CN110579454B - Fluorescent group identification method and device based on electric field modulation fluorescence correlation spectroscopy - Google Patents
Fluorescent group identification method and device based on electric field modulation fluorescence correlation spectroscopy Download PDFInfo
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- CN110579454B CN110579454B CN201910763403.4A CN201910763403A CN110579454B CN 110579454 B CN110579454 B CN 110579454B CN 201910763403 A CN201910763403 A CN 201910763403A CN 110579454 B CN110579454 B CN 110579454B
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Abstract
The invention discloses a fluorescent group identification method based on electric field modulation fluorescence correlation spectroscopy, which comprises the following steps: adding an aqueous solution containing a fluorescent group substance into the microfluidic channel to enable the liquid to flow in the microfluidic channel; increasing alternating electric fields on two sides of the microfluidic channel in the liquid flowing direction, and setting the alternating electric fields as detection areas; exciting a fluorescent group in the detection area by exciting light, recording a photon occurrence event of the fluorescent group by a time-resolved single photon detector, and calculating a fluorescence correlation spectrum; and distinguishing the fluorescent group substances with different charge-mass ratios by comparing the diffusion coefficient variation of fluorescence correlation spectra obtained when different voltages are generated by the alternating electric field. The invention also discloses a fluorescent group identification device based on the electric field modulation fluorescence correlation spectroscopy. The method can avoid the fluorescent group identification error caused by high flow and liquid flow nonuniformity, and improve the identification precision.
Description
Technical Field
The invention relates to a method and a device for identifying a fluorescent group, in particular to a method and a device for identifying a fluorescent group based on an electric field modulation fluorescence correlation spectroscopy.
Background
Fluorescence Correlation Spectroscopy (FCS) is commonly used to evaluate brownian motion of fluorophores of different diffusion coefficients D. The photon emission events of the fluorophores are recorded by a time-resolved single photon detector along a time axis, from which fluorescence correlation spectra can be calculated. The moving speed of the heavy fluorophore is slower than that of the light fluorophore, so the decay time of the fluorescence correlation spectrum is longer, and different fluorophores can be distinguished through the fluorescence correlation spectrum. In the prior art, the acquisition of the photon emission event of a fluorophore is performed by passing an aqueous solution containing the fluorophore through a microchannel device, and then recording the aqueous solution by a time-resolved single photon detector, whereas in a typical microchannel device, the fluorophore in the aqueous solution moves along the microchannel in two different ways, the first is irregular brownian motion, and the second is the flow along with the liquid in the flow direction of the liquid in the microchannel. To achieve high throughput, the fluid flow in the microchannel must be high, which may cause the fluorescence correlation spectrum generated by the fluid flow to overwhelm the fluorescence correlation spectrum corresponding to brownian motion, and spatial non-uniformity in the fluid flow in the microchannel will also result in erroneous fluorophore identification based on the fluorescence correlation spectrum.
Disclosure of Invention
The invention aims to provide a fluorescent group identification method based on electric field modulation fluorescence correlation spectroscopy, so as to avoid fluorescent group identification errors caused by high flow and liquid flow nonuniformity and improve the classification identification precision of fluorescent groups. Another object of the present invention is to provide a fluorophore identification device based on electric field modulated fluorescence correlation spectroscopy.
The technical scheme of the invention is as follows: a fluorescent group identification method based on electric field modulation fluorescence correlation spectroscopy comprises the following steps:
s1, adding the aqueous solution containing the fluorescent group substance into the microfluidic channel to enable the liquid to flow in the microfluidic channel;
s2, adding alternating electric fields on two sides of the microfluidic channel in the liquid flowing direction, and setting the alternating electric fields as detection areas;
s3, exciting a fluorescent group in the detection area by exciting light, recording a photon occurrence event of the fluorescent group by a time-resolved single photon detector, and calculating a fluorescence correlation spectrum;
s4, distinguishing the fluorophore substances with different charge-to-mass ratios by comparing the diffusion coefficient variation of fluorescence correlation spectra obtained when different voltages are generated by the alternating electric field.
Further, the electric field direction of the alternating electric field is perpendicular to the liquid flowing direction.
Further, the alternating electric field forms a bias voltage at both sides of the microfluidic channel in the liquid flow direction with a fixed frequency change.
Further, the variation of the bias voltage is 0V or nV, n > 0.
