Anti-photobleaching excitation light-induced fluorescence heavy metal detection system and detection method

Abstract

The anti-photobleaching excitation light-induced fluorescence heavy metal detection system comprises a system control unit, a fluorescence excitation unit, a fluorescence detection unit and a data acquisition card; the system control unit sends a pulse excitation signal to make the fluorescence excitation unit generate pulse fluorescence; the fluorescence detection unit collects the pulse fluorescence and converts it into an electric signal input into the data acquisition card; the system control unit sends a fluorescence detection signal to make the data acquisition card save n fluorescence intensity values; and the data acquisition card averages the n fluorescence intensity values. The application avoids the problem of photobleaching of the fluorescence probe caused by long-time irradiation of the excitation light by shortening the irradiation time of the excitation light, reduces the error of the detection result and improves the service life of the fluorescence probe.

Description

Technical Field

[0001] This invention belongs to the field of light-induced fluorescence detection technology for heavy metal ions, specifically relating to photobleaching, and is used to avoid photobleaching in the detection of heavy metals by light-induced fluorescence. Background Technology

[0002] With rapid global industrial development, heavy metal pollution is widespread in the atmosphere, water bodies, and soil. When the concentration of heavy metals in the natural environment exceeds safety limits, it can cause serious harm to human health and the health of plants and animals. For heavy metals in water, fluorescence detection technology can accurately measure their ion concentration, speciation, and distribution, helping to understand the pollution status and environmental risks of groundwater and providing a scientific basis for water environment management.

[0003] Currently, fluorescence spectrophotometers based on fluorescence detection technology are widely used in the field of heavy metal detection in water. They have advantages such as high sensitivity, speed, and non-invasiveness. However, such devices are complex in structure, expensive, and cannot achieve in-situ detection of heavy metals.

[0004] To address this issue, patent (CN113866141A) provides an in-situ detection device for heavy metals in groundwater based on laser-induced fluorescence technology. The device includes a waterproof cavity, an optical system, a heavy metal fluorescent probe, and a signal processing and control unit. The waterproof cavity is a sealed structure with a waterproof interface at the top. The optical system, housed within the waterproof cavity, includes a laser diode, an excitation light collimating and focusing lens group, an excitation light trap, a fluorescence collimating lens, a filter, a fluorescence focusing lens, and an optical fiber.

[0005] Current fluorescence detection technologies for heavy metal ions generally rely on the fluorescence enhancement or quenching phenomenon caused by the binding of fluorescent probes to heavy metal ions to measure heavy metal ion concentration. During the detection process, the fluorescence intensity of a blank sample with the fluorescent probe is first measured, followed by the fluorescence intensity after the heavy metal ions have fully bound to the probe. The concentration of heavy metal ions can be calculated based on the changes in fluorescence intensity at different concentrations. Therefore, the linear relationship between the relative change in fluorescence and the concentration of heavy metal ions is crucial for the reliability of the detection results. However, the uncontrollable decrease in fluorescence intensity caused by photobleaching significantly interferes with the detection results.

[0006] The existing technology still has the following problems: The device uses a continuous excitation light source. The heavy metal fluorescent probe may be photobleached under long-term irradiation of the excitation light. On the one hand, it is impossible to obtain a stable blank sample fluorescence intensity value. On the other hand, the fluorescence bleaching may be misjudged as fluorescence quenching, resulting in measurement errors or even permanent failure of the fluorescent probe. Summary of the Invention

[0007] To address the shortcomings of the existing technology, this invention provides an excitation light-induced fluorescence heavy metal detection system and method that resists photobleaching. This invention solves the photobleaching problem of fluorescent probes by shortening the excitation light irradiation time and extracting and calculating the average value of the fluorescence signal intensity within the stable irradiation time of the excitation light.

[0008] The technical solution of the present invention is as follows:

[0009] The photobleach-resistant excitation-induced fluorescence heavy metal detection system includes a system control unit, a fluorescence excitation unit, a fluorescence detection unit, and a data acquisition card.

[0010] The fluorescence excitation unit includes a light source driving module, an excitation light source, a focusing collimating lens, a heavy metal fluorescent probe, and a light trap. The light beam emitted from the excitation light source is converged by the focusing collimating lens and obliquely illuminates the heavy metal fluorescent probe, exciting a fluorescence signal. The light beam reflected by the heavy metal fluorescent probe is absorbed by the light trap. The fluorescence detection unit includes an optical receiving module and a photodetector. The fluorescence signal is collimated and filtered by the optical receiving module and focused onto the photodetector. The photodetector converts the optical signal into an electrical signal, which is input to the data acquisition card via a signal line. The above process is pulse-modulated by the system control unit.

