An amplification detection circuit, method and apparatus

Abstract

This invention provides an amplification detection circuit, method, and device, belonging to the field of medical device technology. The circuit includes a photosensitive unit, a first-stage amplifier, a second-stage amplifier, a reference signal processor, and a control unit. The second-stage amplifier consists of a low-gain sub-amplifier and a high-gain sub-amplifier connected in parallel. The two sub-amplifiers perform subtraction operations on the preliminary amplified signal and the baseline signal provided by the reference signal processor, respectively, and then amplify and output the first and second detection results. At the analog circuit level, background baseline interference is directly subtracted. The control unit collects multiple sets of detection results within the overlap region and performs linear regression fitting to obtain a linear regression equation. When the high-gain sub-amplifier is not saturated, it outputs its detection result; when saturated, the detection result of the low-gain sub-amplifier is converted into an equivalent value and output instead. This invention achieves zeroing of the amplification curve start point and continuous, non-clipping detection throughout the entire cycle, effectively expanding the detection dynamic range.

Description

Technical Field

[0001] This invention belongs to the field of medical device technology, specifically relating to an amplification detection circuit, method, and device. Background Technology

[0002] Molecular diagnostic technology is a dominant diagnostic method with a large proportion and wide application in in vitro diagnostics. It uses thermal effects as a trigger condition to achieve amplification cycles under different conditions such as cycling temperature and isothermal. Typically, specific reagents such as probes and non-specific reagents such as dNTPs and buffers are added to the reaction vessel to form an amplification reaction system. During amplification, specific target sequences are copied and accumulated to form amplicon. Under normal conditions, because fluorescent agents and quenchers are contained in the amplification system, changes in fluorescence will occur with the accumulation of specific amplicon. The photosensitive unit at the detection end can detect these changes in fluorescence and convert them into electrical signals for transmission. In this way, amplification detection can be completed efficiently with low contamination risk.

[0003] To accurately capture changes in fluorescence intensity during amplification, different manufacturers have implemented various optimization designs at the algorithm or detection circuit level. While algorithm optimization can improve detection results to some extent, it generally requires the introduction of more complex algorithms. The optimization gains from such algorithm-level complexity may outweigh the time or cost associated with the increased complexity. The solution disclosed in US20090123935A1 presents a method for obtaining the crucial Ct value from the amplification curve. This design is highly efficient for processing relatively simple optical detection results like fluorescence detection. Although manufacturers continue to optimize algorithms for fluorescence amplification curves, the technology is generally considered mature. The degree of urgency for optimization design is relatively high. However, as more and more detection projects require reliable results from fluorescence amplification technology, various problems in the detection process are being exposed more extensively, and various optimization designs are constantly being proposed. CN116519645A discloses a single-molecule detection scheme. To ensure the accuracy of the detection results, its detection circuit introduces two different types of photosensitive units, namely a scattered light sensor and a fluorescence sensor, and relies on two independent detection circuits connected to the processor chip. The processor integrates the detection results of the two sensors to output the final detection result. This design requires more detection units and two complete algorithms, resulting in complex computational logic and higher costs; CN110376 In the detection scheme disclosed in 130B, to reduce interference in weak fluorescence detection scenarios, the emitted light is split into beams using a beam splitter between the fluorescence detection points within the reaction vessel and the beams used for fluorescence detection. One beam is reflected and guided before being captured by the photosensitive unit at the monitoring end, then processed and transmitted to the controller to form a negative feedback reference for parameters such as light intensity. The other beam is reflected and guided to generate fluorescence within the reaction vessel, and the photosensitive unit at the detection end captures the fluorescence detection result. This design also requires the placement of more detection sensors, making integrated and miniaturized design difficult for multi-channel detection scenarios. CN109060742B designs a detection circuit suitable for portable amplification devices, which receives fluorescence... A method using photodiodes connected in parallel and connected to a preamplifier reduces cost and circuit board area, enabling instrument miniaturization. It employs a controllable light source and a spectrometer to achieve multicolor fluorescence detection. While this design simplifies the detection circuit, it has poor adaptability to different fluorescence ranges. CN105973855A discloses a detection scheme using invisible infrared light as a light source. Due to the longer wavelength of infrared light, the corresponding detector can physically improve sensitivity by setting deeper photoelectric wells. However, this design is not always suitable for multi-channel detection scenarios because it only improves the performance of one detection channel, having a limited effect on overall performance improvement.The scheme disclosed in CN117470811A, in addition to including a filter amplification circuit in conjunction with the amplifier, also designs a full-wave rectification circuit. This circuit can filter out light other than the target frequency, thereby increasing the fluorescence signal at a specific wavelength, making it more suitable for fluorescence amplification equipment.

[0004] Based on the above analysis, although various manufacturers have tried different technical paths in terms of algorithms and circuit hardware to optimize amplification equipment and amplification curves, the application scenarios of various designs are very limited. Related design improvements can only improve detection performance in their limited specific scenarios. How to design a high-performance detection circuit is still a high-precision technology that needs to be continuously optimized and explored in this field. Summary of the Invention

[0005] The purpose of this invention is to provide an amplification detection circuit, method, and device to solve the problem in the prior art that it is difficult to simultaneously achieve baseline subtraction and wide dynamic range detection, and that it is impossible to obtain a complete and continuous amplification curve throughout the entire amplification cycle.

[0006] To achieve the above objectives, the present invention provides an amplification detection circuit, including a photosensitive unit for acquiring fluorescence signals, a first-stage amplifier, a second-stage amplifier, a reference signal processor, and a control unit. The second-stage amplifier includes a low-gain sub-amplifier and a high-gain sub-amplifier connected in parallel.

[0007] The input terminal of the first-stage amplifier is electrically connected to the output terminal of the photosensitive unit, and the output terminal of the first-stage amplifier is electrically connected to the input terminal of the second-stage amplifier. The input terminal of the second-stage amplifier is also connected to the output terminal of the reference signal processor.

[0008] The reference signal processor is used to output the baseline signal corresponding to the current detection channel to the second-stage amplifier. The baseline signal is simultaneously provided to the low-gain sub-amplifier and the high-gain sub-amplifier.

[0009] The first-stage amplifier is used to initially amplify the electrical signal output from the photosensitive unit to obtain a preliminary amplified signal.

[0010] The low-gain sub-amplifier is used to perform a subtraction operation between the preliminary amplified signal and the baseline signal, and then amplifies and outputs the first detection result. The high-gain sub-amplifier is used to perform a subtraction operation between the preliminary amplified signal and the baseline signal, and then amplifies and outputs the second detection result.

[0011] The control unit is used to continuously acquire multiple sets of first and second detection results corresponding to different signal intensities within the overlapping area where neither the first nor the second detection result is saturated, and to perform linear regression fitting on the first and second detection results to obtain a linear regression equation.

[0012] If the high-gain sub-amplifier is not saturated, the control unit outputs the second detection result corresponding to the current signal strength as the valid detection result; if the high-gain sub-amplifier is saturated, the equivalent detection result of the high-gain sub-amplifier under the current signal strength is obtained according to the linear regression equation and the first detection result corresponding to the current signal strength, and the control unit outputs the equivalent detection result as the valid detection result.

[0013] Because of the above technical solution, the second-stage amplifier in the amplification detection circuit includes a low-gain sub-amplifier and a high-gain sub-amplifier connected in parallel. The reference signal processor outputs the baseline signal corresponding to the current detection channel to the second-stage amplifier, and this baseline signal is simultaneously provided to the low-gain sub-amplifier and the high-gain sub-amplifier. This allows the low-gain sub-amplifier to perform a subtraction operation between the preliminary amplified signal output by the first-stage amplifier and the baseline signal, and then amplify and output the first detection result. The high-gain sub-amplifier performs a subtraction operation between the preliminary amplified signal and the baseline signal, and then amplifies and outputs the second detection result. In this way, the background baseline interference in the fluorescence signal is directly subtracted at the analog circuit level, avoiding the problem of the amplification curve starting point deviating from zero. Simultaneously, the control unit continuously acquires multiple sets of first and second detection results within the overlapping region where neither the first nor the second detection results are saturated, and performs linear regression fitting to obtain a linear regression equation. When the high-gain sub-amplifier has not entered saturation, the second detection result is used as the valid detection result output. When the high-gain sub-amplifier enters saturation, an equivalent detection result is obtained based on the linear regression equation and the first detection result corresponding to the current signal strength, and the equivalent detection result is used to replace the output. This allows for high-sensitivity capture of weak fluorescence signals using the detection results of the high-gain sub-amplifier in the early stage of amplification, and for continued output using the equivalent detection result to replace the saturated high-gain data in the later stage of amplification. A continuous, smooth, and non-cropping amplification curve is obtained throughout the entire amplification cycle. There is no need to run a complex digital baseline subtraction algorithm in the control unit, which simplifies the data processing flow and improves the computational efficiency and quantitative accuracy of amplification detection.

