CN110095446A - A kind of portable two-channel time-resolved fluorescence device - Google Patents

Abstract

A kind of portable two-channel time-resolved fluorescence device, including light path module, LED driver circuit module, fluorescence signal detection circuit module, data communication module, embedded main control module and power module；Light path module improves the excitating light strength for injecting sample, and improve the fluorescence intensity for reaching silicon photoelectric diode and shorten light path for transmitting exciting light and fluorescence；LED driver circuit module is used to generate light pulse and adjusts the strong size of LED light；Fluorescence signal detection circuit module is used to detect pA grades of fluorescence signal electric current；Data communication module provides two kinds of communication modes of USB, UART；Embedded main control module is controlled for the constant flow regulation of LED and light pulse, the tim e- domain detection control and the processing of fluorescence intensity signals data and transmission work of fluorescence signal；Portable two-channel time-resolved fluorescence device provided by the invention, have the advantages that miniaturization, low cost, it is modular, unitary sampling can meet required precision, substantially reduce detection time.

Description

A portable dual-channel time-resolved fluorescence device

technical field

The invention relates to the biological detection field of immune analysis technology, in particular to a portable dual-channel time-resolved fluorescent device.

Background technique

Time-Resolved Fluorescence Immunoassay (TRFIA) was proposed in the 1980s, which reduces the interference in the sample and greatly improves the sensitivity of the analysis. Compared with other immunoassay methods, TRFIA technology has the following advantages: high sensitivity, good stability, strong specificity, wide range of standard curve, no interference from background fluorescence, non-radioactive, etc., very suitable for medicine, biology, and environmental science ultratrace analysis. With the deepening of research, this technology has been continuously adopted by hospitals and scientific research institutions. At present, TRFIA technology has been widely used in clinical testing abroad, and its application scenarios are also expanding, such as immunohistochemistry, microarray, multi-label immunoassay, etc.

The design scheme of the existing time-resolved fluorescent immunoassay device in Chinese patents and papers is different from the present invention. For example, the patent number: CN201711029384.X, title: "Small Automatic Time-Resolved Fluorescence Immunoassay Analyzer" and patent number: CN201310674089.5, title: "Automatic Time-Resolved Fluorescence Analysis Device and Method of Use" provide all solutions The automatic high-throughput instrument detects multiple samples at one time. It is different from the present invention in terms of mechanical structure and program design ideas, and cannot be truly miniaturized. The present invention detects a single sample at a time, and there is no screw rod or guide rail and other structures; patent number: CN201711260928.3, title: "A Bracket for Time-Resolved Fluorescence Test" provides only the test bracket structure design of the device, and the angle of excitation light and emission light is controlled by external factors to increase the uncertainty , the present invention includes not only structural design but also hardware circuit and program design. At the same time, the structural design of the two is also different. The optical path structure of the present invention is fixed and cannot be controlled by angle transformation; patent number: CN201110354794.8, name: The optical modules used in the "Time-Resolved Fluorescence System" are integrating spheres and xenon lamps, which cannot be truly miniaturized. However, the present invention uses a single- and dual-channel detection method, which not only increases the intensity of excitation light and fluorescence, but also shortens the optical path. The entire actual design scheme is not the same; patent number: CN201610379774.9, name: "A Time-Resolved Fluorescence Detection Method" provides a detection method rather than an instrument; patent number: CN201610116392.7, name: "Photoelectric Detection System and Time-Resolved Fluorescence Immunity Analyzer "The optical path module described in " has only one light source, and the sample and the light source are on the same side so that the entire optical path structure is different from the present invention. There is a reference ultraviolet photodiode to adjust the light intensity; the main optical path in the paper: "340nm pulsed UV LED system for europium based time-resolved fluorescence detection of immunoassays" is similar to the present invention, but it is only for single-channel detection and does not include Dual-channel detection, and the placement of the two reference ultraviolet photodiodes is different, the method of adjusting the current is also different, and the dynamic adjustment range is different; the paper: "Toward sensitive, quantitative point-of-care testing (POCT) of protein markers: miniaturization of ahomogeneous time-resolved fluoroimmunoassay for prostate-specific antigen detection" and "A novel luminescence anal Yser for europium chelates using solid-state excitation and a gated photomultiplier uses different excitation light sources and detection elements from the present invention. It uses lasers and photomultiplier tubes. Although the power and detection accuracy are high, it increases the cost and occupies space.

