CN113670874A - High-resolution single-cell protein quantitative detection method based on light source modulation - Google Patents
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Abstract
The invention provides a high-resolution single-cell protein quantitative detection method based on light source modulation, which comprises the following steps: step 1: injecting the cells marked by the fluorescent antibody or the fluorescent antibody solution into the compressed micro-channel in equal volume; step 2, when the dyed cell or antibody solution passes through a detection area of a compressed micro-channel, the cell or antibody solution is irradiated and excited by an alternating-current laser source; step 3, the cell or antibody solution emits fluorescence under the excitation of the step 2, and a fluorescence signal is detected by a photomultiplier tube (PMT); step 4, demodulating the amplitude of the obtained modulated fluorescence signal into a fluorescence pulse, and obtaining three key time parameters and a fluorescence intensity parameter of a stabilization period according to the rising stabilization and falling phenomena of the fluorescence intensity of the pulse; step 5, converting the original fluorescence parameters into the concentration of the single-cell protein by using a corresponding calibration curve formed by compressing the micro-channel; and calculating the cell stretching length by using the original time parameters, and further calculating the cell diameter. Finally, the number of the single cell protein is obtained by combining the single cell diameter with the single cell protein concentration.
Description
Technical Field
The invention relates to the technical field of protein detection, in particular to a high-resolution single-cell protein quantitative detection method based on light source modulation.
Background
Measuring proteins at the single cell level facilitates cell type classification and cell status assessment, which plays a key role in the field of cellular heterogeneity, including tumor development and immune response.
The traditional method for quantitatively detecting single-cell protein mainly comprises fluorescence flow cytometry and mass spectrometry flow cytometry. Among them, fluorescence flow cytometry is the most common method for characterizing the expression of single cell proteins, when analyzing cellular proteins, dispersed single cells are firstly labeled with specific antibodies with fluorescence, and then are arranged in a line under the action of fluid focusing, and sequentially enter a detection area, and the cellular proteins marked by the fluorescence are excited to fluoresce, so that the relative content of the proteins can be reflected through the relative fluorescence intensity, and the integrity of the single cells can be maintained; mass spectrometry flow cytometry is the combination of flow cytometry and inductively coupled mass spectrometry, and is characterized in that a specific antibody coupled with a metal element is adopted to mark protein on the surface or in a cell, single cell sample introduction is carried out, an inductively coupled plasma mass spectrometry is used for detecting the atomic mass spectrum of a single cell, finally, the data of the atomic mass spectrum is converted into the content of the protein to be detected of the cell, and the obtained mass data is analyzed through professional analysis software.
The method for quantitatively detecting single-cell protein based on microfluidics roughly comprises a micropore/microcavity array detection method and a microfluidics flow cytometry. The micropore/microcavity array detection method mainly comprises the steps of forming a microcavity by processing micropores or controlling opening and closing of a micro-channel, capturing a single cell in the single cavity, culturing or cracking the cell to obtain specific protein of a single cell, and finally detecting the protein in the microcavity to obtain the protein content level of the single cell layer. The micro-flow cytometry is a method for analyzing single-cell protein by using a traditional flow cytometer on a chip by utilizing a micro-fluidic technology, and is characterized in that a cell to be detected and a fluorescence-labeled specific antibody are mixed and then are introduced into a micro-channel, and the detection of the cell specific protein is realized by detecting the fluorescence intensity. However, this method is similar to the conventional flow cytometry, and the reported micro flow cytometry also cannot quantitatively detect intracellular proteins due to the limitation of the calibration method. In addition, there is a micro-fluidic flow cytometer to quantitatively estimate specific intracellular proteins. However, such quantitative microfluidic flow cytometers suffer from high electrical and optical noise, resulting in the performance of detecting less abundant single-cell proteins.
