Biomarker concentration detection device based on time-dependent intensity correlation statistics

Abstract

The invention relates to the technical field of biosensing and detection, in particular to a biomarker concentration detection device based on time-dependent intensity correlation statistics. In order to solve the problem of insufficient clinical biomarker detection sensitivity, the device comprises a laser used for emitting laser, the laser sequentially passes through a half slide, a quarter slide, a laser attenuator, a reflector, a beam expander and a diaphragm to obtain the most uniform laser, the most uniform laser is reflected by a dichroscope to enter an objective lens, and the objective lens is used for detecting the clinical biomarker. The method comprises the following steps: collecting light beams, focusing the light beams on a micro-fluidic device, exciting a fluorescent nano-probe for marking a biomarker in the micro-fluidic device to emit fluorescent light, collecting the fluorescent light by an objective lens, transmitting the fluorescent light through a dichroscope, and focusing the fluorescent light to a single-photon detector through a spatial light filtering device, a long-pass light filter and a lens in sequence, and the single-photon detector transmits a detected electric signal to the time correlation single-photon counting module to calibrate the arrival time of photons, so that the concentration of the biomarker is accurately calibrated.

Description

Technical Field

[0001] The present invention relates to the field of biosensing and detection technology, and in particular to a biomarker concentration detection device based on time-dependent intensity association statistics. Background Art

[0002] Fluorescence sensing technology has been widely used in cytology, laboratory and pathology research, especially in the kinetic indication and concentration calibration based on fluorescence intensity, which is of great value for the study of life dynamics. It is recognized as the most sensitive clinical laboratory sensing technology. In current biological research, fluorescent nanoprobes are mainly organic dye molecules, which have relatively uniform physical and chemical properties, making the sensing based on fluorescence intensity accurate and stable. And its environmental responsiveness and modifiability make it not only applicable to responsive sensing based on physical and chemical environment, but also can be modified on proteins and peptide chains for fluorescence open-type sensing. Recent major biological research progress and clinical research have applied biological fluorescence sensing technology, and all of them are based on the principle of fluorescence intensity indicating the concentration of biomarkers. In early diseases, biomarker detection based on fluorescence has made important progress, especially for the detection of early cancer. Fluorescence sensing technology has outstanding advantages. This is because the concentration of early cancer biomarkers is extremely low and other detection methods are difficult to detect. However, the limit of detection based on fluorescence intensity is 10 -12 mol / L. The concentration of many early cancer biomarkers is less than 10 -12 mol / L, usually around 10 -16 mol / L, which is difficult to detect by intensity-based fluorescence, even as the most sensitive fluorescence sensing technology.

[0003] However, fluorescent molecular detection and research have been fully studied at the single-molecule level in the field of physics, and single molecules can be accurately detected and characterized. However, in clinical laboratory medicine applications, single fluorescent molecules distributed in a certain volume of solution always make it time-consuming and difficult to characterize their concentration. If biological samples are solidified, their physical and chemical properties will be affected, which makes it extremely difficult to achieve extremely low concentration fluorescence sensing in clinical practice. However, the concentration characterization of biomarkers is of great significance for the early detection of diseases. Summary of the invention

[0004] In order to overcome the problem of insufficient sensitivity of existing clinical biomarker detection, the present invention provides a biomarker concentration detection device based on time-dependent intensity correlation statistics, which accurately calibrates the concentration by photon calibration and photon statistics of moving fluorescent molecules in the solution, and then obtains characteristic time. Fluorescence intensity has a direct corresponding relationship with concentration in a linear process, but at extremely low concentrations, the larger biological sample background will always affect the concentration detection based on fluorescence intensity. To this end, we introduce a photon statistical method based on the use of different light field characteristics of background and molecules to measure the concentration of biological sample solutions with fluorescent labels. Because the stray light and autofluorescence background of biological samples are still electronic noise, they all conform to the Poisson light field characteristics, and the photon statistical behavior on different time scales obeys the Poisson characteristics. Single molecules have different light field characteristics on different time scales. They embody sub-Poisson statistical distribution characteristics that conform to single quantum characteristics on a small time scale, and super-Poisson photon statistical distribution characteristics will be embodied on a larger time scale due to the instantaneous burst of fluorescence. For single molecules in solution, their fluorescence detection will also embody fluctuation characteristics in fluorescence detection due to movement, and super-Poisson distribution characteristics in photon statistical characteristics. Then, the time parameters obtained by photon statistics are used to accurately characterize the concentration of the marker. The accuracy and calibration-free characteristics of the concentration characterization are further determined in theoretical research.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] A biomarker concentration detection device based on time-dependent intensity correlation statistics includes a laser for emitting laser light, wherein the laser light passes through a half glass slide, a quarter glass slide, a laser attenuator, a reflector, a beam expander, and an aperture in sequence to obtain the most uniform laser light, wherein the most uniform laser light is reflected by a dichroic mirror into an objective lens, and the light beam is focused on a microfluidic device, wherein a fluorescent nanoprobe for marking a biomarker in the microfluidic device is excited to emit fluorescence, which is collected by the objective lens, and after being transmitted through the dichroic mirror, passes through a spatial filter device, a long-pass filter, and a lens in sequence to be focused on a single-photon detector, wherein the single-photon detector transmits the detected electrical signal to a time-correlated single-photon counting module to calibrate the photon arrival time, thereby accurately calibrating the concentration of the biomarker.