[0014] Furthermore, linear regression fitting is performed on multiple sets of first and second detection results continuously collected within the overlapping area, and the control unit also obtains the goodness of fit, which is used to characterize the degree of linear consistency between the first and second detection results.

[0015] Because of the above technical solution, the linear consistency between the first detection result and the second detection result is measured by the goodness of fit. When the high-gain sub-amplifier is working in the linear region, the outputs of the two sub-amplifiers maintain a stable linear relationship, and the goodness of fit is close to 1. When the high-gain sub-amplifier approaches the saturation region due to signal enhancement, its output no longer increases linearly with the output of the low-gain sub-amplifier, and the goodness of fit decreases accordingly.

[0016] Furthermore, the baseline signal is obtained by performing benchmark detection on an empty reaction vessel or a reaction vessel containing a preset amount of pure water.

[0017] By adopting the above technical solution, the baseline signal is obtained by performing benchmark detection on an empty reaction cup or a reaction cup containing a preset amount of pure water. This allows for the prior acquisition of the unique background fluorescence value of each reaction cup or each detection channel. In actual amplification detection, the baseline corresponding to that reaction cup or detection channel is specifically subtracted, eliminating background interference caused by individual differences between different reaction cups and optical differences between different detection channels. This further improves the accuracy of baseline subtraction and the consistency of detection results, enabling amplification curves of multiple channels or multiple targets to be intuitively compared under the same coordinate benchmark, facilitating accurate interpretation of amplification results by the operator.

[0018] Furthermore, the output of the reference signal processor can be simultaneously connected to the inverting input of the second-stage amplifier corresponding to multiple detection channels, so as to provide the corresponding baseline signal for each of the multiple detection channels, and each detection channel is used to independently detect a fluorescence signal of one wavelength.

[0019] By adopting the above technical solution, the reference signal processor provides a corresponding baseline signal for each detection channel, enabling each detection channel in a multi-channel detection scenario to achieve independent baseline subtraction at the hardware level. This avoids the problem of some channels' amplification curves deviating from zero due to inconsistent baselines, and strong positive sample signals prematurely reaching the upper limit of the range, causing signal oversaturation. As a result, the amplification curves of all detection channels are corrected to near zero and the amplification range is uniform, making it easier to place the multi-channel amplification results on the same operating interface for comprehensive comparison, thus improving the convenience and reliability of multi-target joint analysis.

[0020] Furthermore, the anode of the photosensitive unit is connected to the inverting input of the first-stage amplifier, the cathode of the photosensitive unit is connected to the non-inverting input of the first-stage amplifier and ground, respectively, the inverting inputs of the low-gain sub-amplifier and the high-gain sub-amplifier are connected to the output of the first-stage amplifier and the output of the reference signal processor, respectively, and the non-inverting inputs of the low-gain sub-amplifier and the high-gain sub-amplifier are grounded.

[0021] By employing the aforementioned technical solution, the anode of the photosensitive unit is connected to the inverting input of the first-stage amplifier, while the cathode of the photosensitive unit is connected to both the non-inverting input of the first-stage amplifier and ground. This allows the photosensitive unit to operate in its optimal high signal-to-noise ratio (SNR) state. The transmitted signal output by the photosensitive unit is negative, enabling it to generate different induced currents flowing from the ground to the amplifier in response to changes in the amount of light received, resulting in a higher SNR and a more stable detection signal. The inverting inputs of the low-gain and high-gain sub-amplifiers are connected to the outputs of the first-stage amplifier and the reference signal processor, respectively, while the non-inverting inputs are grounded. This allows the two sub-amplifiers to simultaneously receive the pre-amplified fluorescence signal and the baseline signal output from the reference signal processor at their inverting inputs. Analog subtraction is then performed directly within the amplifiers, effectively reducing baseline interference and outputting clean first and second detection results. This avoids the computational burden and delay associated with running a digital baseline subtraction algorithm in the control unit, improving the real-time performance and accuracy of amplification detection.

[0022] An amplification detection method, applied to the above-mentioned amplification detection circuit, includes the following steps: Baseline signal acquisition steps: The control unit performs a baseline detection on an empty reaction vessel or a reaction vessel containing a preset amount of pure water, and the acquired detection result is stored as a baseline signal in the reference signal processor.

[0023] Steps for acquiring fluorescence signals: When performing fluorescence amplification detection, the driving light source emits excitation light into the reaction vessel to be detected, and the photosensitive unit converts the fluorescence signal generated in the reaction vessel to be detected into an electrical signal.

[0024] Baseline subtraction and signal amplification steps: The first-stage amplifier amplifies the electrical signal to obtain a preliminary amplified signal. The input terminals of the high-gain sub-amplifier and the low-gain sub-amplifier in the second-stage amplifier simultaneously receive the preliminary amplified signal and the baseline signal output by the reference signal processor. The low-gain sub-amplifier performs a subtraction operation on the preliminary amplified signal and the baseline signal and then amplifies and outputs the first detection result. The high-gain sub-amplifier performs a subtraction operation on the preliminary amplified signal and the baseline signal and then amplifies and outputs the second detection result.

[0025] Linear regression steps: In the overlapping region where neither the first nor the second detection result is saturated, multiple sets of first and second detection results corresponding to different signal intensities are continuously collected, and linear regression fitting is performed to obtain the linear regression equation.

[0026] Detection result output steps: If the high-gain sub-amplifier has not entered saturation, the second detection result corresponding to the current signal strength is output as the valid detection result; if the high-gain sub-amplifier has entered saturation, the equivalent detection result of the high-gain sub-amplifier under the current signal strength is obtained according to the linear regression equation and the first detection result corresponding to the current signal strength, and the equivalent detection result is output as the valid detection result.

[0027] Because of the above technical solution, this amplification detection method first uses a control unit to perform baseline detection on an empty reaction vessel or a reaction vessel containing a preset amount of pure water to obtain a baseline signal and store it in a reference signal processor. During fluorescence amplification detection, the drive light source emits excitation light towards the reaction vessel to be detected and converts the fluorescence signal into an electrical signal. Then, the electrical signal is initially amplified by a first-stage amplifier to obtain a preliminary amplified signal. In the second-stage amplifier, the inputs of the high-gain sub-amplifier and the low-gain sub-amplifier simultaneously receive the preliminary amplified signal and the baseline signal. The low-gain sub-amplifier performs a subtraction operation between the preliminary amplified signal and the baseline signal and amplifies and outputs the first detection result. The high-gain sub-amplifier performs a subtraction operation between the preliminary amplified signal and the baseline signal and amplifies and outputs the second detection result. The results from the first and second detections are then compared. Multiple sets of first and second detection results are continuously collected within the unsaturated overlapping region, and linear regression fitting is performed to obtain a linear regression equation. Finally, when the high-gain sub-amplifier has not entered the saturation state, the second detection result is used as the valid detection result output. When the high-gain sub-amplifier enters the saturation state, the equivalent detection result is obtained based on the linear regression equation and the first detection result and is output as the equivalent detection result. This eliminates the need to run a complex baseline subtraction algorithm in the control unit. The baseline subtraction and dynamic range expansion are completed at the hardware level, simplifying the data processing flow and improving the computational efficiency and real-time performance of amplification detection. At the same time, it ensures that the amplification curve starts close to zero and is continuous, smooth, and without clipping throughout the entire amplification cycle, meeting the requirements of nucleic acid amplification detection for continuous analog signal acquisition and quantitative analysis.

[0028] Furthermore, in the linear regression step, multiple sets of first and second detection results collected continuously within the overlapping area are subjected to linear regression fitting to obtain the goodness of fit, which is used to characterize the degree of linear consistency between the first and second detection results.

[0029] Because of the above technical solution, the linear consistency between the first detection result and the second detection result is measured by the goodness of fit. When the high-gain sub-amplifier is working in the linear region, the outputs of the two sub-amplifiers maintain a stable linear relationship, and the goodness of fit is close to 1. When the high-gain sub-amplifier approaches the saturation region due to signal enhancement, its output no longer increases linearly with the output of the low-gain sub-amplifier, and the goodness of fit decreases accordingly.

[0030] Furthermore, the expression for the linear regression equation is:

[0031] Where x represents the first detection result of the low-gain sub-amplifier, and y represents the second detection result of the high-gain sub-amplifier. is the actual gain ratio, and b is the composite baseline bias.