A fluorophore identification device based on electric field modulation fluorescence correlation spectroscopy, comprising:
a microfluidic channel: allowing an aqueous solution containing a fluorescent substance to flow;
alternating electric field: the alternating voltage is formed in the microfluidic channel;
exciting the light emitting device: emitting exciting light for exciting fluorophores in the microfluidic channel of the alternating electric field;
time-resolved single photon detector: recording photon occurrence events of the excited fluorophores;
a calculation module: calculating a fluorescence correlation spectrum of the excited fluorophore;
and a judging module: fluorescent group substances with different charge-to-mass ratios are distinguished by diffusion coefficient variation of fluorescence correlation spectra calculated when different voltages are generated by an alternating electric field.
Further, the electric field direction of the alternating electric field is perpendicular to the liquid flowing direction.
Further, the voltage change frequency of the alternating electric field is fixed.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
the bias voltage generated by the alternating electric field induces the fluorescent group substances with different charge-mass ratios to generate different motion states, and the induced motion can enable fluorescence correlation spectra of the fluorescent group substances with different charge-mass ratios to have obvious difference, so that the phenomenon that the fluorescent correlation spectra corresponding to the brownian motion of the fluorescent group substances are submerged by the fluorescence correlation spectra generated by the liquid flow during high-flow detection, so that the different fluorescent group substances cannot be distinguished is avoided, and the classification precision is improved.
Drawings
FIG. 1 is a schematic diagram of a fluorophore identification device based on electric field modulated fluorescence correlation spectroscopy.
FIG. 2 is a schematic diagram of the movement of a fluorophore species within a microfluidic channel of the method of the invention.
Fig. 3 is fluorescence correlation spectra of quantum dots and quantum dot-vascular cell adhesion molecule 1 measured in culture dishes and microfluidic channels.
FIG. 4 shows fluorescence correlation spectra of quantum dot-vascular cell adhesion molecule 1 measured in a culture dish and in a microfluidic channel by the method of the present invention.
FIG. 5 shows fluorescence correlation spectra of quantum dots in a petri dish and in a microfluidic channel by the method of the invention
Detailed Description
The present invention is further described in the following examples, which are intended to be illustrative only and not to be limiting as to the scope of the invention, which is to be given the full breadth of the appended claims and any and all equivalent modifications thereof which would occur to persons skilled in the art upon reading the present specification and which are intended to be within the scope of the present invention as defined in the appended claims.
Referring to fig. 1 and 2, a fluorophore identification device based on electric field modulated fluorescence correlation spectroscopy includes:
the microfluidic channel 1: allowing an aqueous solution containing a fluorescent substance to flow;
alternating electric field 2: the alternating voltage is formed in the microfluidic channel in a way that the direction of the electric field is vertical to the flowing direction of the liquid, the changing frequency of the alternating voltage is fixed, and the changing value is 0V or 5V;
excitation light emitting device 3: emitting exciting light for exciting fluorophores in the microfluidic channel 1 of the alternating electric field 2;
the time resolution single photon detector 4: recording photon occurrence events of the excited fluorophores;
the calculation module 5: calculating a fluorescence correlation spectrum of the excited fluorophore;
and a judging module 6: and the fluorescent group substances with different charge-to-mass ratios are distinguished by the diffusion coefficient variation of the fluorescence correlation spectrum calculated when the alternating electric field 2 generates different voltages.
The method is suitable for distinguishing various fluorescent particles, and takes the distinction of quantum dots and quantum dot-vascular cell adhesion molecule 1 binding peptide (VQD) as an example, the method is a fluorescent group identification method based on electric field modulation fluorescence correlation spectroscopy:
s1, adding the aqueous solution containing the fluorescent group substance into the microfluidic channel 1 to enable the liquid to flow in the microfluidic channel 1;
s2, adding alternating electric fields 2 with the change value of 0V or 5V at two sides of the liquid flowing direction of the microfluidic channel 1, and setting the alternating electric fields as detection areas; the movement of the fluorophore substance is shown in FIG. 2, which includes random Brownian movement, rightward fluid flow and downward induced movement under bias voltage;
s3, exciting the fluorescent group by exciting light in the detection area, recording the photon occurrence event of the fluorescent group by the time-resolved single photon detector 4, and calculating a fluorescence correlation spectrum;
s4, comparing the diffusion coefficient variation of fluorescence correlation spectrum obtained when different voltages are generated by the alternating electric field 2 to distinguish the fluorophore substances with different charge-to-mass ratios.