Therefore, it is necessary to develop a high-throughput, high-resolution device and method for quantitatively detecting single-cell proteins.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for quantitatively detecting single-cell protein based on a micro-compression channel by using the principle of optical modulation and demodulation. Compared with the prior art, the method has three improved targets:
(1) the resolution of quantitative single-cell protein detection is improved. The existing quantitative single cell protein detection method based on a micro-compression channel can obtain the content of protein in cells, is limited by low-frequency 1/f noise, causes low detection resolution, and is difficult to detect the intracellular protein which has little content in the single cells but has important significance.
(2) The detection efficiency of quantitative single-cell protein detection is improved. The existing quantitative single cell protein detection method such as fluorescence flow cytometry is limited in resolution, and a plurality of single cell fluorescence pulses are submerged in noise, so that data of a plurality of cells are lost in the detection process, and the detection efficiency of the single cell protein is greatly influenced.
The technical scheme of the invention is as follows: a high-resolution single-cell protein quantitative detection method based on light source modulation comprises the following steps:
step 1: injecting the cells marked by the fluorescent antibody or the fluorescent antibody solution into the compressed micro-channel in equal volume;
step 3, the cell or antibody solution emits fluorescence under the excitation of the step 2, and a fluorescence signal is detected by a photomultiplier tube (PMT); at the moment, the fluorescence signal detected by the PMT is a high-frequency fluorescence signal under the action of high-frequency laser, namely a modulated fluorescence signal;
Step 5, converting the original fluorescence parameters into the concentration of the single-cell protein by using a corresponding calibration curve formed by compressing the micro-channel; and calculating the cell stretching length by using the original time parameters, and further calculating the cell diameter. Finally, the number of the single cell protein is obtained by combining the single cell diameter with the single cell protein concentration.
Further, in the step 2, the laser source with the sine wave is used to modulate the single-cell fluorescence signal labeled by the fluorescent antibody passing through the detection area to a high frequency, that is, the high-frequency laser irradiates a single cell for multiple times in the cell passing process, the obtained single-cell fluorescence signal is composed of the high-frequency sine wave, and the amplitude of the sine wave reflects the intensity of the fluorescence.
Further, in step 3, in the demodulation step, the modulation signal is then demodulated by the lock-in amplifier by means of a reference signal having the same frequency as the laser to obtain the fluorescence pulse, wherein a high-order low-pass filter is used to further remove noise and improve the signal-to-noise ratio.
Further, said step 4, based on curve fitting,three corresponding time parameters T are obtainedr，Ts，TdAnd I represents fluorescence intensityfA parameter; compressing cell stretch length L within a microchannelcThe relationship is transformed from the critical time and geometric parameters as follows:
wherein WdIndicates the width of the window, LsRepresenting the side length of a compressed microchannel having a square cross-section;
then further obtaining the cell diameter DcThe following were used:
the mean and standard deviation of the fluorescence intensity for each gradient concentration of the antibody solution form a calibration curve, and the fluorescence intensity is converted into a specific protein concentration C of individual cells based on a calibration equation with the experimentally obtained fluorescence signal intensity as inputp；
Finally, based on cell diameter DcAnd protein concentration CpFurther, the specific protein number n at the single cell level is obtainedp。
Has the advantages that:
the technical scheme is illustrated in the specification, and the invention comprises the following steps: the method for detecting the bioelectrical characteristics of the cells and the cell nucleuses based on the narrow cross channel has the following beneficial effects:
(1) the method of the invention has high resolution of quantitative single-cell protein detection. Compared with the existing method, the electrical noise and the optical noise can be effectively reduced by the optical modulation principle, and the protein which is expressed less in the cell but has important significance is detected.
(2) The method of the invention has high detection efficiency of quantitative single-cell protein detection. Compared with the existing method, by improving the detection resolution, more effective signals can be detected under the condition of the same number of cells to be detected, the loss of samples is reduced, the detection time is shortened, and the detection efficiency is improved.
(3) Due to the ability of optical modulation to effectively reduce electrical and optical noise compared to prior methods, quantitative protein detection using this principle has proven to be effective in increasing the detection resolution from about 1000 intracellular proteins per cell to about 100 intracellular proteins per cell.