At present, commercial TRFIA analyzers are mainly medium and large-scale instruments with high throughput and ultra-high sensitivity, and there are very few portable or even hand-held products, which is not conducive to real-time POCT detection. At the same time, the light source used by large TRFIA analyzers is xenon lamp or laser, which consumes a lot of power and is expensive. Therefore, the handheld TRFIA analyzer still has great research value and market space, and with the promotion of the concept of personalized medicine, its market will be broader.

Contents of the invention

In order to solve the above-mentioned problems in the prior art, the purpose of the present invention is to propose a portable dual-channel time-resolved fluorescence device, which can achieve low cost and miniaturization while ensuring sensitivity, and adopts ultraviolet light-emitting diodes UV-LEDs as excitation light sources. The detection module is replaced by a silicon photodiode PD, which improves the sensitivity and reduces the cost; the miniaturization is reflected in the miniaturization of the module, the miniaturization of the optical path and the miniaturization of the circuit. The miniaturization of the module refers to the use of a pure optical path. Dual-channel acquisition replaces the way that the motor drives the optical module to switch the acquisition channel. At the same time, it innovatively proposes a multi-point sampling fitting method based on the fluorescence excitation-attenuation model and the pulse LED drive circuit, and finds out the MOSFET through the mathematical model. The optimal working range, and optimize the LED constant current drive and pulse control method.

In order to achieve the above object, technical scheme of the present invention is:

A portable dual-channel time-resolved fluorescent device, including an optical path module 1, an LED drive circuit module 2, a fluorescent signal detection circuit module 3, a data communication module 4, an embedded main control module 5 and all the aforementioned components except the optical path module 1 A power supply module 6 for supplying power to the module;

The optical path module 1 is used to transmit and irradiate the excitation light of the LED driving circuit module 2 to the sample, so as to increase the intensity of the excitation light injected into the sample; transmit the excited fluorescence to the silicon photodiode PD8 of the fluorescence signal detection circuit module 3 , increase the fluorescence intensity reaching the silicon photodiode PD8 and shorten the optical path;

The LED driving circuit module 2 drives the UV-LED12 to generate light pulses, and adjusts the light intensity, and then transmits the UV-LED12 excitation light through the optical path of the optical path module 1 to excite the rare earth chelate and make the fluorescence fall in a larger range;

The fluorescent signal detection circuit module 3 has high sensitivity, is used to detect the pA-level fluorescent signal current transmitted to the silicon photodiode PD 8 through the optical path module 1, and has a signal input bandwidth of about 1 MHz, and a relatively low offset current;

Described data communication module 4 standardizes and encapsulates data so that various upper computers (PC machine embedded systems) transmit data instructions to control the embedded main control module 5, and the embedded main control module 5 is controlled by two kinds of communication modes of USB and UART. 5. The processed fluorescence detection data is transmitted to the host computer for display;

The embedded main control module 5 is used to complete the constant current adjustment and light pulse control of the LED of the LED drive circuit module 2; the time domain detection control of the fluorescence signal detection circuit module 3, and the detection of the fluorescence detection circuit module 3 Subsequent processing and transmission of the received fluorescence intensity signal data; operation of the control instructions transmitted by the data communication module 4, and transmission of detection data to the host computer through the data communication module 4.

The power supply module 6 meets the power supply requirements of circuits at all levels.

The optical path module 1 includes a single-channel detection optical path and a dual-channel detection optical path. The single-channel detection optical path is for the UV-LED 12 to output a bunch of excitation light first through the Rf-PD 7 to adjust the output luminous flux, and then the excitation light passes through the combination lens 13 Parallel to the dichroic mirror 9, the excitation light is reflected by the plano-convex lens 11 and concentrated in the detection object for excitation, and the excited fluorescence is parallelly injected into the dichroic mirror 9 through the plano-convex lens 11, and then passes through the plano-convex lens 11 after transmission Concentrate light on the silicon photodiode PD8 for fluorescence detection;

The two-channel detection light path means that the UV-LEDs 12 on both sides output a bunch of excitation light respectively, after the luminous flux is adjusted by the Rf-PD 7 on both sides, it is injected into the combined lens 13 in front of each, and becomes parallel light through the two lights in front of each. Reflect to the color mirror 9, wherein one beam of reflected light passes through the 50% beam splitter 15, and the other beam of reflected light is reflected by the reflector 14 below and reflected again after the 50% beam splitter 15. At this time, the two beams of light overlap through The plano-convex lens 11 converges in the detection object for fluorescence excitation. After the excited fluorescence passes through the plano-convex lens 11, a beam of fluorescence passes through the 50% beam splitter 15 and the dichroic mirror 9. The photodiode PD8 performs fluorescence detection, and another beam of fluorescence is reflected on the 50% beam splitter 15 and reflector 14, and then passes through the dichroic mirror 9, passes through the plano-convex lens 11, and then directly converges on the silicon photodiode PD8 of the second channel for fluorescence. detection.