[0011] The system control unit sends a pulse excitation signal, which, through the light source driving module, controls the excitation light source to quickly turn on and off. The system control unit also sends a fluorescence detection signal, causing the data acquisition card to store n fluorescence intensity values. The data acquisition card then averages these n fluorescence intensity values.

[0012] The pulse width of the pulse excitation signal is greater than or equal to 2ms and less than 10ms; the sampling rate of the fluorescence detection signal is greater than or equal to 100,000Hz; and the fluorescence detection signal is triggered after the pulse excitation signal has stabilized.

[0013] Preferably, the fluorescence detection signal is triggered between 0.5 ms after the rising edge of the pulse excitation signal and 0.5 ms before the falling edge. More preferably, the fluorescence detection signal is triggered between 1 ms after the rising edge of the pulse excitation signal and 1 ms before the falling edge. Most preferably, the fluorescence detection signal is triggered 1 ms after the rising edge of the pulse excitation signal.

[0014] Optionally, the heavy metal fluorescent probe may be a heavy metal sensitive membrane or a heavy metal liquid fluorescent probe contained in a transparent container.

[0015] Optionally, the heavy metal fluorescent probe is only sensitive to a certain type of heavy metal ion and is not affected by other heavy metal ions in the solution. For different types of heavy metal ions in the solution, heavy metal fluorescent probes with different sensitivity characteristics need to be used for detection.

[0016] The detection method using the above-mentioned heavy metal detection system is characterized by including the following steps:

[0017] 1) Adjust the position of the heavy metal fluorescent probe in the detection system.

[0018] Adjust the heavy metal fluorescent probe to the focal position of the excitation optical path.

[0019] 2) Test the variance or root mean square of the fluorescence intensity value within the millisecond excitation time of the light source, and determine its stability.

[0020] The system control unit emits a single rectangular pulse signal, which, through the light source driving module, controls the excitation light source to rapidly emit a stable excitation light pulse. The light pulse excites a fluorescence pulse on the heavy metal fluorescent probe. The fluorescence pulse is converted into a pulsed electrical signal by the fluorescence detection unit. This pulsed electrical signal is input to the data acquisition card via a signal line. The fluorescence intensity value is read from the data acquisition card, and the variance or root mean square of the fluorescence intensity value within the millisecond excitation time of the light source is calculated and compared with the system stability threshold. If the error exceeds the threshold range, the driving circuit or the fluorescent probe is checked for compliance.

[0021] 3) Take the average of multiple sets of continuous fluorescence intensity values ​​in the middle of the excitation time as the fluorescence intensity at that concentration.

[0022] The sample solution is added to the heavy metal fluorescent probe, the light source is turned on, and after a period of time after the rising edge of the pulse excitation signal is emitted, the system control unit emits a fluorescence detection signal, so that the data acquisition card (4) continuously saves the fluorescence intensity value at the same time interval, and calculates the average value of multiple fluorescence intensity values ​​as the fluorescence intensity value of this measurement.

[0023] 4) Use a heavy metal ion solution of known concentration to calibrate the detection data.

[0024] Obtain multiple sets of fluorescence intensity values ​​with different heavy metal ion concentrations according to steps 1-3 above, and plot the relationship curve between the relative change in fluorescence value and the concentration of heavy metal ions.

[0025] 5) Detect the concentration of heavy metal ions in the unknown solution based on the calibration results.

[0026] Test the solution according to steps 1-3 to obtain the fluorescence intensity value, and calculate the concentration of the target heavy metal ions in the solution based on the working curve.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] 1. This invention avoids photobleaching during the excitation light-induced fluorescence heavy metal detection process by shortening the excitation light irradiation time. Photobleaching occurs in the sub-second range or longer, while the detection time of this invention is within 10ms, effectively preventing fluorescence bleaching.

[0029] 2. This invention avoids the influence of photobleaching on fluorescence detection results, thus improving the stability of the detection results. Compared with detection systems that rely on continuous excitation light, repeatability errors are significantly reduced, and the stability of the detection results is improved by 50%.

[0030] 3. This invention avoids the loss of fluorescent probes caused by photobleaching, thus improving the reliability of the system. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the structure of the excitation light-induced fluorescence heavy metal detection system of the present invention.

[0032] Figure 2 This is a schematic diagram of the control signal and excitation light output waveform of the present invention, wherein... Figure 2 a is a schematic diagram of the pulse excitation signal. Figure 2 b is a schematic diagram of the laser output signal. Figure 2 c is a schematic diagram of the fluorescence detection signal. Figure 2 a, Figure 2 b、 Figure 2 c illustrates the temporal relationship between the pulse excitation signal, the laser output signal, and the fluorescence detection signal.

[0033] Figure 3 This is a schematic diagram of the working curve of the present invention.

