber with the conical head 2 、Ag 2 S, DNA aptamer
Step 3-3: assembling an optical fiber with a conical head part to the tail end of the scanning system, and coupling debugging excitation light into the optical fiber;
step 4: building a first signal collection detection system and a second signal collection detection system, and specifically comprising the following substeps:
step 4-1: a third filter plate, a fourth filter plate and a third reflecting mirror are arranged; the emergent light paths of the third filter plate, the fourth filter plate and the third reflecting mirror correspond to three detection positions of the fluorescence spectrometer;
step 4-2: connecting the fluorescence spectrometer in the step 4-1 with a confocal scanning unit through an optical fiber;
step 4-3: and connecting the optical fiber with the conical head with a reference electrode, an auxiliary electrode and an electrochemical workstation to finally form the intracellular and extracellular photoelectric integrated detection platform.
The invention also provides application of the intracellular and extracellular photoelectric integrated detection platform constructed by the method in vivo and/or in vitro to respectively or simultaneously collect intracellular fluorescence signals and/or extracellular photocurrent signals
The extracellular and intracellular photoelectric integrated detection platform can collect in-vitro intracellular fluorescence signals and extracellular photocurrent signals respectively or simultaneously under excitation of excitation light;
fluorescent molecules used for fluorescent signal detection in the detection platform are developed ratio type nano fluorescent probes;
the fluorescence emission wavelengths of the ratio type nano fluorescent probe are 700nm,600nm and 515nm respectively,
excitation light of the fluorescent molecule is 488nm;
the concentration of the fluorescent molecules is 1mM;
the detection wavelength of the fluorescence detector is 700nm,600nm and 515nm;
the DNA aptamer is a DNA aptamer of dopamine.
The intracellular and extracellular photoelectric integrated detection platform can collect intracellular fluorescence signals and extracellular photocurrent signals of different brain areas in a body respectively or simultaneously under excitation of excitation light;
the different brain regions are cortex, hippocampus, striatum and thalamus;
fluorescent molecules used for fluorescent signal detection in the detection platform are developed ratio type nano fluorescent probes;
the fluorescence emission wavelengths of the ratio type nano fluorescent probe are 700nm,600nm and 515nm respectively,
excitation light of the fluorescent molecule is 488nm;
the concentration of the fluorescent molecules is 1mM;
the detection wavelength of the fluorescence detector is 700nm,600nm and 515nm;
after the DNA aptamer is combined with a corresponding target molecule, linear DNA can be bent, when the electrode detects the target molecule, ferrocene at the far end of the DNA is closer to the surface of the electrode, more electrons can be provided, the phenomenon of photocurrent increase occurs, and the target molecule is quantified by the value of photocurrent, and specifically, the target molecule can be the DNA aptamer of dopamine.
The invention has the beneficial effects that: the method constructs an intracellular and extracellular photoelectric integrated detection platform, can realize the simultaneous collection of intracellular fluorescence signals and extracellular photocurrent signals in a multi-brain region of a living animal, is suitable for quantitative analysis of detection substances, and has the application function which is not possessed by the current commercial instrument
1. The diameter of the tip of the conical fiber electrode is about 500nm, so that the conical fiber electrode can be accurately positioned to the synaptic cleft of the neuron cell, which is not possessed by other technologies.
2. The method for detecting dopamine by photoelectricity can accurately quantify the concentration of dopamine at 0.1-500nM, the linear range is better than the micromolar level of the prior art, and the method is more suitable for quantitatively analyzing dopamine at cell and living body levels.
3. The invention combines the existing fluorescence technology and photoelectric technology, and the required instrument and equipment are simple to assemble and have excellent performance. And the fluorescent probe can be widely applied to fluorescent probes with other wavelengths by replacing the optical filter, and has low cost and high benefit.
Drawings
FIG. 1 is a schematic diagram of an intracellular and extracellular photoelectric integrated detection platform of the invention.
Fig. 2 is a schematic diagram of a scanning system of the present invention.
FIG. 3 is a schematic diagram of a first signal collection and detection system according to the present invention.
Fig. 4 is a schematic diagram of a first detection system of the present invention. The diameter of the tip of the conical optical fiber is about 500 nanometers and is far smaller than 20 micrometers in the prior art, and the conical optical fiber can be used for detecting neurotransmitters between synapses of neurons.