[0007] Furthermore, the laser is a continuous laser or a high-frequency pulsed laser with stable laser output, and its wavelength is selected according to the excitation spectrum of the fluorescent nanoprobe.

[0008] Furthermore, the microfluidic device contains a reaction solution of the biomarker and the fluorescent nanoprobe, and circulates under the drive of an external peristaltic pump.

[0009] The half glass slide and the quarter glass slide can adjust the polarization of the laser so that the laser that excites the fluorescent nanoprobe is circularly polarized light, to prevent uneven excitation efficiency caused by the unstable orientation of the fluorescent nanoprobe, and to ensure uniform excitation effect on each fluorescent molecule.

[0010] The laser attenuator can adjust the laser power to avoid the situation where the laser power can only be adjusted within the threshold range, resulting in excessive power and causing optical bleaching of the fluorescent nanoprobe.

[0011] The reflector, beam expander and aperture will select the beam part to obtain a more uniform beam, so that the focused spot is uniform, thereby ensuring the effective excitation of the fluorescent nanoprobe. The laser beam passes through the reflector and enters the beam expander, making the spot diameter larger, and then the most uniform laser part is selected through the aperture for the excitation of the fluorescent nanoprobe.

[0012] The dichroic mirror can reflect the laser into the objective lens, while ensuring that the fluorescent photons collected by the objective lens can be transmitted through the objective lens into the detection system.

[0013] The objective lens can focus the laser into the microfluidic device to ensure that the laser effectively excites the fluorescent nanoprobes in the microfluidic system and collects fluorescent photons at the same time.

[0014] The spatial filtering system can filter out other environmental stray photons outside the detection window to ensure that the fluorescent photons are purer.

[0015] The filter will allow the fluorescence to pass through and filter out the laser light again.

[0016] The lens can focus the fluorescence onto the detection area of ​​the single-photon detector, so that the optical signal of the fluorescence photon is converted into an electrical signal.

[0017] The time-correlated single-photon counting module can record the arrival time of each photon, and what it detects is the electrical pulse signal emitted by the single-photon detector.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] The present invention is used for high-precision detection of biomarkers, and is particularly suitable for the fields of life sciences, medical diagnosis, environmental monitoring, and food safety testing. Through innovative signal processing and data analysis methods, it achieves efficient and accurate identification of extremely low-level biological molecules, filling the gap in the existing technology for low-concentration biomarker detection.

[0020] The present invention, by deeply mining the information entropy contained in the fluorescence signal and using the statistical theory of time-dependent intensity correlation to enhance the recognition of weak signals, significantly improves the detection limit and broadens the application boundary of biosensor technology, especially in situations requiring extremely high sensitivity and specificity. This technological innovation has important strategic significance and practical value in promoting precision medicine, public health safety prevention and control, and ecological environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of a biomarker concentration detection device based on time-dependent intensity association statistics;

[0022] Figure 2 It is a microfluidic device, and the peristaltic pump drives the liquid to be tested to circulate in the 3D-printed microfluidic channel;

[0023] Figure 3 It is the time-dependent photon statistical sensing spectrum;

[0024] Figure 4 The experimental measurement results of fluorescence detection of biomarker solutions with different concentrations, where (a) to (e) are the concentrations of 10 -13 , 10 -14 , 10 -15 , 10 -16 , 10 -17 The fluorescence detection trajectory under different concentrations, and the corresponding time-dependent photon statistical spectrum trajectory on the right, are fitted with a single exponential to obtain the key time parameter τ S ;

[0025] Figure 5 Numerical simulation of fluorescence detection of biomarker solutions with different concentrations, where (a) to (d) are concentrations of 10 -14 , 10 -15 , 10 -16 , 10 -17 The fluorescence detection trajectory under different concentrations, and the corresponding time-dependent photon statistical spectrum trajectory on the right, are fitted with a single exponential to obtain the key time parameter τ S ;

[0026] Figure 6 Correlation between key time parameters and biomarker concentrations. DETAILED DESCRIPTION

[0027] In order to further illustrate the technical solution of the present invention, the present invention is further described below through embodiments.