[0034] In the diagram: 1: System control unit; 2: Fluorescence excitation unit; 201: Light source driving module; 202: Excitation light source; 203: Focusing collimating lens; 204: Heavy metal fluorescence probe; 205: Light trap; 3: Fluorescence detection unit; 301: Optical receiving module; 302: Photodetector; 4: Data acquisition card. Detailed Implementation

[0035] The present invention will be further described below through specific embodiments and in conjunction with the accompanying drawings.

[0036] like Figure 1 As shown, the photobleach-resistant excitation light-induced fluorescence heavy metal detection system includes a system control unit 1, a fluorescence excitation unit 2, a fluorescence detection unit 3, and a data acquisition card 4.

[0037] The fluorescence excitation unit 2 includes a light source driving module 201, an excitation light source 202, a focusing collimating lens 203, a heavy metal fluorescence probe 204, and a light trap 205. The light beam emitted by the excitation light source 202 is converged by the focusing collimating lens 203 and obliquely illuminates the heavy metal fluorescence probe 204 to excite a fluorescence signal. The light beam reflected by the heavy metal fluorescence probe 204 is absorbed by the light trap 205. The fluorescence detection unit 3 includes an optical receiving module 301 and a photodetector 302. The fluorescence signal is collimated and filtered by the optical receiving module 301 and focused onto the photodetector 302. The photodetector 302 converts the optical signal into an electrical signal and inputs it to the data acquisition card 4 through a signal line. The above process is pulse-modulated by the system control unit 1.

[0038] The system control unit 1 sends a pulse excitation signal, which controls the excitation light source 202 to turn on and off rapidly through the light source driving module 201; the system control unit 1 sends a fluorescence detection signal, which causes the data acquisition card 4 to save n fluorescence intensity values; the data acquisition card 4 averages the n fluorescence intensity values.

[0039] like Figure 2 As shown, the pulse excitation signal has a pulse width of 2ms and a rising edge width of less than or equal to 0.01ms; the fluorescence detection signal has a sampling rate of 100000Hz; n is 10; and the fluorescence detection signal is triggered 1ms after the rising edge of the pulse excitation signal is emitted.

[0040] The excitation light source 202 is a semiconductor laser with a center wavelength of 405nm.

[0041] The light source driving module 201 adopts a driver adapted to a 405nm semiconductor laser.

[0042] The optical receiving module 301 includes a collimating lens, a filter, and a focusing lens. An example of an optical receiving module with fluorescence acquisition function can be found in patent (CN113866141A), which is incorporated herein by way of incorporation to the extent that it is not inconsistent with this document.

[0043] The photodetector 302 is a photomultiplier tube.

[0044] The heavy metal fluorescent probe 204 is a hexavalent chromium quenching solution fluorescent probe.

[0045] like Figure 3 As shown, the detection method using the above-described excitation light-induced fluorescence heavy metal detection system follows the following procedure:

[0046] 1) Add fluorescent probes for heavy metal solutions

[0047] The hexavalent chromium quenching solution fluorescent probe is placed in a sample cup, ensuring that the solution probe covers the area near the focal point of the excitation optical path.

[0048] 2) Test system stability

[0049] The excitation time of the light source is 2ms. Fluorescence intensity values ​​are collected uniformly throughout the entire irradiation period. The variance is calculated to be 1.72, which is less than the set value of 2.00, indicating that the light source has good stability and the probe has no significant bleaching phenomenon.

[0050] 3) Measure the stable fluorescence intensity value

[0051] Add 0 ppb hexavalent chromium blank sample solution, excite the light source for 2 ms, take 10 sets of fluorescence intensity values ​​from 1 ms, and calculate the average value I0;

[0052] 4) Calibrate the working curve

[0053] Following steps 1-3 above, obtain the fluorescence intensity values ​​I corresponding to hexavalent chromium sample solutions of 10ppb, 20ppb, 40ppb, 60ppb, 80ppb, and 100ppb respectively, and plot the relationship curve between the relative change value of fluorescence I0 / I-1 and the concentration of hexavalent chromium ions.

[0054] 5) Detection of heavy metal ion concentration in unknown solutions

[0055] Test the test solution containing an unknown concentration of hexavalent chromium ions according to steps 1-3, obtain the relative change value of fluorescence intensity, and calculate the concentration of hexavalent chromium ions in the solution based on the working curve;

[0056] 6) Results Analysis

[0057] The test results show that after adopting this improved method, the repeatability error decreased from 1.39% to 0.70%, and the stability of the test results improved by 49.77%.

