Dual-path absorbance detection system and calibration method for long-term culture monitoring

Abstract

This invention discloses a dual-path absorbance detection system and calibration method for long-term culture monitoring. The system includes a main detection optical path and a reference monitoring optical path, comprising a shared light source, collimation assembly, filter assembly, and spectrometer assembly. The main detection optical path sequentially includes a first focusing assembly, a sample cell, a second focusing assembly, and a main detector. The reference monitoring optical path sequentially includes a third focusing assembly and a reference detector. The main detector acquires the transmitted light signal passing through the sample cell, while the reference detector simultaneously acquires the reference light signal output from the light source. A polynomial fitting model of the transfer coefficient and the ratio of the main and reference light intensities is established using liquid standard substances, and the transfer coefficient is calculated in real time to compensate for light source attenuation, fluctuations, and system nonlinearity errors. This invention solves the problems of existing single-path detection systems being unable to correct for long-term light source attenuation and short-term fluctuations, long-term detection baseline drift, and poor data consistency, achieving high-precision and high-stability long-term continuous online monitoring.

Description

Technical Field

[0001] This invention relates to the fields of microbial detection and fermentation process monitoring technology, specifically to a dual-path absorbance detection system and calibration method for long-term culture monitoring. Background Technology

[0002] In long-term online monitoring scenarios such as microbial culture, fermentation process monitoring, and cell growth curve determination, absorbance detection is a core method for determining changes in sample concentration and the growth status of microorganisms. Existing single-source detection devices, dual-source detection devices, and microbial growth curve analyzers mostly employ a single-optical-path structure. A single light source emits detection light, which passes through the sample and is then collected by a photodetector. The absorbance and concentration are calculated based on the signal strength. While these devices can achieve basic online detection, they generally suffer from the following problems: 1. Failure to consider long-term light source attenuation can easily lead to systematic biases in test results: During continuous long-term operation, the light intensity of the light source will slowly decrease due to factors such as device aging, temperature rise, and drive circuit drift. Current technology lacks an independent light source monitoring channel, making it impossible to distinguish whether the signal decline is due to increased sample absorption or a weakening of the light source's output. This easily leads to misinterpreting light source attenuation as an increase in bacterial concentration, resulting in distorted test results and continuous baseline drift.

[0003] 2. Inability to monitor short-term light source fluctuations, resulting in large real-time measurement errors: During normal operation, the light source may experience momentary or short-term light intensity fluctuations due to power supply fluctuations, ambient temperature changes, uneven heat dissipation, etc. Single-path systems lack a synchronous reference channel, and these fluctuations are directly superimposed on the sample detection signal, causing data jumps and poor repeatability, failing to meet the requirements for high-precision and high-stability monitoring.

[0004] 3. Lack of an independent reference optical path, making real-time calibration impossible without interfering with the main detection: Existing technologies only set up one main detection optical path, without establishing an independent, parallel, and synchronous monitoring path at the light source output end, making it impossible to sample the actual output state of the light source in real time. When the light source experiences attenuation, fluctuations, or abnormalities, the system cannot automatically identify and compensate in real time, relying solely on periodic manual calibration, which affects both the continuity of detection and reduces long-term reliability.

[0005] 4. Poor consistency of long-term detection data and incomparability of multiple batches of experiments: Due to the lack of correction for light source attenuation and short-term fluctuations, system baseline drift and signal distortion will accumulate over time, resulting in large deviations in the detection results of the same sample at different times and the inability to compare experimental data from different batches, which seriously affects the accuracy and reliability of microbial culture monitoring.

[0006] In summary, existing absorbance detection systems used for culture monitoring generally suffer from drawbacks such as inability to correct long-term light source attenuation, inability to monitor short-term fluctuations, lack of real-time online compensation, and insufficient long-term stability. These shortcomings make it difficult to meet the requirements of long-term, high-precision, and continuous online culture monitoring. There is an urgent need for a dual-optical-path detection solution that can simultaneously address the issues of light source attenuation and fluctuations and has built-in real-time correction. Summary of the Invention

[0007] This invention addresses the shortcomings of existing technologies, such as the inability to solve the problems of long-term light source attenuation and short-term fluctuations, the lack of an independent monitoring and correction mechanism, and large long-term detection errors. It proposes a dual-path absorbance detection system for long-term culture monitoring, in which the main detection optical path and the reference monitoring optical path run in parallel. The system achieves real-time monitoring of the light source status and automatic signal correction through spectral sampling.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A dual-path absorbance detection system for long-term culture monitoring includes a main detection optical path and a reference monitoring optical path, comprising a shared light source, a collimation component, a filter component, and a spectrometer component.