The combined lens 13 is close to the same direction by two plano-convex lenses 11, and one side of its plane faces the convex surface of the UV-LED12. The wavelength range of the UV-LED12 is 395-405nm; the plano-convex lens 11 is K9; the model of the dichroic mirror 9 is FF506-Di03, which performs well in the 350-400 low frequency band.

An Rf-PD7 is placed obliquely above the UV-LED12, which can adjust the output luminous flux of the UV-LED12 through the LED drive circuit module 2, and avoid system errors caused by aging or process differences of the UV-LED12.

The LED driving circuit module 2 adopts a PID software feedback controller to realize current regulation, and the gate-source voltage V _GS can realize regulation in the range of 0-3.3V, and can turn off the UV-LED 12 . The LED driving circuit module 2 is the front end of fluorescence excitation detection, and the whole LED driving process can be divided into constant current regulation and light pulse control. Constant current regulation can adjust the size of the UV-LED12 light intensity, so that the fluorescence falls within a larger range; light pulse control generates light pulses for exciting rare earth chelates; the LED drive circuit module 2 uses a CSD15571Q2 chip, the Under the power supply of 4.2V power supply, the MOSFET drives the UV-LED12 with a constant current power of 3W, and maintains a low heating power. The dynamic range of the DAC voltage output is 1.45-2.25V. In the case of short LED lighting time and negligible temperature rise, the LED driving system is approximately a time-invariant causal system, and the open-loop control of UV-LED12 is performed to further shorten the pulse width of the LED light pulse. The dimming of UV-LED12 is carried out by using the look-up table method, and a table of the relationship between the DAC jump setting value and the UV-LED12 current is established to manually calibrate the current of UV-LED12, and then the UV-LED12 is controlled by the look-up table method. According to the actual measurement, a graph of the relative light intensity of the UV-LED 12 measured at each gear and the measured current of the reference PD8 is obtained. When the UV-LED12 current is in the range of 0-0.8A, the MOSFET always works in the saturation region, refer to Figure 3; the UV-LED12 current and luminous flux basically have a linear relationship, refer to Figure 4, that is, the UV-LED12 sink current and UV-LED12 The optical power is basically linear.

The fluorescent signal detection circuit module 3 adopts a double op-amp amplifier circuit and a silicon photodiode PD8, and the detection method is a pulse method, and the dual op-amp amplifier circuit is used to expand the bandwidth and reduce the output resistance and output noise of the circuit; The above-mentioned silicon photodiode PD8 is used as a current signal source when used for optical signal detection, and adopts zero bias to obtain an optimal linear relationship. Since the input signal of the on-chip ADC on the main control chip of the embedded main control module 5 is usually a voltage signal, a current-voltage conversion circuit is used to convert the current signal output by the silicon photodiode PD8 into a voltage signal before outputting. The maximum output voltage of the fluorescence detection circuit module 3 is 2.048V, and the maximum photocurrent of the photodiode PD8 is about 2.56uA. The stable bandwidth that the fluorescent detection circuit module 3 can achieve is 0.65 MHz, which is determined by the feedback R _F , the gain-bandwidth product f _c (65 MHz) of the amplifier and the total capacitance C _IN at the input end of the amplifier.

Described data communication module 4 adopts and the mode of external communication has CAN and serial port; Described CAN is mainly used for communicating with embedded system; Described serial port is used for communicating with PC, and the used data structure of two kinds of communication methods is A structure in a union.

The main control chip adopted by the embedded main control module 5 is STM32F405RGT6, and an on-chip DAC is used. The control algorithm used mainly includes PID controller and multi-point sampling data fitting method. The PID controller is an incremental PID, which is used for dimming UV-LED12. Its data structure is in the form of a structure, and the output maximum value and output minimum value are added to the parameters to limit the output. The expression of incremental PID is Δu _k ＝u _k -u _k-1 ＝Ae _k +Be _k-1 +Ce _k-2 , where k—the serial number used; T—the interval between two samples. Therefore, it is only necessary to use K _P , K _i and K _d to determine the three parameters A, B, and C, and obtain three consecutive sets of measurement deviation values to obtain the control quantity.