FIG. 5 is a graph showing extracellular photoelectric detection of dopamine and intracellular fluorescence detection of pH and Ca 2+ Is a schematic diagram of the principle of (a). The DNA aptamer is bent after being combined with dopamine serving as an object to be detected, so that more ferrocene is closer to the surface of an electrode after the DNA aptamer is combined with the dopamine during detection, electrons flowing into a silver sulfide valence band are increased, photocurrent is increased, the increase of photocurrent and the concentration of the dopamine are in a linear relationship between 0.1nM and 500nM, the linear range is better than the micromolar level of the prior art, and the quantitative analysis of the dopamine is more suitable for cell and living body levels. Intracellular fluorescence detection uses existing ratiometric nano-fluorescenceProbe, pH linear range: 8.0-5.73, ca 2+ Linear range: 1-300 mu M.
FIG. 6 is a fluorescence spectrum of the ratio-type nano-fluorescent probe of example 1.
FIG. 7 is a graph of change in 700nm,600nm,515nm fluorescence signal and a graph of photoelectric signal in the solution of example 1 detected in vitro at 488nm excitation wavelength.
FIG. 8 is a schematic of the fiber targeting of example 2 to the cell gap.
FIG. 9 is a graph of the change in fluorescence signal and extracellular photoelectric signal of blank optical fibers at 700nm,600nm,515nm in vitro detected at 488nm excitation wavelength of example 2.
FIG. 10 is a schematic of example 3 fiber targeting different brain regions including cortex, hippocampus, striatum, thalamus.
FIG. 11 is a graph of change in fluorescence signal of 700nm,600nm,515nm optical fibers and an extracellular photoelectric signal of example 3 detected in vivo under excitation at 488nm wavelength.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and drawings. The procedures, conditions, experimental methods, etc. for carrying out the present invention are common knowledge and common knowledge in the art, except for the following specific references, and the present invention is not particularly limited.
The invention discloses a method for building an intracellular and extracellular photoelectric integrated detection platform and application thereof. Firstly, a portable fluorescence signal acquisition device is built, and on the basis, an intracellular fluorescence detection system and an extracellular photoelectric detection system are further built, so that the intracellular fluorescence signal and the extracellular photoelectric signal are collected simultaneously. Seven parts of the intracellular and extracellular photoelectric integrated detection platform are built and comprise a light source part, a scanning system, a first detection system, a first signal collection detection system, a second detection system and a second signal collection detection system. The invention also discloses a method for simultaneously collecting the intracellular fluorescence signal and/or the extracellular photocurrent signal of the cell and brain slice by using the intracellular and extracellular photoelectric integrated detection platform.
Example 1 collection of blank Signal for intracellular and extracellular optoelectronic Integrated detection platform
FIG. 6 shows the fluorescence spectrum of a ratio-type nano-fluorescent probe for quantitatively analyzing an analyte by the ratio of fluorescence intensities. Wherein the 700nm position is a reference signal peak, and the intensity is constant; ca at 600nm 2+ Signal peak, intensity with Ca 2+ Increasing and decreasing the concentration; at 515nm is a pH signal peak, the intensity decreases with decreasing pH. F (F) 600/700 With Ca 2+ The concentration is in a linear relation between 1 and 300 mu M; f (F) 515/700 In linear relationship with a pH of 8.0-5.73.
As shown in fig. 7, the fluorescence signal and the photoelectric signal of the optical fiber of the intracellular and extracellular photoelectric integrated detection platform under 488nm light excitation can be seen that the optical fiber does not have autofluorescence at 515nm,600nm and 700nm, and the weak photoelectric signal is from electrons (as background signal) provided by a small amount of ferrocene on the electrode.
Example 2 intracellular and extracellular photoelectric Integrated detection platform for Simultaneous collection of intracellular fluorescence Signal and extracellular photocurrent Signal in cell hierarchy
In order to evaluate the effect of the intracellular and extracellular optoelectronic integration detection platform on the simultaneous collection of the intracellular fluorescent signal and the extracellular photocurrent signal in the cell layer of the solution, the ratio-based nano fluorescent probe (1 mM) was used for signal collection after 30 minutes incubation with the cells.
As shown in fig. 8, which is a schematic diagram of the fiber-targeted cell gap, the tip diameter of the fiber electrode is about 500nm, and the fiber electrode can be accurately inserted into the neuronal synaptic gap for measurement.
As shown in FIG. 9, after the fiber electrode is positioned in the region to be measured, the fluorescence signal at 515nm,600nm and 700nm can be rapidly enhanced and reached to a plateau within 30s by opening the excitation light, and the pH and Ca can be quantified by the fluorescence intensity ratio 2+ Concentration; meanwhile, the rise of the photocurrent can be observed in the electrochemical workstation, and the concentration of the dopamine can be quantified through the rise of the photocurrent.