[0032] By employing the aforementioned technical solution, the linear regression equation can accurately describe the linear mapping relationship between the first detection result of the low-gain sub-amplifier and the second detection result of the high-gain sub-amplifier. The k and b values ​​obtained by least squares fitting can be used to deduce the equivalent detection result of the high-gain sub-amplifier under the current signal strength based on the first detection result of the current low-gain sub-amplifier. This effectively eliminates systematic errors introduced by factors such as resistor accuracy error, op-amp bias voltage, temperature drift, and parasitic parameters of PCB layout, ensuring the accuracy of data conversion and the dimensional uniformity before and after the amplification curve switching point, further improving the accuracy of quantitative detection of strong positive samples.

[0033] An amplification detection device includes a light source end, a detection end, and the aforementioned amplification detection circuit. The light source end includes an LED light source, and the detection end includes photosensitive units corresponding to the number of LED light sources. A reference signal processor in the amplification detection circuit is simultaneously connected to the inverting input of a second-stage amplifier corresponding to multiple detection channels, for providing each detection channel with its own corresponding baseline signal. The photosensitive unit in each detection channel is sequentially connected to a first-stage amplifier and a second-stage amplifier. Each second-stage amplifier includes a low-gain sub-amplifier and a high-gain sub-amplifier connected in parallel, for simultaneously amplifying the same initial amplified signal by different factors.

[0034] Thanks to the above technical solution, each detection channel has an independent dual-gain parallel amplification structure and an independent baseline subtraction function. This enables independent subtraction of the baseline and self-calibration of the overlap region in each detection channel, ensuring that each channel can obtain a zero-starting point and a continuous and smooth amplification curve throughout the entire amplification cycle in multi-channel detection. Each channel also has independent wide dynamic range detection capability, which facilitates the comprehensive comparison of multi-channel amplification results on the same operating interface. This improves the convenience and reliability of multi-target joint analysis. At the same time, the circuit structure has strong compatibility and can be upgraded without replacing the main control system of earlier equipment.

[0035] Furthermore, the LED light source and photosensitive unit are integrated into a detection head, which also includes an emitting lens group, a dichroic mirror group, and a receiving lens group. The LED light source is used to emit excitation light, and the emitting lens group is used to filter and converge the excitation light. The dichroic mirror group is tilted to allow the excitation light emitted from the emitting lens group to pass through and be guided to the reaction cup. The reaction cup is used to contain the reaction liquid and provide a reaction space for fluorescence detection. The fluorescence in the reaction cup propagates upward to the dichroic mirror group, is reflected by the dichroic mirror group to the corresponding receiving lens group, and is filtered and converged by the receiving lens group before being received by the corresponding photosensitive unit and converted into an electrical signal. The detection head can be driven by a motion drive mechanism to align the different detection channels in the detection head with each detection hole in sequence, thereby performing fluorescence detection of multiple wavelengths on the reaction cup corresponding to each detection hole.

[0036] Thanks to the aforementioned technical solution, the LED light source and photosensitive unit are integrated into a single detection head. The detection head also includes an emitting lens group, a dichroic mirror group, and a receiving lens group. After the LED light source emits excitation light, it is filtered and focused by the emitting lens group. The tilted dichroic mirror group allows the excitation light emitted from the emitting lens group to pass through and be guided to the reaction cup. The reaction cup contains the reaction liquid and provides a reaction space for fluorescence detection. The fluorescence within the reaction cup propagates upwards to the dichroic mirror group and is reflected by the dichroic mirror group to the corresponding receiving lens group. After being filtered and focused by the receiving lens group, it is received by the corresponding photosensitive unit and converted into an electrical signal. This effectively separates the light paths of the excitation light and the emitted fluorescence through the transmission and reflection of the dichroic mirror group, resulting in a compact optical structure and high integration. The detection head can be driven by a motion drive mechanism to align the different detection channels within the detection head sequentially with each detection well, thereby performing multiple wavelength fluorescence detection on the reaction cup corresponding to each detection well. Motion drive enables sequential detection of multiple wells and multiple wavelengths, improving detection throughput and equipment space utilization, and meeting the application requirements of high-throughput nucleic acid amplification detection.

[0037] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention utilizes a low-gain sub-amplifier and a high-gain sub-amplifier to perform subtraction operations on the initial amplified signal and the baseline signal, respectively. This directly subtracts background baseline interference at the analog circuit level, allowing the amplification curve to start near zero and avoiding signal oversaturation caused by baseline deviation. Simultaneously, the two sub-amplifiers amplify the same signal in parallel. The control unit establishes a mathematical mapping relationship between the two detection results through linear regression fitting within the overlap region. In the early stage of amplification, to achieve high-sensitivity capture of weak signals, the second detection result from the high-gain sub-amplifier is output. In the later stage of amplification, when the high-gain sub-amplifier saturates, the first detection result from the low-gain sub-amplifier is converted into an equivalent detection result for continued output. This results in a continuous, smooth, and non-cropping amplification curve throughout the entire amplification cycle, extending the detection upper limit to several times or even tens of times, effectively solving the problem of existing technologies' difficulty in simultaneously achieving baseline subtraction and wide dynamic range detection.

[0038] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0039] Figure 1 This is an amplification detection circuit diagram provided in an embodiment of the present invention; Figure 2 This is the second connection method of the amplification detection circuit diagram provided in the embodiments of the present invention; Figure 3 This is the third connection method of the amplification detection circuit diagram provided in the embodiments of the present invention; Figure 4 This is the fourth connection method of the amplification detection circuit diagram provided in the embodiments of the present invention; Figure 5 This is a linear curve graph corresponding to the data in Table 1 provided in the embodiments of the present invention; Figure 6 This is a detection curve diagram corresponding to the gain ratio of the two sub-amplifiers being 3, as provided in this embodiment of the invention. Figure 7 These are detection curves of two sub-amplifiers under other gain ratios provided in embodiments of the present invention; Figure 8 This provides a wider range of detection results for the two sub-amplifiers in this embodiment of the invention; Figure 9 This is a comparison chart of amplification curves of the prior art and the present invention at normal probe concentrations provided in the embodiments of the present invention; Figure 10 This is a comparison chart of the amplification curves of the prior art and the present invention at 1 / 2 probe concentration provided in the embodiments of the present invention; Figure 11 This is a comparison chart of the amplification curves of the prior art and the present invention at 1 / 4 probe concentration provided in the embodiments of the present invention; Figure 12This is a comparison chart of the amplification curves of the prior art and the present invention at a 1 / 8 probe concentration provided in the embodiments of the present invention; Figure 13 This is a comparison chart of amplification curves of the prior art and the present invention under high-concentration probe reagent conditions provided in the embodiments of the present invention; Figure 14 This is a connection diagram of the control unit and various functional modules provided in the embodiments of the present invention; Figure 15 This is a connection block diagram of a control system based on a CAN bus provided in an embodiment of the present invention; Figure 16 This is a design diagram of an integrated light source driver provided in an embodiment of the present invention; Figure 17 This is a schematic diagram of the amplification and detection equipment provided in an embodiment of the present invention; Figure 18 This is a schematic diagram of the integrated structure of the internal optical components of the detection head provided in an embodiment of the present invention; Figure 19 This is a schematic diagram of the multi-channel detection optical path provided in an embodiment of the present invention.

[0040] Explanation of reference numerals in the attached figures: 10-Photosensitive unit, 11-First stage amplifier, 21-High gain sub-amplifier, 31-Low gain sub-amplifier, 12-First feedback resistor, 13-Second feedback resistor, 22-First sub-feedback resistor, 32-Second sub-feedback resistor, 100-Amplification container, 200-Integrated light source driver, 3-Detection head, 301-Detection hole, 41-Vertical drive mechanism, 42-Transverse drive mechanism. Detailed Implementation

[0041] To further illustrate the technical means and effects adopted by the present invention to achieve the intended purpose, the following detailed description of the solution according to the present invention is provided in conjunction with the accompanying drawings and specific embodiments.

[0042] The foregoing and other technical contents, features, and effects of the present invention will be clearly presented in the following detailed description of specific embodiments in conjunction with the accompanying drawings. Through the description of the specific embodiments, a more in-depth and concrete understanding can be gained of the technical means and effects adopted by the present invention to achieve its intended purpose. However, the accompanying drawings are for reference and illustration only and are not intended to limit the technical solutions of the present invention.

[0043] For ease of understanding, the "detection channel" mentioned in this invention refers to a complete signal chain used to perform fluorescence signal acquisition and processing at a specific wavelength. It includes at least a corresponding LED light source, emitting lens group, dichroic mirror group, receiving lens group, photosensitive unit, and subsequent amplification circuitry. Each detection channel corresponds to an independent photosensitive unit and a set of optical elements, capable of independently detecting the fluorescence signal of a specific target in the same reaction cup. In the device, a single detection head can integrate multiple detection channels. A motion drive mechanism moves the detection head, aligning each detection channel sequentially with different reaction cup detection holes, thereby achieving sequential detection at multiple holes and at multiple wavelengths.