The multi-point sampling data fitting method can calculate two parameters in the decay equation: relative fluorescence intensity and fluorescence lifetime. The multi-point sampling data fitting method includes steps:

S1. There is a relationship between the fluorescence concentration and the fluorescence detection intensity Taking the natural logarithm on both sides, we get Wherein αC is the relative fluorescence intensity at time t=0.

S2. After taking the natural logarithm of the acquired data, a linear regression analysis is performed to obtain the fluorescence lifetime and the relative initial fluorescence intensity.

S3, use the least squares method to fit the converted data, where ei is the error of the sample (Xi, Yi), and Indicates the parameters to be fitted. existential relation In the formula: ei - the error of the sample (Xi, Yi); - fitting parameter 1; - Fitting parameter 2. So the error e _i can be expressed as: Then the square loss function can be expressed as Equation 5-9, where Q is the residual sum of squares, we get When the Q value is the smallest, the determined straight line is the best fitting straight line. The partial derivative equation of Q with respect to the two parameters to be estimated is: Solving the above formula, we can get and for: The data fitting can be completed, the fluorescence lifetime and the relative fluorescence intensity at time t=0 are obtained, and the fluorescence decay equation is obtained.

Beneficial effect

The portable dual-channel time-resolved fluorescence device provided by the embodiment of the present invention makes up for the deficiency of the contrast technology, and specifically has the following advantages:

(1) Two-degree-of-freedom dimming method using current peak plus current pulse width;

(2) A single sampling can meet the accuracy requirements, which can greatly shorten the sampling time;

(3) The measurement and control module realizes miniaturization, low cost and modularization;

(4) Fluorescence light intensity variation coefficient CV<3.4%, fluorescence lifetime variation coefficient CV<5.8%;

(5) With Eu(TTA)3phen fluorescent probe as the detection object, the detection limit is <0.185mg/L.

Description of drawings

Table 1 is the DAC setting value and LED pouring current measurement table utilizing the look-up table method according to the embodiment of the present invention;

1 is a schematic diagram of a module of a portable dual-channel time-resolved fluorescence device of the present invention;

Fig. 2 is the internal structure diagram of the portable dual-channel time-resolved fluorescence device of the present invention;

Fig. 3 is the relationship between the DAC set value and the LED1 pouring current utilizing the look-up table method according to an embodiment of the present invention;

Fig. 4 is the relationship diagram between LED1 pouring current and relative light intensity utilizing the look-up table method according to an embodiment of the present invention;

Among them: 1: Optical path module; 2: LED drive circuit module; 3: Fluorescent signal detection circuit module; 4: Data communication module; 5: Embedded main control module; 6: Power module; 7: Rf PD; 8: Silicon photoelectric Diode (PD); 9: dichroic mirror; 10: optical filter; 11: K9 plano-convex lens; 12: ultraviolet light-emitting diode (UV-LED); 13: combined lens; 14: reflector; 15: 50% light splitting mirror.

Detailed ways

The specific implementation manners of the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

As shown in Figure 1, a portable dual-channel time-resolved fluorescence device includes an optical path module 1, an LED drive circuit module 2, a fluorescent signal detection circuit module 3, a data communication module 4, an embedded main control module 5 and the aforementioned optical path Power supply module 6 for supplying power to all modules except module 1;

The optical path module 1 is used to transmit and irradiate the excitation light of the LED driving circuit module 2 to the sample, so as to increase the intensity of the excitation light injected into the sample; transmit the excited fluorescence to the silicon photodiode PD8 of the fluorescence signal detection circuit module 3 , increase the fluorescence intensity reaching the silicon photodiode PD8 and shorten the optical path;

The LED driving circuit module 2 drives the UV-LED12 to generate light pulses, and adjusts the light intensity, and then transmits the UV-LED12 excitation light through the optical path of the optical path module 1 to excite the rare earth chelate and make the fluorescence fall in a larger range;

The fluorescent signal detection circuit module 3 has high sensitivity, is used to detect the pA-level fluorescent signal current transmitted to the silicon photodiode PD8 through the optical path module 1, and has a signal input bandwidth of about 1 MHz, and a low offset current;