Example 3 intracellular and extracellular photoelectric Integrated detection platform for Simultaneous collection of intracellular fluorescence Signal and extracellular photocurrent Signal in living hierarchy
The intracellular and extracellular photoelectric integrated detection platform can be used for simultaneously collecting intracellular fluorescence signals and extracellular photocurrent signals of different brain regions, and the capacity of optical fibers for targeting brain regions is evaluated at first. As shown in fig. 10, fig. 10 is a schematic diagram of the optical fiber targeting different brain regions of the present embodiment, including cortex, hippocampus, striatum, thalamus. The dotted circle indicates the location of the electrode, and the optical fiber can effectively target the cerebral cortex, the hippocampus, the striatum, the thalamus and other brain areas. And incubating the ratio-type nano fluorescent probe and the brain slice for 30 minutes, and then simultaneously collecting the intracellular fluorescent signals and the extracellular photocurrent signals of different brain areas by using an extracellular and intracellular photoelectric integrated detection platform. As shown in FIG. 11, the intracellular and extracellular photoelectric integrated detection platform can be used for simultaneously collecting intracellular fluorescence signals and extracellular photocurrent signals of different brain regions, which is a device for realizing the simultaneous collection of the intracellular fluorescence signals and the extracellular photocurrent signals of different brain regions, for the first time. In this example, an intracellular 700nm,600nm,515nm fiber optic fluorescence signal profile and an extracellular photoelectric signal profile were detected in vivo under excitation at 488nm wavelength. After the optical fiber electrode is positioned in the region to be detected, the fluorescence signal at 700nm,600nm and 515nm can be rapidly enhanced and reached to a platform in 30 seconds by opening the excitation light, and the pH and Ca can be quantified by the ratio of the fluorescence intensity 2+ Concentration; meanwhile, the rise of the photocurrent can be observed in the electrochemical workstation, and the concentration of the dopamine can be quantified through the rise of the photocurrent.
The protection of the present invention is not limited to the above embodiments. Variations and advantages that would occur to one skilled in the art are included within the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is defined by the appended claims.
Claims (10)
1. An extracellular and intracellular photoelectric integrated detection platform, which is characterized by comprising: the system comprises a light source part, a scanning system, a detection system, a first signal collection detection system and a second signal collection detection system;
the light source part, the scanning system, the detection system and the first signal collection and detection system form an intracellular fluorescence detection system; the light source part, the detection system and the second signal collection and detection system form an extracellular photoelectric detection system;
wherein, the liquid crystal display device comprises a liquid crystal display device,
the light source part is a fiber laser (1);
the scanning system is based on a laser confocal scanning unit and comprises a first filter (2), a second filter (3), a first reflecting mirror (4), a second reflecting mirror (5), a scanner (9) and an objective lens (10);
the detection system is an optical fiber (11) with a conical head based on a multimode optical fiber, and ITO and TiO are sequentially modified on the surface of the optical fiber 2 、Ag 2 S, DNA aptamer, simultaneously as photoelectrochemical sensor;
the first signal collecting and detecting system is partially overlapped with the scanning system and comprises an objective lens (10), a scanner (9), a second reflecting mirror (5), a second filter (3), a third filter (6), a fourth filter (7), a third reflecting mirror (8) and a fluorescence detector (12);
the second signal collecting and detecting system is a loop formed by a three-electrode system formed by a working electrode, a reference electrode (13) and an auxiliary electrode (14) based on an optical fiber (11) and an electrochemical workstation (15) through wires, and a photocurrent signal is output from the electrochemical workstation (15).
2. The intracellular and extracellular optoelectronic integrated detection platform according to claim 1, wherein the excitation center wavelength of the fiber laser (1) of the light source part is 488nm ±0.5nm; the setting range of the output power is 0-500mW; line width less than 0.1nm; the output interface is SMA905 or FC/PC; the operating voltage is 220V.
3. The intracellular and extracellular optoelectronic integrated detection platform according to claim 1, wherein the first filter (2) in the scanning system is a band-pass filter, which is only used for light transmitted through 488nm; the second filter (3) is a notch filter and is used for filtering 488 and nm incident light; the first reflecting mirror (4) and the second reflecting mirror (5) are all total reflecting mirrors; the scanner (9) is a laser confocal scanning unit; the magnification of the objective lens (10) is 10×, and the numerical aperture NA is 0.25.