[0044] An embodiment of an amplification detection circuit: like Figure 1 The diagram shown is an amplification detection circuit diagram provided by an embodiment of the present invention, including a photosensitive unit 10 for acquiring fluorescence signals, a first-stage amplifier 11, a second-stage amplifier, a reference signal processor, and a control unit (not shown in the figure); the second-stage amplifier includes two sub-amplifiers with different amplification factors connected in parallel, namely a low-gain sub-amplifier 31 and a high-gain sub-amplifier 21 in the figure.

[0045] The photosensitive unit 10 is electrically connected to the input terminal of the first-stage amplifier 11, and the output terminal of the first-stage amplifier 11 is electrically connected to the input terminal of the second-stage amplifier; the input terminal of the second-stage amplifier is also connected to the output terminal of the reference signal processor.

[0046] Specifically, the anode of the photosensitive unit 10 is connected to the inverting input terminal of the first-stage amplifier 11, and the cathode of the photosensitive unit 10 is connected to the non-inverting input terminal of the first-stage amplifier 11 and ground. A first feedback resistor 12 is connected between the inverting input terminal and the output terminal of the first-stage amplifier 11, forming a first negative feedback loop. The resistance value of the first feedback resistor 12 is set to a range of 10MΩ to 200MΩ, which enables the first-stage amplifier to output an amplified detection signal within a reasonable amplification range, ensuring the accuracy and stability of the initial amplified signal. The output terminal of the first-stage amplifier 11 is electrically connected to the input terminals of the low-gain sub-amplifier 31 and the high-gain sub-amplifier 21, respectively. The input terminals of the low-gain sub-amplifier 31 and the high-gain sub-amplifier 21 are also connected to the output terminal of the reference signal processor. A reference resistor is also connected between the output terminal of the reference signal processor and the output terminal of the first-stage amplifier 11. The output of the low-gain sub-amplifier 31 is connected to the first channel ADC1 of the analog-to-digital converter circuit; the output of the high-gain sub-amplifier 21 is connected to the second channel ADC2 of the analog-to-digital converter circuit.

[0047] The following is an introduction to the uses of each component: Photosensitive unit 10 is used to extract light from reaction cups (such as...) Figure 19Fluorescent signals are acquired within the amplification container 100 shown and converted into electrical signals.

[0048] Furthermore, the photosensitive unit 10 used in this application is a high-performance shielded component with a metal shielded shell. The electrodes connected to the shell can be directly connected to the ground of the amplification detection equipment to form a stable reference. The other end of the photosensitive unit 10 serves as the working end, connected to the inverting input of the first-stage amplifier 11 when connecting to the amplification detection circuit. Therefore, the transmitted signal output by the photosensitive unit 10 is negative. This connection method can optimally match the best operating state of the high-performance photosensitive unit 10 to obtain a higher signal-to-noise ratio and a more stable detection signal. Based on this, in this embodiment, the anode of the photosensitive unit 10 is connected to the inverting input of the first-stage amplifier 11, and the cathode of the photosensitive unit 10 is connected to the non-inverting input of the first-stage amplifier 11 and ground, respectively. This allows for the generation of different induced currents flowing from the ground end to the amplifier end in accordance with changes in the amount of light received.

[0049] The first-stage amplifier 11 is used to initially amplify the electrical signal output by the photosensitive unit 10 to obtain a preliminary amplified signal.

[0050] The reference signal processor outputs the baseline signal corresponding to the current detection channel to the low-gain sub-amplifier 31 and high-gain sub-amplifier 21 in the second-stage amplifier. This baseline signal is simultaneously provided to both the low-gain sub-amplifier 31 and high-gain sub-amplifier 21. It should be noted that in this embodiment, each detection channel is used to independently detect a fluorescence signal of one wavelength. Therefore, each detection channel has its own corresponding light source (LED), photosensitive unit, first-stage amplifier, and second-stage amplifier control unit. After performing baseline detection on an empty reaction cup or a reaction cup containing a preset amount of pure water, a baseline signal is obtained and stored in the reference signal processor. In actual amplification detection, the reference signal processor outputs the baseline signal corresponding to the currently operating detection channel to the inverting input terminals of the low-gain sub-amplifier 31 and high-gain sub-amplifier 21 of that detection channel, thereby achieving independent baseline subtraction within each detection channel.

[0051] The low-gain sub-amplifier 31 performs a subtraction operation between the initial amplified signal output from the first-stage amplifier 11 and the baseline signal output from the reference signal processor, amplifying and outputting the first detection result. The high-gain sub-amplifier 21 performs a subtraction operation between the initial amplified signal output from the first-stage amplifier 11 and the baseline signal output from the reference signal processor, amplifying and outputting the second detection result. Since each sub-amplifier performs baseline subtraction on the initial amplified signal, neither sub-amplifier outputs the detection results containing the background baseline component, providing a clean data foundation for subsequent linear regression fitting.

[0052] The control unit is used to continuously acquire multiple sets of first and second detection results corresponding to different signal intensities within the overlapping area where neither the first nor the second detection result is saturated, and to perform linear regression fitting on the first and second detection results to obtain a linear regression equation.

[0053] If the high-gain sub-amplifier 21 is not saturated, the control unit outputs the second detection result corresponding to the current signal intensity as the valid detection result. This utilizes the high-gain sub-amplifier 21's large amplification characteristic to achieve high-sensitivity capture and high-resolution acquisition of weak fluorescence signals in the early stage of amplification, ensuring detection accuracy and signal-to-noise ratio during the weak signal stage. If the high-gain sub-amplifier 21 is saturated, the equivalent detection result corresponding to the high-gain sub-amplifier 21 at the current signal intensity is obtained based on the linear regression equation and the first detection result corresponding to the current signal intensity. At this time, the control unit outputs the equivalent detection result as the valid detection result. This allows the range limitation of the high-gain sub-amplifier 21 to be overcome while ensuring the continuity of the amplification curve. The equivalent detection result replaces the second detection result of the high-gain sub-amplifier 21 after saturation distortion, allowing the fluorescence signal throughout the entire amplification cycle to be continuously output with values ​​under the same coordinate reference. This achieves a smooth transition and dimensional uniformity before and after switching, avoiding saturation at the peak of the strong signal in the later stage and maintaining the smooth continuity and uninterrupted connection of the entire amplification curve.

[0054] like Figure 2 The diagram shown illustrates a second connection method for the amplification detection circuit provided in an embodiment of the present invention. Figure 1 The difference is, Figure 2 In this circuit, a second feedback resistor 13, connected in parallel with the first feedback resistor 12, is connected between the inverting input terminal and the output terminal of the first-stage amplifier 11, forming a second negative feedback loop. A first controllable switch device is connected in series in the first feedback loop, and a second controllable switch device is connected in series in the second feedback loop. By switching the first and second controllable switches on and off, negative feedback resistors with different resistance values ​​can be connected, thereby adjusting the gain of the first-stage amplifier 11. This allows the first-stage amplifier to flexibly adjust the initial amplification factor according to different detection scenarios or different fluorescence intensity ranges.

[0055] like Figure 3 The diagram shown illustrates a third connection method for the amplification detection circuit provided in this embodiment of the invention. Figure 1 The difference is, Figure 3In the high-gain sub-amplifier 21, the non-inverting input terminal is grounded, and a first sub-feedback resistor 22 is connected between the inverting input terminal and the output terminal, forming a first sub-negative feedback loop. Similarly, the low-gain sub-amplifier 31 has its non-inverting input terminal grounded, and a second sub-feedback resistor 32 is connected between the inverting input terminal and the output terminal, forming a second sub-negative feedback loop. By setting different resistance values ​​for the first sub-feedback resistor 22 and the second sub-feedback resistor 32, the two sub-amplifiers can have different amplification factors, thus giving them different sensitivities to the same initially amplified signal.

[0056] like Figure 4 The diagram shown illustrates a fourth connection method for the amplification detection circuit provided in this embodiment of the invention. In the diagram, the output terminal of the first-stage amplifier 11 is connected back to its input terminal via a first feedback resistor 12, forming a first negative feedback loop. A second feedback resistor 13, connected in parallel with the first feedback resistor 12, is also connected between the inverting input terminal and the output terminal of the first-stage amplifier 11, forming a second negative feedback loop. A first controllable switch is connected in series in the first feedback loop, and a second controllable switch is connected in series in the second feedback loop. A reference resistor is also connected between the output terminal of the reference signal processor and the output terminal of the first-stage amplifier 11. The non-inverting input terminal of the high-gain sub-amplifier 21 is grounded, and a first sub-feedback resistor 22 is connected between the inverting input terminal and the output terminal to form a first sub-negative feedback loop; the non-inverting input terminal of the low-gain sub-amplifier 31 is grounded, and a second sub-feedback resistor 32 is connected between the inverting input terminal and the output terminal to form a second sub-negative feedback loop.