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FIG. 1: the detection method of the invention is schematically shown in principle;
fig. 2 (a): modulating a cell signal;
fig. 2 (B): demodulating the cell pulse;
FIG. 3: calibration curves.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments, and all other embodiments obtained by a person skilled in the art based on the embodiments of the present invention belong to the protection scope of the present invention without creative efforts. The embodiment of the invention provides a device and a method for quantifying protein in a single-cell with high resolution based on a compressed micro-channel and optical modulation.
The principle schematic diagram and the operation flow of the invention are shown in figure 1. According to one embodiment of the present invention, a device for high resolution quantification of proteins in single-cell cells based on compressed micro-channels and optical modulation comprises: microfluidic chips, microscope stages, pressure controllers, and the like; a micro-fluidic chip with a compression channel with a rectangular cross section is stably placed on an object stage of an inverted fluorescence microscope, a pressure controller applies certain negative pressure to the channel under the connection of a hose and the channel, the compression channel can enable cells to be properly compressed to pass through the channel, when the cells pass through the compression channel, only a slit formed by a shading film can transmit light, a laser emits high-frequency alternating-current laser with specific power and frequency to irradiate a detected object through a light-transmitting slit under the control of a signal generator, so that the excited fluorescence can be detected through the light-transmitting slit, the excited fluorescence is an alternating-current fluorescence signal modulated by the high-frequency laser, the signal is collected by a photomultiplier and sent to a phase-locked amplifier for demodulation, meanwhile, a signal used for controlling the laser is also sent to the phase-locked amplifier to be used as a reference signal for demodulation, and the demodulated signal can be sent to a signal collection card for collection, thereby analyzing the detected signal. FIG. 1 shows a schematic diagram of a high-resolution method for quantifying proteins in single-cell cells based on compact micro-channels and optical modulation, which can be used for high-resolution and high-throughput quantification of single-cell proteins using fluorescence modulation. The method specifically comprises the following steps:
step 1: injecting equal volumes of cells labeled with a fluorescent antibody (see (a) in fig. 1) or a fluorescent antibody solution (see (B) in fig. 1) into the compressed microchannel;
step 3, the cell or antibody solution fluoresces under the excitation of step 2, and the fluorescence signal is detected by a photomultiplier tube detector tube (PMT) (see (C) in fig. 1). At the moment, the fluorescence signal detected by the PMT is a high-frequency fluorescence signal under the action of high-frequency laser, namely a modulated fluorescence signal;
Step 5, using the corresponding calibration curve formed by compressing the microchannel (see (F) in fig. 1), the raw time and fluorescence parameters are converted into the cell stretch length and single-cell protein concentration (see (G) in fig. 1), and then further converted into the cell diameter and the number of single-cell proteins (see (H) in fig. 1).
The key component of the microfluidic platform is the process of modulating and demodulating the fluorescence pulse of a single cell, thereby improving the detection resolution of single cell protein analysis (see (D) and (E) in fig. 1). In order to avoid low frequency 1/f noise, in the modulation step, a laser source with a sine wave is used to modulate the single-cell fluorescence signal labeled by the fluorescent antibody passing through the detection region to a high frequency, i.e., a high-frequency laser irradiates a single cell for multiple times during cell passing, and the obtained single-cell fluorescence signal is composed of a high-frequency sine wave, and the amplitude of the sine wave reflects the intensity of fluorescence (see (D) in fig. 1). In a demodulation step, the modulated signal is then demodulated by a lock-in amplifier with a reference signal at the same frequency as the laser to obtain the fluorescence pulses, with a high order low pass filter to further remove noise and improve the signal-to-noise ratio.