4. The integrated intracellular and extracellular optoelectronic testing platform according to claim 1, wherein the optical fiber in the detection system is a multimode optical fiber, the diameter of the fiber core is 200 microns, the thickness of the cladding is 12.5 microns, the NA of the numerical aperture is 0.22, the transmission range is 400-1100nm, the taper length of the end of the optical fiber is 500 microns, and the diameter of the tip of the optical fiber is 500nm.
5. The intracellular and extracellular photoelectric integrated detection platform according to claim 1, wherein the third filter (6) and the fourth filter (7) in the first signal collection and detection system are bandpass filters, and the third reflector (8) is a total reflector; the third filter (6) is used for separating out a fluorescence signal of 700nm, the fourth filter (7) is used for separating out a fluorescence signal of 600nm, and the third reflector (8) is used for reflecting a fluorescence signal of 515 nm.
6. The integrated intracellular and extracellular optoelectronic testing platform of claim 1, wherein the optical fiber surface modified ITO in the detection system is used to make the optical fiber conductive, tiO 2 And Ag 2 S is the electron transmission part in the photoelectrochemical sensor, and the DNA aptamer is used for specifically identifying the substance to be detected.
7. The intracellular and extracellular optoelectronic integrated detection platform according to claim 1, wherein the working electrode in the second signal collection detection system is an electrode based on an optical fiber (11), the reference electrode (13) is a silver/silver chloride electrode, the auxiliary electrode (14) is a platinum electrode, and the electrochemical workstation (15) is a Chen Huachi 660 electrochemical workstation.
8. The intracellular and extracellular optoelectronic integrated detection platform according to claim 1, wherein fluorescent molecules used for fluorescent signal detection in the detection platform are developed ratio type nano fluorescent probes; the fluorescence emission wavelength of the ratio-type nano fluorescent probe is 700nm,600nm,515nm respectively; excitation light of the fluorescent molecule is 488nm; the fluorescent molecule concentration is 1mM; the detection wavelength of the fluorescence detector (12) is 700nm,600nm,515nm; after the DNA aptamer is combined with a corresponding target molecule, linear DNA can be bent, when the electrode detects the target molecule, ferrocene at the far end of the DNA is closer to the surface of the electrode, more electrons can be provided, the phenomenon of photocurrent increase occurs, and the target molecule is quantified by the value of the photocurrent.
9. A method for constructing the intracellular and extracellular photoelectric integrated detection platform according to any one of claims 1 to 8, comprising the steps of:
step 1: assembling a laser confocal scanning unit, which specifically comprises the following substeps:
step 1-1: a first filter (2), a first reflecting mirror (4), a second filter (3) and a second reflecting mirror (5) are sequentially arranged;
step 1-2: installing a scanner (9) and an objective lens (10);
step 2: setting up a scanning system, which specifically comprises the following substeps:
step 2-1: connecting an optical fiber laser (1) with a confocal scanning unit through an optical fiber;
step 2-2: debugging the optical path so that the test excitation light can pass through the confocal scanning unit and be transmitted out by the objective lens (10);
step 3: the method specifically comprises the following substeps of:
step 3-1: the optical fiber is tapered to obtain an optical fiber (11) with a tapered head, so that the optical fiber signal collection efficiency is improved;
step 3-2: the surface of the optical fiber (11) with the conical head is sequentially modified with ITO and TiO 2 、Ag 2 S, DNA aptamer；
Step 3-3: assembling an optical fiber (11) with a conical head to the end of the scanning system, and coupling debugging excitation light into the optical fiber;
step 4: building a first signal collection detection system and a second signal collection detection system, and specifically comprising the following substeps:
step 4-1: a third filter (6), a fourth filter (7) and a third reflector (8) are arranged; the emergent light paths of the third filter (6), the fourth filter (7) and the third reflector (8) correspond to three detection positions of the fluorescence detector (12);
step 4-2: connecting the fluorescence detector (12) in the step 4-1 with a confocal scanning unit through an optical fiber;
step 4-3: and connecting the optical fiber (11) with the conical head with a reference electrode (13), an auxiliary electrode (14) and an electrochemical workstation (15) to finally form the intracellular and extracellular photoelectric integrated detection platform.
10. Use of an intracellular and extracellular optoelectronic integration detection platform constructed by the method of claim 9 for collecting an intracellular fluorescence signal and/or an extracellular photocurrent signal in vivo and/or in vitro, respectively or simultaneously.
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