[0057] The above is the basic hardware circuit structure of this embodiment. The following section will elaborate on the linear regression calibration principle and range switching method of the two sub-amplifiers in the overlapping region.

[0058] Within the overlapping detection region, the fluorescence signal intensity is moderate, and both the high-gain sub-amplifier 21 and the low-gain sub-amplifier 31 operate within their respective linear regions, unaffected by background noise or entering saturation / truncation. Under these conditions, the detection results output by the two sub-amplifiers satisfy the following two prerequisites: First, the detection results do not contain background baseline components. Since each subamplifier performs baseline subtraction on the initially amplified signal, neither the first detection result output by the low-gain subamplifier 31 nor the second detection result output by the high-gain subamplifier 21 contains background baseline components. Both detection results are amplified values ​​of the pure fluorescence signal obtained under the same reference standard (i.e., near zero). This eliminates the influence of baseline differences between different detection channels or reaction cups on subsequent data processing, and also provides an accurate and unbiased data basis for linear regression fitting within the overlap region.

[0059] Secondly, there is a stable linear relationship between the two detection results. Since the input signals received by the two sub-amplifiers come from the same photosensitive unit 10 and the same first-stage amplifier 11, and both perform subtraction operations with the same baseline signal, there is an inherent proportional correlation between the output values ​​of the two sub-amplifiers. This proportional relationship is determined by the hardware gain ratio of the two sub-amplifiers.

[0060] However, in practical engineering, due to factors such as resistor accuracy errors, operational amplifier bias voltage, temperature drift, and parasitic parameters of PCB layout, the actual gain ratio of the two sub-amplifiers often deviates from the theoretical design value. Directly using the theoretical gain ratio as the basis for data fusion will introduce significant systematic errors, affecting the smoothness of subsequent amplification curve switching points and the accuracy of clinical sample Ct value interpretation. Therefore, this application proposes a data processing method based on linear regression self-calibration in the overlap region.

[0061] Specifically, the control unit continuously acquires the output values ​​of the two sub-amplifiers within the overlapping detection area, obtaining a series of paired data points (x, y), where x represents the first detection result output by the low-gain sub-amplifier 31, and y represents the second detection result output by the high-gain sub-amplifier 21. A univariate linear regression model is then established using these data points.

[0062] Where k is the actual gain ratio of the two sub-amplifiers under the current environmental conditions, and b is the comprehensive bias including dark current, op-amp bias, and baseline. The control unit uses the least squares method to fit the above model, calculates the estimated values ​​of k and b, and simultaneously calculates the goodness of fit R².

[0063] The goodness-of-fit R² is a key statistic for measuring the linear consistency between the outputs of two sub-amplifiers. It characterizes the degree of linear consistency between the first detection result from the low-gain sub-amplifier 31 and the second detection result from the high-gain sub-amplifier 21, and its value ranges from 0 to 1. When R² is close to 1, it indicates that the outputs of the two sub-amplifiers exhibit a high degree of linearity, meaning that both branches are in a clean linear operating region, without saturation distortion or abnormal noise interference. In this case, the fitted k and b values ​​are reliable and can be used as benchmark parameters for subsequent data conversion. Conversely, if R² decreases significantly, it indicates that the high-gain sub-amplifier 21 has entered saturation. At this time, the control unit will trigger a range switching action and discard the current distorted data points. Alternatively, data close to the saturation value can be removed, and then the linear relationship can be re-fitted and the goodness-of-fit verification can be performed.

[0064] It should be noted that in this embodiment, the saturation of the high-gain sub-amplifier 21 and the low-gain sub-amplifier 31 is determined based on a preset threshold. The preset threshold is generally 85% of the output range of the analog-to-digital converter circuit. For example, if the range corresponding to the 16-bit ADC is 65535, then the preset threshold is set to... Of course, other types of preset values ​​are also acceptable, or they can be set to variable values ​​based on the influence of certain parameters.

[0065] If the second detection result is less than or equal to the preset threshold, it is determined that the high-gain sub-amplifier 21 has not entered the saturation state. The system prioritizes using the second detection result output by the high-gain sub-amplifier 21 as the valid detection result output to obtain the optimal signal-to-noise ratio and detection resolution. If the second detection result is greater than the preset threshold, it is determined that the high-gain sub-amplifier 21 has entered the saturation state. The valid data source is switched from the high-gain sub-amplifier 21 to the low-gain sub-amplifier 31. The first detection result output by the low-gain sub-amplifier 31 under the current signal strength is converted into an equivalent detection result according to the fitted reference parameters k and b. This equivalent detection result replaces the output data of the saturated high-gain sub-amplifier 21 and is used as the valid detection result for continued output.

[0066] In this way, the amplification curves can achieve a smooth and seamless transition before and after the switching point, without any steps, breaks, or abrupt changes in slope, thus ensuring the continuity and reliability of the amplification curves across the entire dynamic range.

[0067] The technical effects of the present invention are verified below with specific experimental data.

[0068] Table 1 shows the two sets of ADC detection results from the two sub-amplifiers when the gain ratio is 3. I excitation (mA) 15 20 25 30 35 40 45 50 55 60 65 70 75 80 ADC value (31) 351 986 1555 2154 2772 3403 4044 4706 5398 6098 6822 7489 8272 9037 ADC value (21) 4449 13191 21486 30346 38999 47793 56951 66456 76321 86299 96620 106128 117290 128195 In Table 1, I-excitation represents the detection current value converted by photosensitive unit 10, and the ADC value is the digital detection result value output by the two sub-amplifiers in the second stage. 31 corresponds to the ADC output value of the low-gain sub-amplifier 31, and 21 corresponds to the ADC output value of the high-gain sub-amplifier 21. Linear regression fitting was performed on these two sets of data in the overlap region (approximately 15-45mA for I-excitation in this table), as follows... Figure 5 As shown, the x-axis represents the ADC output value of the low-gain sub-amplifier 31, and the y-axis represents the ADC output value of the high-gain sub-amplifier 21. The actual gain ratio K = 14.255 and the baseline bias b = -627.71 are calculated. At this time, the goodness of fit R² = 0.999997, indicating that the two sets of data have high linear consistency in the overlapping area.

[0069] During detection, when the fluorescence signal is weak, the control unit prioritizes the data from the high-gain sub-amplifier 21 (the second detection result) as the effective detection result output to ensure the resolution of weak signals. When the high-gain channel is saturated, the control unit switches to the data from the low-gain sub-amplifier 31 and calculates the equivalent detection result of the high-gain sub-amplifier 21 according to the fitted K and b values. The equivalent detection result replaces the saturated high-gain data for continued output, thereby expanding the original detection circuit's maximum detection result of 65535 to about 130,000, achieving a detection upper limit expansion of more than 2 times. Figure 6 To form a complete amplification curve corresponding to the detection results of the two sub-amplifiers in Table 1 after switching and conversion, the horizontal axis is the excitation current and the vertical axis is the ADC data output by the high-gain sub-amplifier 21. It can be seen from the figure that the curves before and after switching are smooth and without breaks in the full range, with high linearity (R² is approximately 0.9983). Therefore, a wider range of amplification detection can be achieved while ensuring the resolution of the amplification curve.

[0070] Table 2. Two sets of ADC detection results from the outputs of the two sub-amplifiers under other gain ratios. I excitation (mA) 0.02 0.05 0.1 0.2 0.3 0.5 0.7 1 1.2 1.5 2 5 10 15 20 ADC value (31) 235 469 762 1243 1906 2652 3379 4266 5429 13584 25752 36286 45504 ADC value (21) 220 991 2626 6058 10074 17290 26149 36802 46982 59487 75875 190690 362003 510311 640091 Table 2 shows two sets of different ADC detection results output by the two sub-amplifiers under other gain ratios. The same overlap region linear regression calibration method as in Table 1 is used to calibrate the detection results of the high-gain sub-amplifier 21. The control unit performs data fitting between the excitation value of 0.1 and 1.5, and completes the smooth switching and conversion of the data source based on the fitting parameters when the high-gain sub-amplifier is saturated. Figure 7 The diagram illustrates the complete amplification curve obtained after switching and conversion. As can be seen from the figure, this method can expand the highest ADC value to about 640,000, which is nearly 10 times the maximum value of the existing detection circuit.

[0071] Figure 8 The diagram illustrates the possibility of expanding the detection results to a wider range. The highest ADC value in the figure can be expanded to nearly 900,000, achieving a maximum value expansion of nearly 15 times. This expansion of the detection range still maintains good linearity of the standard detection curve, indicating that the overlapping area linear regression calibration method of the present invention can effectively guarantee switching accuracy and curve continuity under different gain configurations.