The data communication module 4 standardizes the data packaging so that various upper computers (PC embedded systems) transmit data instructions to control the embedded main control module 5, and the embedded main control module 5 can be controlled by two communication modes of USB and UART. The fluorescence detection data processed by module 5 is transmitted to the host computer for display;

The embedded main control module 5 is used for constant current regulation and light pulse control of the LED of the LED drive circuit module 2; time-domain detection control of the fluorescence signal detection circuit module 3, and detection of the fluorescent signal detection circuit module 3 Follow-up processing and transmission of the fluorescence intensity signal data; operate the control instructions transmitted by the data communication module 4, and transmit the detection data to the host computer through the data communication module 4.

The power supply module 6 meets the power supply requirements of circuits at all levels.

The optical path module 1 includes a single-channel detection optical path and a dual-channel detection optical path. The single-channel detection optical path is for the UV-LED 12 to output a bunch of excitation light first through the Rf-PD 7 to adjust the output luminous flux, and then the excitation light passes through the combination lens 13 Parallel to the dichroic mirror 9, the excitation light is reflected by the plano-convex lens 11 and concentrated in the detection object for excitation, and the excited fluorescence is parallelly injected into the dichroic mirror 9 through the plano-convex lens 11, and then passes through the plano-convex lens 11 after transmission Concentrate light on the silicon photodiode PD8 for fluorescence detection;

The two-channel detection light path means that the UV-LEDs 12 on both sides output a bunch of excitation light respectively, after the luminous flux is adjusted by the Rf-PD 7 on both sides, it is injected into the combined lens 13 in front of each, and becomes parallel light through the two lights in front of each. Reflect to the color mirror 9, wherein one beam of reflected light passes through the 50% beam splitter 15, and the other beam of reflected light is reflected by the reflector 14 below and reflected again after the 50% beam splitter 15. At this time, the two beams of light overlap through The plano-convex lens 11 converges in the detection object for fluorescence excitation. After the excited fluorescence passes through the plano-convex lens 11, a beam of fluorescence passes through the 50% beam splitter 15 and the dichroic mirror 9. The photodiode PD8 performs fluorescence detection, and another beam of fluorescence is reflected on the 50% beam splitter 15 and reflector 14, and then passes through the dichroic mirror 9, passes through the plano-convex lens 11, and then directly converges on the silicon photodiode PD8 of the second channel for fluorescence. detection.

Described combination lens 13 is close to in the same direction by two plano-convex lenses 11, and one side of its plane is facing the convex surface of UV-LED12, and UV-LED12 is placed in the object space focal point of combination lens 13, and the numerical aperture of excitation light path is increased 0.36 times; the wavelength range of the UV-LED 12 is 395-405nm; the plano-convex lens 11 is K9; the model of the dichroic mirror 9 is FF506-Di03, which performs well in the 350-400 low frequency band. An Rf-PD7 is placed obliquely above the UV-LED12, which can adjust the output luminous flux of the UV-LED12 through the LED drive circuit module 2, and avoid system errors caused by aging or process differences of the UV-LED12.

The LED driving circuit module 2 adopts a PID software feedback controller to realize current regulation, and the gate-source voltage V _GS can realize regulation in the range of 0-3.3V, and can turn off the UV-LED 12 . The LED driving circuit module 2 is the front end of fluorescence excitation detection, and the whole LED driving process can be divided into constant current regulation and light pulse control. The constant current regulation can adjust the size of the UV-LED12 light intensity, so that the fluorescence falls within a larger range; the light pulse control generates light pulses for exciting rare earth chelates; the LED drive circuit module 2 uses a CSD15571Q2 chip, the Under the power supply of 4.2V power supply, the MOSFET drives the UV-LED12 with a constant current power of 3W, and maintains a low heating power. The dynamic range of the DAC voltage output is 1.45-2.25V. In the case of short LED lighting time and negligible temperature rise, the LED driving system is approximately a time-invariant causal system, and the open-loop control of UV-LED12 is performed to further shorten the pulse width of the LED light pulse. The dimming of UV-LED12 is carried out by using the table look-up method, and a table of the relationship between the DAC jump setting value and the UV-LED12 current is established to manually calibrate the current of UV-LED12, and then the UV-LED12 is controlled by the table look-up method, refer to the table 1,

Table 1

According to the actual measurement, a graph of the relative light intensity of the UV-LED 12 measured at each gear and the measured current of the reference PD8 is obtained. When the UV-LED12 current is in the range of 0-0.8A, the MOSFET always works in the saturation region, refer to Figure 3; the UV-LED12 current and luminous flux basically have a linear relationship, refer to Figure 4, that is, the UV-LED12 sink current and UV-LED12 The optical power is basically linear.