[0028] like Figure 1As shown, a biomarker concentration detection device based on time-dependent intensity correlation statistics includes a laser 1 for emitting laser light, and the laser light passes through a half glass slide 2, a quarter glass slide 3, a laser attenuator 4, a reflector 5, a beam expander 6, and an aperture 7 in sequence to obtain the most uniform laser light, and the most uniform laser light is reflected by a dichroic mirror 8 into an objective lens 9, and the light beam is focused on a microfluidic device 10, and the fluorescent nanoprobe used to mark the biomarker in the microfluidic device 10 is excited to emit fluorescence, and the fluorescence is collected by the objective lens 9, and after being transmitted through the dichroic mirror 8, it passes through a spatial filter device 11, a long-pass filter 12, and a lens 13 in sequence, and is focused on a single-photon detector 14, and the single-photon detector 14 transmits the detected electrical signal to a time-correlated single-photon counting module 15 to calibrate the photon arrival time, thereby accurately calibrating the concentration of the biomarker.

[0029] The laser 1 of this embodiment is a continuous laser or a high-frequency pulse laser that outputs laser light stably, and its wavelength is selected according to the excitation spectrum of the fluorescent nanoprobe.

[0030] The microfluidic device 10 of this embodiment is as follows Figure 2 As shown, a reaction solution containing biomarkers and fluorescent nanoprobes circulates under the drive of an external peristaltic pump, and only the nanoprobes marked with biomarkers can be illuminated by the laser, and they are randomly illuminated within the focusing detection window of the objective lens 9.

[0031] The fundamental principle of photon statistical sensing in the present invention is that when a mixed solution without biomarkers is exposed to the action of laser, the luminescence of the ensemble solution is generated, which is a uniform luminescence process, and does not generate a burst of photons due to the opening of the fluorescence of the nanoprobe. Therefore, when performing photon statistical analysis, no matter how strong the detected fluorescence signal is, it shows a Poisson photon statistical distribution process. Figure 3 As shown in (a), the consistent statistical characteristics of the background signal across different time scales are shown, which indicates the characteristics of a Poisson distribution. When a biomarker is present, the nanoprobe circulates under the drive of a peristaltic pump, which will cause the intensity of the detected fluorescence photons to burst at irregular intervals. This photon burst makes its photon statistical distribution present super-Poisson statistical distribution characteristics. Figure 3 As shown in (b), the time-dependent photon statistics curve corresponding to the dotted trajectory shows different characteristics, which suggests the existence of a super-Poissonian distribution. By fitting with an exponential function (e-exponential), two key time parameters τ can be obtained: S and τ T Among them, τ SIt represents the statistical time constant of the photon burst event, reflects the effect of biomarker concentration on the photon burst time rate, and can directly reveal the characteristics of the biomarker concentration.

[0032] To this end, different concentrations of biomarkers were measured, where the concentration of biomarkers and fluorescent nanoprobes was kept at 10 -10 After the biomarker and the mixed solution of the fluorescent nanoprobe are mixed, the biomarker fully responds to turn on the fluorescence of a certain number of fluorescent nanoprobes, which are placed in a Figure 2 In the microfluidic device shown in FIG. 1 , the fluorescence trajectory detected in the detection window is shown in FIG. Figure 4 Intuitively, a decrease in concentration will directly reduce the number of bright states, but the bright state and the dark state are often difficult to distinguish, and different integration times will produce different results. Here, the integration time used for the fluorescence trajectory measurement is 10 ms. -16 and 10 -17 It is difficult to obtain an obvious bright state display for biomarkers with a concentration of mol / L, but the time-dependent photon statistics of fluorescent photon sequences of different concentrations were measured, which all showed super-Poisson photon statistical behavior, and the key time parameter τ was obtained by fitting them. S Among them, the key parameters increase as the biomarker concentration decreases.