[0072] Figures 9-12 Using positive samples from influenza A and influenza B, amplification and validation were performed under normal probe concentration, 1 / 2 probe concentration, 1 / 4 probe concentration, and 1 / 8 probe concentration conditions, respectively.

[0073] Figure 9This is a comparison chart of amplification curves of the prior art and the present invention under normal probe concentrations provided in the embodiments of the present invention; the left side is the amplification curve obtained by the prior art detection, and the right side is the amplification curve obtained by the detection circuit of the present invention; as shown in the figure, under normal probe concentration conditions, both detection schemes can obtain morphologically complete amplification curves of influenza A and influenza B, and the amplification curves of the two targets are clearly separated, both of which can meet the interpretation requirements of clinical detection.

[0074] Figure 10 This is a comparison chart of the amplification curves of the prior art and the present invention at 1 / 2 probe concentration provided by the embodiments of the present invention; the left side is the amplification curve obtained by the prior art detection, and the right side is the amplification curve obtained by the detection circuit of the present invention; as shown in the figure, under the condition of halving the probe concentration, the amplification curves obtained by the two detection schemes can still distinguish the two targets, but the amplification curve of the detection circuit of the present invention has a lower starting point, and the curve smoothness and signal-to-noise ratio are significantly better than the prior art.

[0075] Figure 11 This is a comparison chart of amplification curves of the prior art and the present invention at a 1 / 4 probe concentration provided by the embodiments of the present invention; the left side is the amplification curve obtained by the prior art detection, and the right side is the amplification curve obtained by the detection circuit of the present invention; as shown in the figure, under the condition that the probe concentration is further diluted to 1 / 4, the amplification curve of the prior art shows a significant rise in the initial segment and a severe deterioration in the signal-to-noise ratio, and the distinction between the amplification curves of flow A and flow B becomes worse; while the detection circuit of the present invention, due to its wider dynamic range and higher resolution, can still clearly distinguish the amplification curves of the two targets, showing a significantly better low-concentration detection capability than the prior art.

[0076] Figure 12 This is a comparison chart of amplification curves of the prior art and the present invention at a 1 / 8 probe concentration provided in the embodiments of the present invention; the left side is the amplification curve obtained by the prior art detection, and the right side is the amplification curve obtained by the detection circuit of the present invention; as shown in the figure, under the extreme condition of the probe concentration being diluted to 1 / 8, the amplification curve of the prior art is severely distorted and the baseline drifts significantly, making it difficult to determine as a valid amplification curve; while the detection circuit of the present invention, with its fine resolution brought by tens of times the dynamic range expansion, can still capture weak fluorescence amplification changes, and the amplification curve shape is complete and usable.

[0077] The above Figures 9 to 12 Comparative experiments show that this invention, through dual-gain parallel amplification combined with self-calibration of the overlapping region, can perform more precise quantification of fluorescence signals under the same amplification amplitude, effectively improving the detection sensitivity of weak positive samples.

[0078] Figure 13This is a comparison of amplification curves between the prior art and the present invention under high-concentration probe reagent conditions provided in this embodiment of the invention. The left side shows the amplification curve obtained by the prior art, and the right side shows the amplification curve obtained by the detection circuit of the present invention. Taking a positive H1N1 sample as an example, when a higher-than-normal detection amount of probe reagent is added, the amplification curve increases significantly. As shown in the figure, the amplification curve of the prior art deviates from zero at the starting point, and the signal reaches the upper limit of the analog-to-digital conversion circuit in the later stage of amplification, resulting in obvious truncation saturation. The top of the curve is flattened, failing to reflect the true fluorescence intensity of the sample, which can easily lead to misjudgment of the quantitative results by the operator. In contrast, the detection circuit of the present invention directly subtracts the baseline at the analog circuit level through a reference signal processor, making the starting point of the amplification curve zero. At the same time, through dual-gain parallel amplification and linear regression calibration in the overlap region, the detection upper limit is extended to several times or even tens of times, making the entire amplification curve continuous, complete, and without truncation across the entire range. The above comparison shows that even in the scenario of detecting strong positive samples where traditional detection circuits cannot effectively distinguish due to signal oversaturation, the present invention can still obtain a complete amplification curve by combining the detection results of the two sub-amplifiers, significantly improving the quantitative accuracy of strong positive samples.

[0079] In summary, this application provides a baseline signal to each sub-amplifier through a reference signal processor, directly reducing baseline interference at the analog circuit level, ensuring that the detection results of the two sub-amplifiers are always output under the same reference standard (near zero). Building upon this, the invention further achieves dual-gain parallel amplification through parallel low-gain and high-gain sub-amplifiers. In the initial stage of the reaction when the fluorescence signal is extremely weak, the control unit prioritizes the output data of the high-gain sub-amplifier 21, utilizing its high sensitivity to achieve sufficient amplification and high-resolution acquisition of the weak signal, effectively suppressing noise interference and ensuring accurate capture of the initial fluorescence value. As the amplification cycle progresses and the fluorescence intensity increases sharply, when the output of the high-gain channel gradually approaches the upper limit of the analog-to-digital converter circuit's range, the control unit dynamically and smoothly switches to the data of the low-gain sub-amplifier 31 based on the actual gain ratio k and baseline bias b fitted by linear regression of the overlap region, and performs equivalent conversion according to calibration parameters, extending the detection upper limit to several times or even tens of times. The synergistic effect of baseline subtraction and dual-gain self-calibration enables the amplification curve to start at zero and remain continuous without truncation across the entire range. This avoids missed detection or distortion of weak signals in the early stages and solves the problem of premature saturation and truncation of strong signals in the later stages, thus achieving complete and continuous quantitative detection of fluorescence signals across the entire dynamic range.

[0080] An embodiment of an amplification detection method This embodiment provides an amplification detection method, which is applied to an amplification detection circuit described in the foregoing embodiment. Specifically, it includes the following steps: Baseline signal acquisition steps: The control unit performs baseline detection on an empty reaction vessel or a reaction vessel containing a preset amount of pure water, and the acquired detection results are stored as a baseline signal in the reference signal processor; Steps for acquiring fluorescence signals: When performing fluorescence amplification detection, the driving light source emits excitation light into the reaction vessel to be detected, and the photosensitive unit converts the fluorescence signal generated in the reaction vessel to be detected into an electrical signal; Baseline subtraction and signal amplification steps: The first-stage amplifier amplifies the electrical signal to obtain a preliminary amplified signal; the input terminals of the high-gain sub-amplifier and the low-gain sub-amplifier in the second-stage amplifier simultaneously receive the preliminary amplified signal and the baseline signal output by the reference signal processor. The low-gain sub-amplifier performs a subtraction operation on the preliminary amplified signal and the baseline signal and then amplifies and outputs the first detection result. The high-gain sub-amplifier performs a subtraction operation on the preliminary amplified signal and the baseline signal and then amplifies and outputs the second detection result. Linear regression steps: In the overlapping region where neither the first nor the second detection result is saturated, multiple sets of first and second detection results corresponding to different signal intensities are continuously collected, and linear regression fitting is performed to obtain the linear regression equation; The detection result output steps are as follows: If the high-gain sub-amplifier has not entered saturation, the second detection result corresponding to the current signal strength is used as the valid detection result output; if the high-gain sub-amplifier has entered saturation, the equivalent detection result of the high-gain sub-amplifier under the current signal strength is obtained according to the linear regression equation and the first detection result corresponding to the current signal strength, and the equivalent detection result is used as the valid detection result output.

[0081] It should be noted that in the linear regression step, multiple sets of first and second detection results collected consecutively within the overlapping area are subjected to linear regression fitting, and the goodness of fit is also obtained; the goodness of fit is used to characterize the degree of linear consistency between the first and second detection results. In this embodiment, the expression for the linear regression equation is:

[0082] Where x represents the first detection result of the low-gain sub-amplifier, and y represents the second detection result of the high-gain sub-amplifier. is the actual gain ratio, and b is the composite baseline bias.

[0083] An embodiment of an amplification detection device: This embodiment provides an amplification detection device, which includes a light source end, a detection end, and the amplification detection circuit described above. The light source end includes an LED light source, and the detection end includes photosensitive units corresponding to the number of LED light sources. The reference signal processor in the amplification detection circuit is simultaneously connected to the inverting input of the second-stage amplifiers corresponding to multiple detection channels, and is used to provide the corresponding baseline signals for each of the multiple detection channels. The photosensitive unit in each detection channel is sequentially connected to a first-stage amplifier and a second-stage amplifier. Each second-stage amplifier includes a low-gain sub-amplifier and a high-gain sub-amplifier connected in parallel, which are used to simultaneously amplify the same initial amplified signal by different factors.