Experiments were performed in the prior art method by first placing Quantum Dots (QD) and quantum dot-vascular cell adhesion molecule 1 binding peptide (VQD) in a petri dish where the liquid flow was almost zero and measuring the Fluorescence Correlation Spectrum (FCS) of the two with a fluorescence microscope. Then, the quantum dots and the quantum dot-vascular cell adhesion molecule 1 aqueous solution were placed in the microfluidic channel to measure fluorescence correlation spectra, and the results are shown in fig. 3. FCS of QD and VQD in petri dishes clearly shows that QD diffuses faster than VQD because it has a smaller mass than VQD, thus VQD and QD are easily distinguished by FCS. However, fluid flow within the microfluidic channel accelerated VQD, shifting the FCS spectrum of VQD to the left, overlapping the FCS of the QDs in the petri dish, affecting the work of distinguishing VQD from the QDs.
Referring to FIG. 4 and FIG. 5, the method of the present invention is adopted because of the bias voltage VbThe FCS of VQD is different for the induced motion and the bias voltage, but the change is tauVQDVery small because the charge-to-mass ratio of VQD is small. In the case of two different bias voltages, the FCS variation τ of QD is large because of the large charge-to-mass ratio of QDQDIs very large. Therefore, VQD and QD can be distinguished by distinguishing the amount of change in FCS, and fluorophore substances having different charge-to-mass ratios can be distinguished.
Claims (7)
1. A fluorescent group identification method based on electric field modulation fluorescence correlation spectroscopy is characterized by comprising the following steps:
s1, adding the aqueous solution containing the fluorescent group substance into the microfluidic channel to enable the liquid to flow in the microfluidic channel;
s2, adding alternating electric fields on two sides of the microfluidic channel in the liquid flowing direction, and setting the alternating electric fields as detection areas;
s3, exciting a fluorescent group in the detection area by exciting light, recording a photon occurrence event of the fluorescent group by a time-resolved single photon detector, and calculating a fluorescence correlation spectrum;
s4, distinguishing the fluorophore substances with different charge-to-mass ratios by comparing the diffusion coefficient variation of fluorescence correlation spectra obtained when different voltages are generated by the alternating electric field.
2. The method of claim 1, wherein the direction of the alternating electric field is perpendicular to the direction of the liquid flow.
3. The method of claim 1, wherein the alternating electric field forms a bias voltage at a fixed frequency variation on both sides of the microfluidic channel in the direction of liquid flow.
4. The method of claim 3, wherein the bias voltage is varied to 0V or nV, and n > 0.
5. A fluorophore identification device based on electric field modulation fluorescence correlation spectroscopy, characterized by comprising:
a microfluidic channel: allowing an aqueous solution containing a fluorescent substance to flow;
alternating electric field: the alternating voltage is formed in the microfluidic channel;
exciting the light emitting device: emitting exciting light for exciting fluorophores in the microfluidic channel of the alternating electric field;
time-resolved single photon detector: recording photon occurrence events of the excited fluorophores;
a calculation module: calculating a fluorescence correlation spectrum of the excited fluorophore;
and a judging module: fluorescent group substances with different charge-to-mass ratios are distinguished by diffusion coefficient variation of fluorescence correlation spectra calculated when different voltages are generated by an alternating electric field.
6. The apparatus according to claim 5, wherein the direction of the electric field of the alternating electric field is perpendicular to the flow direction of the liquid.
7. The apparatus of claim 5, wherein the frequency of the voltage change of the alternating electric field is fixed.
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| CN109520982A (en) * | 2018-11-20 | 2019-03-26 | 东南大学 | A kind of fluorescence correlation spectroscopy measuring system |
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| GB2408628A (en) * | 2003-11-28 | 2005-06-01 | Imp College Innovations Ltd | Gated image intensifier |
| WO2005121864A2 (en) * | 2004-06-07 | 2005-12-22 | Fluidigm Corporation | Optical lens system and method for microfluidic devices |
| EP2163899A1 (en) * | 2008-09-10 | 2010-03-17 | Koninklijke Philips Electronics N.V. | Sensitive fluorescence correlation measurements of soluble adhesion molecules |
| WO2015095546A1 (en) * | 2013-12-19 | 2015-06-25 | Luminex Corporation | Crosstalk reduction |
| CN109187450A (en) * | 2018-08-01 | 2019-01-11 | 傅英 | A kind of biomolecule concentration detection method based on quantum dot |
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