Specifically, the cell passing frequency is 1kHz, the laser carrier frequency is 20kHz, the carrier frequency is guaranteed to be more than 10 times higher than the cell passing frequency, the fluorescence signal of a single cell can be effectively modulated to a higher frequency domain, low-frequency noise is far away, meanwhile, due to the fact that PMT is used for subsequent collection, the bandwidth of an amplifying circuit is 20kHz/200kHz at most, and the optical signal cannot be effectively amplified if the carrier frequency exceeds the frequency. In order to effectively improve the detection resolution, the protein detection part based on optical modulation needs to optimize the voltage setting of the signal generator for controlling the high-frequency excitation light, including the dc bias and the ac voltage amplitude of the control voltage, which are gradually increased within a suitable range by means of a control variable, the increase of the ac amplitude means the enhancement of the laser intensity, resulting in the improvement of the signal-to-noise ratio, therefore, the ac amplitude should be as high as possible, and simultaneously, considering the potential thermal damage of the high laser power to the compressed microchannel, the ac amplitude of 1Vpp is selected as the optimum parameter, corresponding to which, when the dc bias is half of the ac amplitude, the carrier wave keeps a complete sinusoidal waveform, the dc component is minimum, producing the lowest level of noise, therefore, in the present invention, at the ac amplitude of 1Vpp, a dc bias of 0.5V is selected. And then amplifying the modulated fluorescent signal with a specific wavelength by a photomultiplier with a band-pass filter, estimating the duration of moving a single cell to 10ms, demodulating by a lock-in amplifier with a time constant set to 0.5ms to ensure a complete single-cell waveform, sampling the demodulated signal by a data acquisition card at a sampling rate of 80kHz, estimating PMT noise to 10ns, and further removing the noise by a low-pass filter with a time constant of 0.5ms and a 40-point median filter after acquiring single-cell pulses.
The raw data processing method of the embodiment is as follows: the raw data is the modulated fluorescence signal (see fig. 2(a)), the left image is a series of single cell signals, the right image shows the amplified image of the left image with single cell signals in a rectangular frame, the single cell pulses obtained by the demodulation of the modulated signals (see fig. 2(B)), similarly, the left image is a series of single cell signals, the right image is an amplified image of single cell pulses in a rectangular frame of the left image, and each pulse representing a single cell is divided into an ascending domain, a stable domain and a descending domain by fitting the acquired single cell pulses to a trapezoid (see fig. 2 (B)). More specifically, based on curve fitting, three corresponding time parameters T are obtainedr，Ts，TdAnd I represents fluorescence intensityfAnd (4) parameters. Compressing cell stretch length L within a microchannelcThe relationship is transformed from the critical time and geometric parameters as follows:
wherein WdIndicates the width of the window, LsShowing the side length of a compressed microchannel having a square cross-section.
Then further obtaining the cell diameter DcThe following were used:
the mean value and standard deviation of the fluorescence intensity for each gradient concentration of the antibody solution form a calibration curve (see fig. 3). In these calibration curves, the fluorescence intensity is correlated with the fluorescence labelThe concentration of the antibody is linearly related, so a linear fit is used to obtain the corresponding calibration equation. Based on the calibration equation, the fluorescence intensity is converted into a specific protein concentration (C) of a single cell using the experimentally obtained fluorescence intensity as an inputp) (see (G) in FIG. 1).
Finally, based on cell diameter (D)c) And protein concentration (C)p) Further, a specific protein (n) at the single cell level was obtainedp) Number (see (H) in fig. 1).
According to an embodiment of the present invention, the detection requirement can be met based on the laser source controlled by the signal generator, without limitation to the instrument for controlling the light source, the type, number or optical path structure of the light source, and without limitation to the form, shape or depth of laser focusing, as long as the formation of a suitable laser spot region is satisfied.
According to an embodiment of the present invention, the compressed micro-channel in the present invention is not limited to the shape, size or material of the micro-fluidic channel, and the detection requirement can be met as long as the cells can be properly deformed in the compressed channel and can conform to the calibration model.
According to one embodiment of the invention, negative pressure is used to drive the cell solution through the channel, but other means, such as applying positive pressure to the end of the cell solution injection channel, may be used.
Although illustrative embodiments of the present invention have been described above to facilitate the understanding of the present invention by those skilled in the art, it should be understood that the present invention is not limited to the scope of the embodiments, but various changes may be apparent to those skilled in the art, and it is intended that all inventive concepts utilizing the inventive concepts set forth herein be protected without departing from the spirit and scope of the present invention as defined and limited by the appended claims.