The fluorescent signal detection circuit module 3 adopts a double op-amp amplifier circuit and a silicon photodiode PD8, and the detection method is a pulse method, and the dual op-amp amplifier circuit is used to expand the bandwidth and reduce the output resistance and output noise of the circuit; The above-mentioned silicon photodiode PD8 is used as a current signal source when used for optical signal detection, and adopts zero bias to obtain an optimal linear relationship. Since the input signal of the on-chip ADC on the main control chip of the embedded main control module 5 is usually a voltage signal, a current-voltage conversion circuit is used to convert the current signal output by the silicon photodiode PD8 into a voltage signal before outputting. The maximum output voltage of the fluorescence detection circuit module 3 is 2.048V (determined by the reference voltage of ADS8328), and the maximum photocurrent of the photodiode PD8 is about 2.56uA. The stable bandwidth that the fluorescent detection circuit module 3 can achieve is 0.65 MHz, which is determined by the feedback R _F , the gain-bandwidth product f _c (65 MHz) of the amplifier and the total capacitance C _IN at the input end of the amplifier.

Described data communication module 4 adopts and the mode of external communication has CAN and serial port; Described CAN is mainly used for communicating with embedded system; Described serial port is used for communicating with PC, and the used data structure of two kinds of communication methods is A structure in a union.

The main control chip adopted by the embedded main control module 5 is STM32F405RGT6, and an on-chip DAC is used. The control algorithms used mainly include PID controller and multi-point sampling data fitting method. The PID controller is an incremental PID, which is used for dimming UV-LED12. Its data structure is in the form of a structure, and the output maximum value and output minimum value are added to the parameters to limit the output. The expression of incremental PID is Δu _k ＝u _k -u _k-1 ＝Ae _k +Be _k-1 +Ce _k-2 , where k—the serial number used; T—the interval between two samples. Therefore, it is only necessary to use K _P , K _i and K _d to determine the three parameters A, B, and C, and obtain three consecutive sets of measurement deviation values to obtain the control quantity.

The external power supply of the power supply module 6 is 5V, the 4.2V voltage is supplied to each circuit through the TLV62084 step-down converter, and the 3.3V voltage is supplied to the operational amplifier circuit through the LP5907 voltage regulator and the RT9193 voltage regulator. The power supply type of the power supply module involves a linear power supply, a charge pump and a switching power supply.

The multi-point sampling data fitting method makes up for the long sampling time problem of the traditional detection method, and two parameters in the decay equation can be deduced through calculation: relative fluorescence intensity and fluorescence lifetime. The multi-point sampling data fitting method includes steps:

S1. There is a relationship between the fluorescence concentration and the fluorescence detection intensity Taking the natural logarithm on both sides, we get Wherein αC is the relative fluorescence intensity at time t=0.

S2. After taking the natural logarithm of the acquired data, a linear regression analysis is performed to obtain the fluorescence lifetime and the relative initial fluorescence intensity.

S3, use the least squares method to fit the converted data, where ei is the error of the sample (Xi, Yi), and Indicates the parameters to be fitted. existential relation In the formula: ei - the error of the sample (Xi, Yi); - fitting parameter 1; - Fitting parameter 2. So the error e _i can be expressed as: Then the square loss function can be expressed as Equation 5-9, where Q is the residual sum of squares, we get When the Q value is the smallest, the determined straight line is the best fitting straight line. The partial derivative equation of Q with respect to the two parameters to be estimated is: Solving the above formula, we can get and for: The data fitting can be completed, the fluorescence lifetime and the relative fluorescence intensity at time t=0 are obtained, and the fluorescence decay equation is obtained.