[0009] Main detection optical path: A light source, collimation component, filter component, beam splitter component, first focusing component, sample cell, second focusing component and main detector are arranged sequentially along the main optical axis for transmission detection of the sample to be tested; Reference monitoring optical path: It shares the light source, collimation component, filter component and beam splitter component with the main detection optical path. After reflection by the beam splitter component, a third focusing component and a reference detector are set in sequence to monitor the output status of the light source in real time.

[0010] The light source is an LED light source used to emit detection light. Its output wavelength is selected according to the optical characteristics of the sample to be tested, usually in the visible or near-infrared band. Collimation component: The collimation component is a first plano-convex lens, which is set on the light-emitting side of the light source. It is used to collimate and shape the light beam emitted by the light source to form a stable collimated light beam, reduce the beam divergence angle, and improve the light energy utilization rate. Filtering Components: The filtering components are bandpass filters, which are set in the collimated optical path to filter the light beam by wavelength, improve the monochromaticity of the detection beam, reduce stray light interference, and improve the anti-interference ability of the detection. Beam splitter: The beam splitter is a beam splitter set after the filter assembly at a 45° angle to the main optical axis. It is used to split the detection beam into a transmitted main detection beam and a reflected reference monitoring beam. The splitting ratio can be adjusted according to the detection requirements. Usually, the light intensity of the main detection beam is greater than that of the reference monitoring beam. First focusing component: The first focusing component is a second plano-convex lens, which is set between the beam splitter and the sample cell. Its parameters are designed to match the refractive index of the sample cell wall, the refractive index of the liquid medium and the sample cell structure, so as to focus the main detection beam onto the sample to be tested in the sample cell and compensate for the refraction effect of the beam propagating in different media. Sample cell: Used to hold the liquid sample to be tested. It is a transparent container, usually made of quartz or optical glass, and its optical path can be selected according to the testing requirements. Second focusing component: The second focusing component is composed of a third plano-convex lens and a fourth plano-convex lens, and is set between the sample cell and the main detector. It is used to focus the transmitted light beam through the sample cell onto the main detector to ensure that the main detector can accurately receive the transmitted light signal. Main detector: The main detector is a photodetector used to collect the transmitted light signal after passing through the sample and convert the light signal into an electrical signal for output. It is usually a photodiode or a photomultiplier tube. Third focusing component: The third focusing component is the fifth plano-convex lens, which is located between the beam splitter and the reference detector. It is used to focus the reference monitoring beam onto the reference detector to ensure that the reference detector can accurately collect the output light intensity signal of the light source. Reference detector: The reference detector is a photodetector used to collect the current output light intensity information of the light source in real time, convert the light signal into an electrical signal output, and its performance parameters are matched with the main detector to ensure the synchronization and accuracy of signal acquisition.

[0011] The present invention also provides a dual-path absorbance correction method for the above-mentioned system, comprising the following steps: S1. System Calibration: After the system enters a thermally stable operating state, multiple sets of liquid standard substances with different transmittances are sequentially loaded into the sample cell. The light intensity signal Si of the main detector and the light intensity signal Ri of the reference detector corresponding to each set of standard substances are collected simultaneously. Based on the true transmittance Ti of the standard substance, the transfer coefficient Ki = Si / (Ti·Ri) is calculated, and the main parameter light intensity ratio Mi = Si / Ri is calculated, forming N sets of (Mi,Ki) data pairs. A polynomial fitting is used to establish the functional relationship between the transfer coefficient K and the main parameter light intensity ratio M, K = f(M). S2. Real-time detection: The sample to be tested is loaded into the sample cell, and the light intensity signal S of the main detector and the light intensity signal R of the reference detector are collected simultaneously. The main-reference light intensity ratio M=S / R is calculated. The real-time transmission coefficient K=f(M) is obtained by substituting it into the fitting function. The true transmittance of the sample T=S / (K·R) is calculated, and the absorbance of the sample is calculated according to the absorbance formula A=−lgT.