[0058] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A photobleaching resistant excitation light induced fluorescence heavy metal detection system characterized by, The detection system comprises a system control unit (1), a fluorescence excitation unit (2), a fluorescence detection unit (3) and a data acquisition card (4), wherein the fluorescence excitation unit (2) comprises a light source driving module (201), an excitation light source (202), a focusing collimating lens (203), a heavy metal fluorescent probe (204) and a light trap (205), and the fluorescence detection unit (3) comprises an optical receiving module (301) and a photodetector (302), The system control unit (1) sends a pulse excitation signal to the light source driving module (201), the light source driving module (201) drives the excitation light source (202) to generate a laser beam, the laser beam is converged by the focusing collimating lens (203) and is obliquely irradiated to the heavy metal fluorescent probe (204), the reflected laser beam of the heavy metal fluorescent probe (204) is absorbed by the light trap (205), the excited fluorescence signal is collimated, filtered and focused on the photodetector (303) by the optical receiving module (301), the photodetector (303) converts the optical signal into an electrical signal and inputs the electrical signal to the data acquisition card (4), and the data acquisition card (4) reads the fluorescence intensity value; The system control unit (1) sends a fluorescence detection signal to the data acquisition card (4), the data acquisition card (4) saves the fluorescence intensity value according to the fluorescence detection signal, and then takes an average, and the fluorescence detection signal is triggered after the pulse excitation signal is relatively stable; The fluorescence detection signal is triggered between 0.5 ms after the rising edge of the pulse excitation signal is sent and 0.5 ms before the falling edge ends. The pulse width of the pulse excitation signal is greater than or equal to 2 ms and less than 10 ms, and the sampling rate of the fluorescence detection signal is greater than or equal to 100000 Hz.

2. A photobleaching resistant excitation light induced fluorescence heavy metal detection system as claimed in claim 1, wherein, The fluorescence detection signal is triggered between 1 ms after the rising edge of the pulse excitation signal is sent and 1 ms before the falling edge ends.

3. A photobleaching resistant excitation light induced fluorescence heavy metal detection system as claimed in claim 1, wherein, The fluorescence detection signal is triggered at 1 ms after the rising edge of the pulse excitation signal is sent.

4. A photobleaching resistant excitation light induced fluorescence heavy metal detection system as claimed in claim 1, wherein, The excitation light source (202) is a semiconductor laser with a central wavelength of 405 nm.

5. A photobleaching resistant excitation light induced fluorescence heavy metal detection system as defined in claim 1, wherein, The heavy metal fluorescent probe (204) is a heavy metal sensitive film or a heavy metal liquid fluorescent probe contained in a transparent container.

6. A photobleaching resistant excitation light induced fluorescence heavy metal detection system as defined in claim 1, wherein, The photodetector (302) is a photomultiplier tube.

7. A detection method of the anti-photobleaching excitation light-induced fluorescence heavy metal detection system according to any one of claims 1-6, characterized in that the method comprises the following steps: S1: adjusting the position of the heavy metal fluorescent probe in the detection system, adjusting the heavy metal fluorescent probe (204) to the focal point position of the excitation light path; S2: testing the variance or root mean square of the fluorescence intensity value in the millisecond excitation time of the light source, and judging the stability, S21: the system control unit (1) sends a single rectangular pulse signal, and the light source driving module (201) controls the excitation light source (202) to quickly send an excitation light pulse with stable power; S22: the light pulse is converged by the focusing collimating lens (203) and is obliquely irradiated on the heavy metal fluorescent probe (204) to excite a fluorescence pulse; S23: the fluorescence pulse is converted into a pulse electrical signal by the fluorescence detection unit (3); S24: the pulse electrical signal is input into the data acquisition card (4) through the signal line, the fluorescence intensity value is read out from the data acquisition card (4), the variance or root mean square of the fluorescence intensity value in the millisecond excitation time of the light source is calculated, and compared with the threshold value, if the error exceeds the threshold value range, check whether the driving circuit or the fluorescence probe is qualified; S3: average calculation of multiple groups of continuous fluorescence intensity values in the middle section of the excitation time as the fluorescence intensity at this concentration, In the heavy metal fluorescence probe (204), the sample solution is filled, the excitation light source (202) is turned on, after a period of time after the rising edge of the pulse excitation signal, the system control unit (1) sends out the fluorescence detection signal, so that the data acquisition card (4) continuously saves the fluorescence intensity value at the same time interval, and calculates the average value of multiple fluorescence intensity values as the fluorescence intensity value of this measurement; S4: calibrate the detection data by using the known concentration of heavy metal ion solution, According to the above steps S1-S3, multiple groups of fluorescence intensity values of different heavy metal ion concentrations are obtained, and the relationship curve between the relative change value of fluorescence and the concentration of heavy metal ion is drawn; S5: according to the calibration result, the concentration of heavy metal ion in the unknown solution is detected, According to steps S1-S3, the fluorescence intensity value of the solution to be tested is obtained, and the concentration of the target heavy metal ion in the solution is calculated according to the working curve.

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