The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that it can still be described in the foregoing embodiments The recorded technical solutions are modified, or some of the technical features are replaced equivalently; and these modifications or replacements do not make the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (9)

1. A portable dual-channel time-resolved fluorescent device, characterized in that it comprises an optical path module (1), an LED drive circuit module (2), a fluorescent signal detection circuit module (3), a data communication module (4), an embedded host control module (5) and a power supply module (6) for supplying power to all modules except the optical path module (1); The optical path module (1) is used to transmit and irradiate the excitation light of the LED drive circuit module (2) to the sample, so as to increase the intensity of the excitation light injected into the sample; transmit the excited fluorescence to the fluorescence signal detection circuit module (3) On the silicon photodiode PD8, improve the fluorescence intensity reaching the silicon photodiode PD8 and shorten the optical path; The LED drive circuit module (2) drives the UV-LED (12) to generate light pulses, and adjusts the light intensity, and then transmits the UV-LED (12) excitation light through the optical path of the optical path module (1), for Excite the rare earth chelate and make the fluorescence fall in a larger range; The fluorescent signal detection circuit module (3) is used to detect the pA-level fluorescent signal current transmitted to the silicon photodiode PD (8) through the optical path module (1), and has a signal input bandwidth of about 1 MHz, and a relatively low offset current; The data communication module (4) standardizes and encapsulates the data so that various upper computers transmit data instructions to control the embedded main control module (5), and the embedded main control module (5) The processed fluorescence detection data is transmitted to the host computer for display; The embedded main control module (5) is used to complete the constant current adjustment and light pulse control of the LED of the LED drive circuit module (2); the time domain detection control of the fluorescent signal detection circuit module (3), and the The fluorescence intensity signal data detected by the fluorescence detection circuit module (3) is subjected to subsequent processing and transmission; the control instruction transmitted by the data communication module (4) is operated, and the detection data is transmitted to the host computer through the data communication module (4). 2. A portable dual-channel time-resolved fluorescence device according to claim 1, wherein said optical path module (1) comprises a single-channel detection optical path and a dual-channel detection optical path, and said single-channel detection optical path is UV - LED (12) outputs a beam of excitation light firstly through Rf-PD (7) to adjust the output luminous flux, then the excitation light passes through the combined lens (13) and shoots on the dichroic mirror (9) in parallel, and after the excitation light is reflected, it passes through the plano-convex lens (11) Concentrate light in the detection object for excitation, and the excited fluorescence passes through the plano-convex lens (11) and enters the dichroic mirror (9) in parallel, and after transmission, the light is concentrated on the silicon photodiode PD (8 ) for fluorescence detection; The two-channel detection light path means that the UV-LEDs (12) on both sides output a beam of excitation light, after the luminous flux is adjusted by the Rf-PD (7) on both sides, and then enter the combined lens (13) in front of each, and become parallel The light is reflected through the dichroic mirrors (9) in front of each other, wherein one beam of reflected light passes through the 50% beam splitter (15), and another beam of reflected light is reflected in the 50% beam splitter (14) through the reflector (14) below. 15) and then reflect again, at this time, the two beams of light overlap and converge in the detection object through the plano-convex lens (11) for fluorescence excitation, and the excited fluorescence passes through the plano-convex lens (11), and then a beam of fluorescence passes through the 50% beam splitter (15) And the dichroic mirror (9) directly converges on the silicon photodiode PD (8) of the first passage through the plano-convex lens (11) to carry out fluorescence detection, and another beam of fluorescence is detected at the 50% beam splitter (15) and reflector (14) ) after being reflected by the dichroic mirror (9), passing through the plano-convex lens (11), and then directly converging on the silicon photodiode PD (8) of the second channel for fluorescence detection. 3. A kind of portable two-channel time-resolved fluorescence device according to claim 2, characterized in that, the combined lens (13) is close to the same direction by two plano-convex lenses (11), and one side of its plane faces the UV - the convex surface of the LED (12), the UV-LED (12) is placed at the focal point of the combined lens (13); the wavelength range of the UV-LED (12) is 395-405nm; the plano-convex lens (11) is K9; the model of the dichroic mirror (9) is FF506-Di03. 4. A kind of portable dual-channel time-resolved fluorescent device according to claim 2, characterized in that, an Rf-PD (7) is placed obliquely above the UV-LED (12), which can pass through the LED driver circuit module ( 2) Adjusting the output luminous flux of the UV-LED (12) to avoid systematic errors caused by aging or process differences of the UV-LED (12). 