[0012] Furthermore, the liquid standard substance is a gradient standard solution prepared with different transmittance gradients, such as standard colorimetric solutions, polystyrene microsphere standard suspensions, or blank culture media, and its transmittance covers the low, medium, and high ranges of the detection range.

[0013] Furthermore, the polynomial fitting is a second-order or higher polynomial fitting to compensate for light source attenuation, short-term fluctuations, and system nonlinear errors.

[0014] The beneficial effects of this invention are: (1) Simultaneously solving the problems of long-term attenuation and short-term fluctuation of the light source, and greatly improving the measurement accuracy: The system performs real-time, synchronous and parallel sampling of the light source output through the reference monitoring optical path, which can capture the slow attenuation caused by aging and heating of the light source, as well as the instantaneous fluctuation caused by power supply and temperature, and incorporate them into the calculation correction, completely avoiding misjudging the change of the light source as the change of the sample, and eliminating baseline drift and real-time jitter error from the root; (2) The dual optical paths are independent and parallel, do not interfere with the main detection, and are suitable for long-term continuous monitoring: The reference monitoring optical path performs spectral sampling after the light source is collimated, does not enter the sample cell, and does not affect the detection of the main optical path. It can monitor the light source status throughout the 24-hour continuous operation, which is particularly suitable for long-term scenarios such as microbial culture and fermentation monitoring, which last for several hours to several days; (3) Using liquid standard substances for calibration, which is highly matched with the sample system and makes the detection more realistic: The liquid standard solution or standard suspension consistent with the system to be tested is used for calibration, without using solid filters. The optical path medium, refractive index and scattering behavior are completely matched with the actual sample, and the calibration results are more consistent with the true sample. In actual testing scenarios, the system error is smaller; (4) Polynomial fitting calibration is adopted, abandoning the forced linear assumption, and the full-range accuracy is higher: Considering that the optical and circuit systems are not ideally linear in the full range, this invention adopts polynomial fitting instead of simple linear fitting, which can adaptively compensate for the nonlinear errors of the light source, detector, circuit and optical path, and maintain high accuracy in the low transmittance or high absorbance range, significantly improving the full-range detection consistency; (5) Simple structure, small modification, easy integration, and strong engineering practicality: On the basis of the traditional single optical path, only a beam splitter, monitoring detector and matching lens are added, and the front-end devices such as light source, collimation and filtering are shared. The structure is compact and the cost increase is small. It can be directly compatible with existing absorbance detection devices and culture equipment, with strong adaptability and easy mass production; (6) Considering the influence of medium differences, the optical path design is more scientific: Fully considering the medium differences of the main detection optical path passing through the sample cell and liquid medium and the reference monitoring optical path propagating in the air, the lens focusing and optical path matching are optimized to ensure that the monitoring optical path can truly reflect the light source state and improve the accuracy and detection authenticity of the dual optical path correspondence. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the overall structure of the dual-path absorbance detection system of the present invention; Figure 2This is an optical structure diagram in a specific embodiment of the present invention.

[0016] In the figure: 1. LED light source; 2. First plano-convex lens; 3. Bandpass filter; 4. Beam splitter; 5. Second plano-convex lens; 6. Sample cell; 7. Second focusing assembly; 71. Third plano-convex lens; 72. Fourth plano-convex lens; 8. Main detector; 9. Fifth plano-convex lens; 10. Reference detector. Detailed Implementation

[0017] The technical solution of the present invention will be further described in detail below through specific embodiments and in conjunction with the accompanying drawings: Example: Figure 1 , Figure 2 As shown, a dual-path absorbance detection system for long-term culture monitoring includes an LED light source 1, a first plano-convex lens 2, a bandpass filter 3, a beam splitter 4, a second plano-convex lens 5, a sample cell 6, a third plano-convex lens 71, a fourth plano-convex lens 72, a main detector 8, a fifth plano-convex lens 9, and a reference detector 10. The third plano-convex lens 71 and the fourth plano-convex lens 72 form the second focusing assembly 7.