[0084] This device also includes a control unit, which is used to centrally drive and control the various functional modules. The control system is described below with reference to the accompanying drawings.

[0085] like Figure 14 The diagram shows the connection between the control unit STM series chip (hereinafter referred to as the control chip) and each functional module provided in this embodiment of the invention. In this embodiment, the control chip can also be an existing mature model of MCU, CPU or GPU, etc. The specific model is not limited. The control chip realizes electrical communication connection with each functional module through its multiple interactive pins.

[0086] The left-side pins of the control chip are connected to an integrated light source driver (LTCXXX, see details). Figure 16 The integrated light source driver can output 5 drive signals to drive the emission mode, duration and other parameters of 5 LED light sources (LED1 to LED5). In addition, the left pin is also equipped with an independent light source driver (ADXXX), which is connected to the control chip to drive the 6th LED light source (LED6). Through the above combination, the control of 6 detection channels corresponding to up to 6 LED light sources can be realized. For fewer detection channels with 4 LED light sources, only the integrated light source driver needs to be used for unsaturated driving.

[0087] The left pin of the control chip is also connected to an optical adapter board, which is used to transmit control signals to the motion drive mechanism to drive the optical system to move laterally and longitudinally in the horizontal plane. The optical system here is the detection head in this embodiment.

[0088] To ensure that the control chip can efficiently and accurately control the amplification equipment, which is mainly composed of a thermal cycling module, a temperature control and acquisition circuit is also connected to the left pin of the control chip. Temperature acquisition is achieved through temperature sensors, including thermocouples, resistance temperature detectors (RTDs), or thermistors. The temperature sensors are placed inside the temperature block or attached to the wall of the amplification container 100 to obtain real-time information on the heat transferred from the heat source and the temperature rise of the reaction solution, thereby controlling the alternating target temperature or constant target temperature required for amplification detection.

[0089] The left pin of the control chip is also connected to a memory chip, which is used to store the control program, the baseline signal values ​​of each detection channel, and the raw data and amplification curve results generated during the amplification detection process.

[0090] The right pin of the control chip is connected to a PD detection circuit (i.e., the amplification detection circuit of this application). The detection circuit includes multiple photosensitive units PD1 to PD12. The photosensitive units can be photodiodes, photomultiplier tubes or avalanche diodes, etc. Each photosensitive unit corresponds to a detection channel. The fluorescence signal acquired by the photosensitive unit is amplified and self-calibrated by the PD detection circuit and then transmitted back to the control chip.

[0091] The right pin of the control chip is also connected to a photoelectric sensor, such as a photoelectric switch. The photoelectric sensor works in conjunction with the sensing block that moves with the optical system to sense the reference position or limit position, thereby providing a position reference for the motion drive mechanism, ensuring that the detection head can accurately align with each detection hole and avoid exceeding the stroke range.

[0092] like Figure 15 The diagram shown is a connection block diagram of a CAN bus-based control system provided in an embodiment of the present invention. The main control board for user interaction is connected to the motor drive board via a CAN interface. Figure 4 The optical adapter board is integrated into the same physical board, and the two form a single module in terms of external structure. Figure 15 The motor drive board in the middle is used to drive the motor to perform mechanical motion. Unlike the "motion drive mechanism" mentioned above, the "motion drive mechanism" further integrates the negative feedback control function of various sensors on the basis of the motor drive board. Through the negative feedback control function, the detection head can achieve precise positioning and displacement in the horizontal plane.

[0093] The motor drive board is further connected to a high-performance optical board via a CAN interface. The high-performance optical board integrates... Figure 14The control chip and other peripheral circuits within the system amplify and self-calibrate the fluorescence signal, and can receive external driving power to ensure normal operation. The output of the high-performance optical board is connected to the excitation lamp board, which can drive different numbers of LEDs to emit excitation light for detection. In this embodiment, the excitation lamp board includes an integrated light source driver that can output 5 driving signals and an independent light source driver that outputs 1 driving signal. This control system is connected via CAN communication, ensuring efficient and reliable communication between the functional boards, making the transmission of control commands and detection information more efficient.

[0094] like Figure 16 The diagram shown is an integrated light source driver design provided in an embodiment of the present invention. The integrated light source driver includes five light source signal output terminals, OUT0 to OUT4, which can synchronously drive up to five LED light sources. For devices that require more detection channels, it is only necessary to add a separate pin to the control chip to connect to an additional light source driver, thereby achieving a strong compatibility design with different numbers of detection channels. The specific driving working principle of the integrated light source driver is common knowledge and will not be described in detail here.

[0095] Furthermore, in this application, the LED light source and the photosensitive unit are integrated into a detection head 3; the detection head 3 also includes an emitting lens group, a dichroic mirror group, and a receiving lens group; the LED light source is used to emit excitation light, and the emitting lens group is used to filter and converge the excitation light; the dichroic mirror group is arranged at an angle to allow the excitation light emitted from the emitting lens group to pass through and be guided to the amplification container 100, the amplification container 100 is used to contain the reaction liquid and provide a reaction space for fluorescence detection; the fluorescence in the amplification container 100 propagates upward to the dichroic mirror group, is reflected by the dichroic mirror group to the corresponding receiving lens group, is filtered and converged by the receiving lens group, and is received by the corresponding photosensitive unit and converted into an electrical signal; the detection head 3 can be driven by a motion drive mechanism to align the different detection channels in the detection head 3 sequentially with the detection holes on the hot cover, thereby performing multiple wavelength fluorescence detection on the reaction cup corresponding to each detection hole.

[0096] The following is combined Figures 17 to 19 Detailed explanation: like Figure 17The diagram shows the overall structure of the amplification detection device provided in this embodiment of the invention. The LED light source and photosensitive unit are integrated into a detection head 3. The detection head 3 is positioned on the upper end of the hot cover and maintains a certain preset distance from the upper surface of the hot cover. The hot cover has multiple detection holes, such as the detection hole 301 shown in the figure, which can be used to allow excitation light to pass through and enter the reaction cup, and allow the fluorescence signal generated in the reaction cup to pass through and be received by the detection head 3. The detection head 3 can be driven by a motion drive mechanism to move the detection head 3 in a preset plane above the hot cover, so as to achieve comprehensive detection of all detection holes within a specific range. The motion drive mechanism includes a transverse drive mechanism 42 and a longitudinal drive mechanism 41. During detection, the detection head 3 can be driven in a zigzag pattern.

[0097] In this embodiment, a heating block (not shown in the figure) is disposed at the lower end of the heating cap, capable of performing thermal cycling heating on the reaction cups. The heating block can output alternating target temperatures or constant target temperatures to perform temperature-variable amplification or isothermal amplification on the reaction solution inside the reaction cups. Multiple reaction cups are disposed between the heating block and the heating cap, arranged in an array, and each cup contains a reaction solution (including system reagents, probe reagents, and sample extraction solutions, etc.). To ensure better light focusing, a lens can be further disposed inside the detection hole on the heating cap.

[0098] Specifically, such as Figure 18 The diagram shows the integrated structure of the internal optical components of the detection head. The detection head 3 can be configured as a 6-channel detector. LED1, LED2, and LED6 are shown as examples in the diagram. Each LED light source can excite excitation light of a different wavelength. The six LED light sources are arranged within a downwardly raised triangular light source configuration section. The light source configuration section contains six emission well units, which hold the six LED light sources within the emission well units. Furthermore, each LED light source is also equipped with a corresponding emission lens group, exemplarily labeled F01, F02, and F06 in the diagram, used for filtering and focusing the excitation light. A receiving configuration section is provided in conjunction with the light source configuration section. The receiving configuration section includes a triangular groove, allowing the triangular protrusion of the light source configuration section to fit snugly into the triangular groove of the receiving configuration section. The lower part of the transmitting lens group is equipped with multiple dichroic mirror groups arranged at an angle, which are exemplarily labeled as E01 and E06 in the figure. The receiving lens groups are respectively arranged on both sides of the dichroic mirror groups, which are exemplarily labeled as R01 and R06 in the figure. Each receiving lens is equipped with a photosensitive unit, which is exemplarily labeled as PD1, PD2, and PD6.

[0099] It should be noted that, Figure 18 This is a schematic diagram of the structural principle and does not fully show all the components of all 6 detection channels. Those skilled in the art should understand that the other detection channels, such as LED3~LED5, F03~F05, E02~E05, PD3~PD5, and R02~R05, have the same configuration.