5. A kind of portable dual-channel time-resolved fluorescent device according to claim 1, characterized in that, said LED drive circuit module (2) adopts a PID software feedback controller to realize current regulation, and the gate-source voltage _VGS realizes The regulation in the range of 0-3.3V can turn off the UV-LED (12); the LED driving circuit module (2) is the front end of the fluorescence excitation detection, and the whole LED driving process can be divided into constant current regulation and light pulse control; constant current The adjustment can adjust the light intensity of the UV-LED (12), so that the fluorescence falls within a larger range; the light pulse control generates light pulses for exciting rare earth chelates; the LED drive circuit module (2) adopts CSD15571Q2 Chip, under the power supply of 4.2V power supply, the MOSFET can drive the UV-LED (12) with a constant current power of 3W, and maintain a low heating power. The dynamic range of the DAC voltage output is 1.45-2.25V; during the LED lighting time Short and under the condition of negligible temperature rise, the LED drive system is approximately a time-invariant causal system, and the open-loop control of the UV-LED (12) is performed to further shorten the pulse width of the LED light pulse; the dimming of the UV-LED (12) utilizes The look-up table method is carried out, and a DAC jump setting value and UV-LED (12) current relationship table is established to manually calibrate the current of the UV-LED (12), and then the UV-LED (12) is controlled by the look-up table method. 6. A kind of portable dual-channel time-resolved fluorescent device according to claim 1, characterized in that, the fluorescent signal detection circuit module (3) adopts a double operational amplifier amplifier circuit and a silicon photodiode PD (8) to detect The method is a pulse method, and the double op-amp amplifier circuit is used to expand the bandwidth and reduce the output resistance and output noise of the circuit; the silicon photodiode PD (8) is used as a current signal source when the optical signal is detected, and the zero bias is adopted. Obtain the optimal linear relationship; use the current-voltage conversion circuit to convert the current signal output by the silicon photodiode PD (8) into a voltage signal and then output it; the maximum output voltage of the fluorescence detection circuit module (3) is 2.048V, and the photodiode The maximum photocurrent of the PD (8) is about 2.56uA; the stable bandwidth that can be realized by the fluorescent detection circuit module (3) is 0.65MHz, which is determined by the feedback R _F , the gain-bandwidth product f _c of the amplifier and the total capacitor C _IN determines. 7. A kind of portable dual-channel time-resolved fluorescent device according to claim 1, characterized in that, the mode of external communication adopted by the data communication module (4) has CAN and serial ports; the CAN is mainly used for Communicate with the embedded system; the serial port is used to communicate with the PC, and the data structures used by the two communication methods are structures in the union. 8. A kind of portable dual-channel time-resolved fluorescence device according to claim 1, characterized in that, the main control chip adopted by the embedded main control module (5) is STM32F405RGT (6), and an on-chip DAC is used. The control algorithm includes PID controller and multi-point sampling data fitting method; PID controller is incremental PID, used for UV-LED (12) dimming, its data structure adopts the form of structure, and the output is added to the parameters The maximum value and the minimum output value are used to limit the output; the expression of incremental PID is Δu _k ＝u _k -u _k-1 ＝Ae _k +Be _k-1 +Ce _k-2 , where k—the serial number; T—the interval between two samples; therefore, it is only necessary to use K _P , K _i and K _d to determine the three parameters A, B, and C, and obtain three consecutive sets of measurement deviation values, namely Amount of control can be obtained. 9. A kind of portable dual-channel time-resolved fluorescence device according to claim 8, characterized in that, the multi-point sampling data fitting method comprises the steps of: S1. There is a relationship between the fluorescence concentration and the fluorescence detection intensity Taking the natural logarithm on both sides, we get Wherein αC is the relative fluorescence intensity at time t=0; S2. After taking the natural logarithm of the collected data, perform a linear regression analysis to obtain the fluorescence lifetime and the relative initial fluorescence intensity; S3, use the least squares method to fit the converted data, where ei is the error of the sample (Xi, Yi), and Indicates the parameters to be fitted; there is a relational expression In the formula: ei - the error of the sample (Xi, Yi); - fitting parameter 1; - fitting parameter 2; thus the error e _i is expressed as: Then the square loss function can be expressed as Equation 5-9, where Q is the residual sum of squares, we get When the Q value is the smallest, the determined straight line is the best fitting straight line; the partial derivative equation of Q with respect to the two parameters to be estimated is: Solving the above formula, we can get and for: The data fitting can be completed, the fluorescence lifetime and the relative fluorescence intensity at time t=0 are obtained, and the fluorescence decay equation is obtained.