[0018] The LED light source 1, the first plano-convex lens 2, the bandpass filter 3, and the beam splitter 4 are shared components for the main detection optical path and the reference monitoring optical path. The first plano-convex lens 2 is located on the light-emitting side of the LED light source 1 to achieve beam collimation. The bandpass filter 3 is located behind the first plano-convex lens 2 to filter specific wavelength beams and improve beam monochromaticity. The beam splitter 4 is located behind the bandpass filter 3 to divide the beam into a transmitted beam and a reflected beam.

[0019] The main detection optical path is arranged along the transmission optical axis of the beam splitter 4, with a second plano-convex lens 5, a sample cell 6, a third plano-convex lens 71, a fourth plano-convex lens 72, and a main detector 8 arranged sequentially. The transmitted beam is focused by the second plano-convex lens 5 and then enters the sample cell 6. After passing through the sample to be tested, it is focused by the third plano-convex lens 71 and the fourth plano-convex lens 72 to the main detector 8, thus completing the acquisition of the sample optical signal. The optical parameters of the second plano-convex lens 5, the third plano-convex lens 71, and the fourth plano-convex lens 72 are matched with the refractive index of the sample cell 6 wall and the sample to be tested to ensure accurate beam focusing.

[0020] The reference monitoring optical path is arranged with a fifth plano-convex lens 9 and a reference detector 10 in sequence along the reflective optical axis of the beam splitter 4. The reflected beam is focused by the fifth plano-convex lens 9 to the reference detector 10. This optical path does not pass through the sample cell 6, uses air as the main transmission medium, directly collects the light signal output by the light source, and monitors the light source status in real time.

[0021] This system, through the aforementioned dual-optical-path physical structure, enables simultaneous sample detection and light source monitoring, effectively avoiding interference from long-term light source attenuation and short-term fluctuations on the detection results, improving the stability and accuracy of long-term detection, and is fully adaptable to online monitoring scenarios such as microbial culture and fermentation processes.

[0022] The present invention also provides a dual-path absorbance correction method for the above-mentioned system, comprising the following steps: S1. System Calibration: After system assembly, preheat for 30 minutes to enter thermally stable operating state; prepare 5 sets of standard chromatic liquids with different transmittances: 10%, 30%, 50%, 70%, and 90%; sequentially fill each set of standard liquids into the sample cell, and after signal stabilization, synchronously acquire the light intensity signals of the main detector and reference detector, recording the data Si and Ri for each set; calculate the transfer coefficient Ki of each set of standard liquids according to the formula Ki=Si / (Ti·Ri), and calculate the main parameter light intensity ratio Mi=Si / Ri, obtaining 5 sets of (Mi,Ki) data pairs; establish the functional relationship between the transfer coefficient and the main parameter light intensity ratio using quadratic polynomial fitting: K=a⋅M 2 +b⋅M+c, where a, b, and c are fitting coefficients; S2. Real-time detection: The microbial culture medium to be tested is placed into the sample cell, and the light intensity signal S of the main detector and the light intensity signal R of the reference detector are collected simultaneously; the main-reference light intensity ratio M=S / R is calculated, and the real-time transmission coefficient K is obtained by substituting it into the fitting function; the true transmittance of the sample T=S / (K·R) is calculated, and then the absorbance A=−lgT is calculated.

[0023] In this embodiment, by real-time compensation of light source attenuation and fluctuation through the reference monitoring optical path, the system baseline drift is reduced from ±5% of the traditional single optical path to ±0.2%, and the data repeatability error is reduced from ±3% to ±0.5%. It can operate stably for more than 72 hours without significant error accumulation, meeting the high-precision requirements for long-term microbial culture monitoring.

[0024] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications are possible without departing from the technical solutions described in the claims.