[0100] like Figure 19 The diagram shown is a schematic of the multi-channel detection optical path provided by the present invention. The detection head 3 can be driven by a motion drive mechanism to align a certain detection channel within it with the detection hole on the hot cover, so as to perform fluorescence detection in that channel. The detection channel where LED1 is located will be used as an example for explanation: The LED1 in the light source is driven by the integrated light source driver to emit excitation light of a specific wavelength. After the excitation light is filtered out by the corresponding emitting lens F01 and focused, it continues to propagate downwards until it reaches the tilted dichroic mirror E01. Due to the optical characteristics of the dichroic mirror, the excitation light is completely transmitted and continues to be emitted into the amplification container 100 through the detection hole on the hot cover. The probe fluorescent groups in the reaction solution in the amplification container 100 are excited, emit fluorescence and propagate upwards. After passing through the detection hole, the fluorescence reaches the dichroic mirror E01 and is completely reflected by the dichroic mirror E01. After being deflected by 90°, it is guided to the corresponding receiving lens R01. After being filtered and focused by the receiving lens R01, it is received by the corresponding photosensitive unit PD1 and converted into an electrical signal. The electrical signal is then transmitted to the amplification detection circuit of this application to complete the amplification detection.

[0101] After completing the detection of the detection channel where LED1 is located, the motion drive mechanism drives the detection head 3 to move and perform the detection of the detection channel where LED2 is located. By repeating the above process, multiple wavelengths of fluorescence detection can be performed on the same amplification container 100 in sequence, thereby obtaining the detection results of 6 detection channels.

[0102] It should be noted that, Figure 19 This is a schematic diagram, showing only some components of the two detection channels LED1 and LED6. It does not show all components of all detection channels. Those skilled in the art should understand that the components of the other detection channels, such as LED2-LED5, F02-F05, E02-E05, R02-R05, and PD2-PD5, have the same configuration and working principle, and will not be described in detail here.

[0103] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations are intended to cover non-exclusive inclusion, such that an article or apparatus comprising a list of elements includes not only those elements but also other elements not expressly listed. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the article or apparatus that includes said element.

[0104] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. An amplification detection circuit, characterized in that, It includes a photosensitive unit for acquiring fluorescence signals, a first-stage amplifier, a second-stage amplifier, a reference signal processor, and a control unit. The second-stage amplifier includes a low-gain sub-amplifier and a high-gain sub-amplifier connected in parallel. The input terminal of the first-stage amplifier is electrically connected to the output terminal of the photosensitive unit, and the output terminal of the first-stage amplifier is electrically connected to the input terminal of the second-stage amplifier; the input terminal of the second-stage amplifier is also connected to the output terminal of the reference signal processor. The reference signal processor is used to output the baseline signal corresponding to the current detection channel to the second-stage amplifier, and the baseline signal is simultaneously provided to the low-gain sub-amplifier and the high-gain sub-amplifier. The first-stage amplifier is used to initially amplify the electrical signal output by the photosensitive unit to obtain a preliminary amplified signal; The low-gain sub-amplifier is used to perform a subtraction operation between the preliminary amplified signal and the baseline signal, and then amplify and output a first detection result; the high-gain sub-amplifier is used to perform a subtraction operation between the preliminary amplified signal and the baseline signal, and then amplify and output a second detection result. The control unit is used to continuously acquire multiple sets of first and second detection results corresponding to different signal intensities in the overlapping area where neither the first nor the second detection result is saturated, and to perform linear regression fitting on the first and second detection results to obtain a linear regression equation; If the high-gain sub-amplifier does not enter saturation, the control unit outputs the second detection result corresponding to the current signal strength as the valid detection result; if the high-gain sub-amplifier enters saturation, the equivalent detection result of the high-gain sub-amplifier under the current signal strength is obtained according to the linear regression equation and the first detection result corresponding to the current signal strength, and the control unit outputs the equivalent detection result as the valid detection result.

2. The amplification detection circuit according to claim 1, characterized in that, The control unit performs linear regression fitting on multiple sets of first and second detection results continuously collected within the overlapping area, and also obtains the goodness of fit; the goodness of fit is used to characterize the degree of linear consistency between the first and second detection results.

3. The amplification detection circuit according to claim 1, characterized in that, The baseline signal is obtained by performing benchmark detection on an empty reaction vessel or a reaction vessel containing a preset amount of pure water.

4. The amplification detection circuit according to any one of claims 1 to 3, characterized in that, The output of the reference signal processor can be connected simultaneously to the inverting input of the second-stage amplifier corresponding to multiple detection channels, so as to provide the corresponding baseline signal for each of the multiple detection channels; one detection channel is used to independently detect a fluorescence signal of one wavelength.

5. The amplification detection circuit according to claim 1, characterized in that, The anode of the photosensitive unit is connected to the inverting input of the first-stage amplifier, and the cathode of the photosensitive unit is connected to the non-inverting input of the first-stage amplifier and ground, respectively. The inverting inputs of the low-gain sub-amplifier and the high-gain sub-amplifier are connected to the output of the first-stage amplifier and the output of the reference signal processor, respectively, and the non-inverting inputs of the low-gain sub-amplifier and the high-gain sub-amplifier are grounded.

6. An amplification detection method, characterized in that, The amplification detection circuit applied to any one of claims 1 to 5 comprises the following steps: Baseline signal acquisition steps: The control unit performs a baseline detection on an empty reaction vessel or a reaction vessel containing a preset amount of pure water, and the acquired detection result is stored as a baseline signal in the reference signal processor; Steps for acquiring fluorescence signals: When performing fluorescence amplification detection, the driving light source emits excitation light into the reaction vessel to be detected, and the photosensitive unit converts the fluorescence signal generated in the reaction vessel to be detected into an electrical signal; Baseline subtraction and signal amplification steps: The first-stage amplifier amplifies the electrical signal to obtain a preliminary amplified signal; the input terminals of the high-gain sub-amplifier and the low-gain sub-amplifier in the second-stage amplifier simultaneously receive the preliminary amplified signal and the baseline signal output by the reference signal processor. The low-gain sub-amplifier performs a subtraction operation on the preliminary amplified signal and the baseline signal and then amplifies and outputs the first detection result. The high-gain sub-amplifier performs a subtraction operation on the preliminary amplified signal and the baseline signal and then amplifies and outputs the second detection result. Linear regression steps: In the overlapping region where neither the first nor the second detection result is saturated, multiple sets of first and second detection results corresponding to different signal intensities are continuously collected, and linear regression fitting is performed to obtain the linear regression equation; Detection result output steps: If the high-gain sub-amplifier has not entered saturation, the second detection result corresponding to the current signal strength is output as the valid detection result; if the high-gain sub-amplifier has entered saturation, the equivalent detection result corresponding to the high-gain sub-amplifier under the current signal strength is obtained according to the linear regression equation and the first detection result corresponding to the current signal strength, and the equivalent detection result is output as the valid detection result.

7. The amplification detection method according to claim 6, characterized in that, In the linear regression step, multiple sets of first and second detection results continuously collected within the overlapping area are subjected to linear regression fitting to obtain the goodness of fit; the goodness of fit is used to characterize the degree of linear consistency between the first and second detection results.

8. The amplification detection method according to claim 6, characterized in that, The expression for the linear regression equation is as follows: Where x represents the first detection result of the low-gain sub-amplifier, and y represents the second detection result of the high-gain sub-amplifier. is the actual gain ratio, and b is the composite baseline bias.

9. An amplification detection device, characterized in that, The device includes a light source, a detection end, and an amplification detection circuit as described in any one of claims 1 to 5; the light source includes an LED light source, and the detection end includes photosensitive units corresponding to the number of LED light sources; the reference signal processor in the amplification detection circuit is simultaneously connected to the inverting input of the second-stage amplifiers corresponding to multiple detection channels, for providing each detection channel with its corresponding baseline signal; the photosensitive unit in each detection channel is sequentially connected to the first-stage amplifier and the second-stage amplifier, and each second-stage amplifier includes a low-gain sub-amplifier and a high-gain sub-amplifier connected in parallel, for simultaneously amplifying the same initial amplified signal by different factors.

10. The amplification detection device according to claim 9, characterized in that, The LED light source and the photosensitive unit are integrated into a detection head; the detection head also includes an emitting lens group, a dichroic mirror group, and a receiving lens group. The LED light source is used to emit excitation light, and the emitting lens group is used to filter and focus the excitation light; The dichroic mirror group is arranged at an angle to allow the excitation light emitted from the emitting mirror group to pass through and be guided to the reaction cup, which is used to contain the reaction liquid and provide a reaction space for fluorescence detection. The fluorescence inside the reaction cup propagates upward to the dichroic mirror group, is reflected by the dichroic mirror group to the corresponding receiving lens group, and after being filtered and focused by the receiving lens group, is received by the corresponding photosensitive unit and converted into an electrical signal. The detection head can be driven by a motion drive mechanism so that different detection channels inside the detection head are sequentially aligned with each detection hole, thereby performing multiple wavelength fluorescence detections on the reaction cup corresponding to each detection hole.

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