[0033] At the same time, the process was numerically simulated by photon statistical sensing:

[0034] Photon statistical sensing performs photon statistics based on the detected photon sequence, and distinguishes the photon density based on the time interval of photon detection. The density characteristics of photons represent the current state of the detected marker. To this end, the entire detection process was numerically simulated based on the experimental parameters, in which the reaction solution of the biomarker and the nanoprobe will circulate in the pipe of the microfluidic device driven by a peristaltic pump. The detection probability was simulated through the concentration and detection window, and the time setting of the detection photon sequence was performed based on the experimentally measured background signal intensity and signal intensity. Figure 5 As shown, the biomarker concentration is 10 -14 , 10 -15 , 10 -16 and 10 -17Photon detection intensity trajectory at a concentration of 1 mol / L. It can be seen that as the concentration of the biomarker decreases, the bright states displayed in the intensity trajectory gradually decrease, and at the current integration time, the number of bright states within a certain time range corresponds to the concentration. In order to ensure that there are enough bright states to be displayed, the numerical simulation time is increased to a certain extent with the concentration. In experimental measurements, it is not necessarily necessary to take too long a measurement time to ensure that the bright state is visible. In order to quantitatively characterize the relationship between concentration characteristics and photon distribution, the photon statistical spectrum of the photon sequence was calculated and analyzed. Statistics of photon sequences of different concentrations will obtain different photon statistical spectral characteristics. E-exponential fitting can obtain the quantitative parameter τ corresponding to the concentration. S It can be seen that in the numerical simulation process, as the concentration of the biomarker decreases, its photon statistical time parameter will increase, which means that the decrease in concentration will directly change the increase in the time interval of the photon burst.

[0035] A systematic investigation was conducted to elucidate the time scale parameter τ S The correlation between the concentration of biomarkers in the samples was significant. The analysis showed that the time interval between consecutive bright states was significantly shortened in samples with higher biomarker concentrations. Figure 5 As shown, the statistical distribution in the box represents the experimental data and numerical simulation results collected at different biomarker concentration levels, which clearly depicts the relationship between the standardized samples and their core parameter τ S These experimental observations were compared with theoretical expectations and confirmed that τ S There was a robust linear relationship with biomarker concentrations.

[0036] In addition, for Figure 5 The statistical distribution results of the boxed area show that a comprehensive thermodynamic analysis has been conducted, and the long-term kinetic parameters are actually deterministic values ​​at a given molecular concentration. Figure 6 As shown by the star in the middle, the results of the thermodynamic analysis show the time parameter τ S A good linear relationship between the time-dependent photon statistical sensing method and the inverse of the concentration (1 / n) is shown. When the simulation results are compared with the measured data, the calibration-free nature of the time-dependent photon statistical sensing method is indisputably demonstrated. The time-dependent photon statistical parameters are found to be intrinsically directly related to the concentration of the biomarker, mainly due to the super-Poisson nature of photon statistics, which is not affected by background noise interference. This deterministic concentration-to-parameter conversion relationship highlights the potential of the time-dependent photon statistical method to achieve unlimited range biomarker concentration measurement. Notably, this method eliminates the need for standard calibration procedures, opening up a new way to simplify clinical testing processes and disease early warning monitoring.

[0037] The above shows and describes the main features and advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes that fall within the meaning and scope of the equivalent elements of the claims are included in the present invention.

[0038] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A biomarker concentration detection device based on time-dependent intensity association statistics, characterized in that: The invention comprises a laser (1) for emitting laser light. The laser light passes through a half glass slide (2), a quarter glass slide (3), a laser attenuator (4), a reflector (5), a beam expander (6), and an aperture (7) in sequence to obtain the most uniform laser light. The most uniform laser light is reflected by a dichroic mirror (8) into an objective lens (9), and the light beam is focused on a microfluidic device (10). A fluorescent nanoprobe for marking a biomarker in the microfluidic device (10) is excited to emit fluorescence. The fluorescence is collected by the objective lens (9), and after being transmitted through the dichroic mirror (8), it passes through a spatial filter device (11), a long-pass filter (12), and a lens (13) in sequence to be focused on a single-photon detector (14). The single-photon detector (14) transmits the detected electrical signal to a time-correlated single-photon counting module (15) to calibrate the arrival time of the photons, thereby accurately calibrating the concentration of the biomarker.

2. A biomarker concentration detection device based on time-dependent intensity association statistics according to claim 1, characterized in that: The laser (1) is a continuous laser or a high-frequency pulse laser that outputs laser light stably, and its wavelength is selected according to the excitation spectrum of the fluorescent nanoprobe.

3. The biomarker concentration detection device based on time-dependent intensity association statistics according to claim 1, characterized in that: The microfluidic device (10) contains a reaction solution of a biomarker and a fluorescent nanoprobe, and circulates under the drive of an external peristaltic pump.

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