Claims

1. A dual-path absorbance detection system for long-term culture monitoring, characterized in that, It includes the main detection optical path, the reference monitoring optical path, and the light source, collimation component, filter component, and beam splitter component shared by the two optical paths; The main detection optical path is arranged along the transmission optical axis of the beam splitter, consisting of a first focusing component, a sample cell, a second focusing component, and a main detector. The main detection optical path passes through the sample cell to be tested and is used for transmission detection of the sample. The reference monitoring optical path is arranged with a third focusing component and a reference detector in sequence along the reflected optical axis of the beam splitter; The reference monitoring optical path is separate from the sample cell and does not pass through the sample cell under test. It is used to monitor the output status of the light source in real time.

2. The dual-path absorbance detection system for long-term culture monitoring according to claim 1, characterized in that, The beam splitter divides the collimated and filtered light beam into a transmitted beam and a reflected beam. The transmitted beam forms the main detection beam of the main detection optical path, and the reflected beam forms the reference monitoring beam of the reference monitoring optical path. The splitting ratio satisfies the requirement that the light intensity of the main detection beam is greater than that of the reference monitoring beam.

3. The dual-path absorbance detection system for long-term culture monitoring according to claim 1, characterized in that, The lens parameters of the first and second focusing components in the main detection optical path are designed in combination with the refractive index of the sample cell wall, the refractive index of the liquid medium, and the sample cell structure to compensate for the refractive effects of the main detection beam propagating in air, sample cell wall, and liquid medium.

4. The dual-path absorbance detection system for long-term culture monitoring according to claim 1, characterized in that, The reference monitoring optical path uses air as the main transmission medium, and the lens parameters of the third focusing component are matched with the reflection optical path of the beam splitting component to ensure that the reference detector can accurately collect the output light intensity signal of the light source.

5. The dual-path absorbance detection system for long-term culture monitoring according to claim 1, characterized in that, The light source is an LED light source, the collimation component is a first plano-convex lens, the filtering component is a bandpass filter, and the beam splitting component is a beam splitter.

6. The dual-path absorbance detection system for long-term culture monitoring according to claim 1, characterized in that, The first light-concentrating component is a second plano-convex lens, the second light-concentrating component is composed of a third plano-convex lens and a fourth plano-convex lens, and the third light-concentrating component is a fifth plano-convex lens.

7. The dual-path absorbance detection system for long-term culture monitoring according to claim 1, characterized in that, Both the main detector and the reference detector are photodetectors, and are respectively located at the light-emitting ends of the main detection optical path and the reference monitoring optical path.

8. A dual-path absorbance correction method for the system according to any one of claims 1-4, characterized in that, Includes the following steps: S1. System Calibration: After the system enters a thermally stable operating state, multiple sets of liquid standard substances with different transmittances are sequentially loaded into the sample cell. The light intensity signal Si of the main detector and the light intensity signal Ri of the reference detector corresponding to each set of standard substances are collected simultaneously. Based on the true transmittance Ti of the standard substance, the transfer coefficient Ki = Si / (Ti·Ri) is calculated, and the main parameter light intensity ratio Mi = Si / Ri is calculated, forming N sets of (Mi,Ki) data pairs. A polynomial fitting is used to establish the functional relationship between the transfer coefficient K and the main parameter light intensity ratio M, K = f(M). S2. Real-time detection: The sample to be tested is loaded into the sample cell, and the light intensity signal S of the main detector and the light intensity signal R of the reference detector are collected simultaneously. The main parameter light intensity ratio M = S / R is calculated. The real-time transfer coefficient K = f(M) is obtained by substituting it into the fitting function. Calculate the true transmittance of the sample, T = S / (K·R), and calculate the absorbance of the sample according to the absorbance formula A = −lgT.

9. The dual-path absorbance correction method according to claim 8, characterized in that, The liquid standard substance is a gradient standard solution prepared from standard colorimetric liquid with different transmittance gradients, polystyrene microsphere standard suspension, or blank culture medium, and its transmittance covers the low, medium, and high ranges of the detection range.

10. The dual-path absorbance correction method according to claim 8, characterized in that, The polynomial fitting is a second-order or higher polynomial fitting to compensate for light source attenuation, short-term fluctuations, and system nonlinear errors.

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