Claims (4)
1. A high-resolution single-cell protein quantitative detection method based on light source modulation is characterized by comprising the following steps:
step 1: injecting the cells marked by the fluorescent antibody or the fluorescent antibody solution into the compressed micro-channel in equal volume;
step 2, when the stained cells or antibody solution passes through a detection area of a compressed micro-channel, irradiating and exciting the cells or antibody solution by using a laser source modulated by alternating current;
step 3, the cell or antibody solution emits fluorescence under the excitation of the step 2, and a fluorescence signal is detected by a photomultiplier tube (PMT); at the moment, the fluorescence signal detected by the PMT is a high-frequency fluorescence signal under the action of high-frequency laser, namely a modulated fluorescence signal;
step 4, the amplitude of the obtained modulated fluorescence signal is demodulated into fluorescence pulses, and according to the rising stability and the falling phenomenon of the pulse fluorescence intensity, three obtained key time parameters are the rising time TrStationary time TsAnd a fall time TdThe fluorescence parameter is fluorescence intensity If；
And 5, converting the original time and the fluorescence parameters into the cell stretching length and the single-cell protein concentration by using a corresponding calibration curve formed by the compressed micro-channel, and further converting the cell stretching length and the single-cell protein concentration into the cell diameter and the single-cell protein quantity.
2. The method for quantitatively detecting the high-resolution single-cell protein based on the light source modulation as claimed in claim 1, wherein the method comprises the following steps:
in the step 2, the laser source with sine wave is used for modulating the single-cell fluorescence signal marked by the fluorescent antibody in the detection area to high frequency, namely, the high-frequency laser irradiates a single cell for multiple times in the cell passing process, the obtained single-cell fluorescence signal is composed of high-frequency sine wave, and the intensity of the sine wave reflects the intensity of the fluorescence.
3. The method for quantitatively detecting the high-resolution single-cell protein based on the light source modulation as claimed in claim 1, wherein the method comprises the following steps:
in said step 3, in the demodulation step, the modulated signal is then demodulated by the lock-in amplifier with the reference signal having the same frequency as the laser to obtain the fluorescence pulse, wherein a high order low pass filter is used to further remove noise and improve the signal-to-noise ratio.
4. The method for quantitatively detecting the high-resolution single-cell protein based on the light source modulation as claimed in claim 1, wherein the method comprises the following steps: in the step 4, three corresponding time parameters T are obtained based on curve fittingr，Ts，TdAnd I represents fluorescence intensityfA parameter; compressing cell stretch length L within a microchannelcThe relationship is transformed from the critical time and geometric parameters as follows:
wherein WdIndicates the width of the window, LsRepresenting the side length of a compressed microchannel having a square cross-section;
then further obtaining the cell diameter DcThe following were used:
the mean and standard deviation of the fluorescence intensity for each gradient concentration of the antibody solution form a calibration curve, and the fluorescence intensity is converted into a specific protein concentration C of individual cells based on a calibration equation with the experimentally obtained fluorescence signal intensity as inputp；
Finally, based on cell diameter DcAnd protein concentration CpFurther, the specific protein number n at the single cell level is obtainedp。
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| CN106290279A (en) * | 2016-08-09 | 2017-01-04 | 中国科学院电子学研究所 | A kind of single cell protein detecting system and application thereof |
| CN107064091A (en) * | 2017-04-19 | 2017-08-18 | 中国科学院电子学研究所 | A kind of micro-fluidic chip, single cell protein quantitative testing device and method |
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| US20090218515A1 (en) * | 2005-10-06 | 2009-09-03 | Nanyang Technological University | Eliminating fluorescence background noise |
| US20080213915A1 (en) * | 2007-03-02 | 2008-09-04 | Gary Durack | System and method for the measurement of multiple fluorescence emissions in a flow cytometry system |
| CN106290279A (en) * | 2016-08-09 | 2017-01-04 | 中国科学院电子学研究所 | A kind of single cell protein detecting system and application thereof |
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