Intelligent sensor-based microfluidic immuno-fluorescence quantitative detection method for polycyclic aromatic hydrocarbons

Abstract

The present application relates to the technical field of intelligent sensing and control equipment, in particular to a PAH microfluidic immunofluorescence quantitative detection method based on intelligent sensors, comprising the following steps: obtaining a blank propulsion reference, determining the front edge and pump step according to the inlet fluorescence rise, determining the residence period according to the channel parameters and single-step volume, correcting the central fluorescence for dark current, background and light source drift, screening the reaction window according to the growth amount, pressure stability, continuous rise and unsaturated conditions, and outputting the content or range state from the window growth amount and low-high reference relationship. In the present application, the continuous calculation link, dark current, background, drift, flow instability, front edge misjudgment, residence deviation and saturation response are simultaneously weakened through the reference correction, flow discrimination, residence constraint and window screening, the corresponding relationship between the fluorescence growth and the real immune response is closer, the low and high range boundaries are clearer, and the stability, repeatability and reliability of the content interpretation are improved.

Description

Technical Field

[0001] This invention relates to the field of intelligent sensing and control equipment technology, and in particular to a microfluidic immunofluorescence quantitative detection method for polycyclic aromatic hydrocarbons based on intelligent sensors. Background Technology

[0002] The field of intelligent sensing and control equipment technology involves equipment systems that utilize sensors, signal conditioning units, embedded processors, and human-machine interfaces to collect, convert, and output external physical or chemical quantities. Among these, the traditional microfluidic immunofluorescence quantitative detection method for polycyclic aromatic hydrocarbons (PAHs) based on intelligent sensors refers to a technical process in which sensors collect the light signal generated by the immunofluorescence reaction within a microfluidic channel, convert the light signal into an electrical signal, input it into a processor, and then a display terminal or host computer completes parameter reading, detection process triggering, and quantitative result display.

[0003] Traditional detection processes focus on optical signal acquisition within the channel and terminal reading display. Detection triggering and result output largely depend on single or overall fluorescence responses. Dark current, background, light source, flow, and temperature disturbances generated during the blank advance phase can easily contaminate the reaction signal. The arrival of the reaction liquid front, the sample residence interval, and the micropump advance state lack closed-loop constraints. Flow fluctuations and saturation responses may still participate in the calculation, resulting in a loose correspondence between fluorescence growth and the actual immune reaction process. The stability of low and high range boundary judgment is insufficient, limiting the repeatability and quantitative reliability of detection. However, existing detection methods mostly focus on the correction of the fluorescence signal itself or the judgment of result thresholds. They lack sufficient constraints on the correlation between the arrival time of the reaction liquid front, the number of micropump advance steps, the actual residence time in the detection zone, and abnormal states of light source, temperature, and pressure. This results in some unstable flow states or abnormal sampling points still participating in the reaction window calculation, thus affecting the stability of polycyclic aromatic hydrocarbon content interpretation. Summary of the Invention

[0004] To achieve the above objectives, the present invention adopts the following technical solution: a microfluidic immunofluorescence quantitative detection method for polycyclic aromatic hydrocarbons based on intelligent sensors, comprising the following steps:

[0005] Obtain the dark current reference, background correction reference, light source drift reference, flow stability reference, and temperature correction reference for the blank propagation stage. Each reference is generated by synchronously acquiring the fluorescence intensity values ​​at the center of the detection area, the fluorescence intensity values ​​at the entrance of the detection area, the intensity values ​​of the reference optical path, the channel pressure values, and the temperature values ​​within the same blank propagation stage.

[0006] Based on the background correction benchmark, the fluorescence intensity value at the entrance of the detection area is continuously compared to determine the arrival time of the reaction liquid front, and the cumulative number of micro-pump advance steps corresponding to the arrival time of the reaction liquid front is determined as the entrance benchmark step number.

[0007] Based on the arrival time of the reaction liquid front, the inlet reference number of steps, the length of the detection zone, the channel cross-sectional area, and the single-step propulsion volume, the number of steps through the detection zone is determined, and the dwell time of the sample in the detection zone is determined according to the inlet reference number of steps and the number of steps through the detection zone.

[0008] For the fluorescence intensity value of the center of the detection area during the dwell time period, dark current subtraction, background subtraction and light source drift ratio correction are performed in sequence. Based on the temperature correction benchmark, the sampling points are screened for temperature status. After removing abnormal light source sampling points and abnormal temperature sampling points, a corrected fluorescence intensity sequence is generated.

[0009] The fluorescence growth of adjacent sampling intervals is calculated based on the modified fluorescence intensity sequence, and the sampling intervals whose fluorescence growth meets the preset minimum growth boundary, whose channel pressure value falls within the flow stability reference, and which do not cross abnormal sampling points are determined as candidate reaction fragments.

[0010] For the candidate reaction fragments that continuously increase, do not reach the upper limit of saturation, and do not cross the flow instability fragment, abnormal sampling point of light source, and abnormal sampling point of temperature, determine the effective reaction window and the amount of fluorescence growth in the window;

[0011] Based on the positional relationship between the fluorescence growth in the window and the fluorescence growth in the low-concentration and high-concentration references, the polycyclic aromatic hydrocarbon content value or range status is output.

[0012] As a further aspect of the present invention, the process of obtaining the dark current reference, the background correction reference, the light source drift reference, the flow stability reference, and the temperature correction reference includes:

[0013] During the blank propulsion phase, the fluorescence intensity values ​​at the center of the detection area, the fluorescence intensity value at the entrance of the detection area, the intensity value of the reference optical path, the channel pressure value, and the temperature value are collected.

[0014] The fluorescence intensity values ​​at the center of the detection area where the sampling timestamp is in the non-propelled range of the micropump are statistically analyzed to generate the dark current reference.

[0015] The fluorescence intensity values ​​at the entrance of the detection area during the blank advancement phase are statistically analyzed to generate an entrance background reference.

[0016] The fluorescence intensity values ​​of the detection area center after subtraction from the dark current reference during the blank advancement phase are statistically analyzed to generate a center background reference.

[0017] Based on the difference between the entrance background reference and the center background reference, an entrance-center blank difference reference is generated;

[0018] The background correction benchmark includes the entrance background benchmark, the center background benchmark, and the entrance-center blank difference benchmark.

[0019] The blank fluctuation range is determined for the reference optical path intensity value, channel pressure value, and temperature value, respectively, and the light source drift reference, the flow stability reference, and the temperature correction reference are generated.

[0020] The process of screening sampling points for temperature status based on the temperature correction benchmark includes:

[0021] Obtain the blank temperature center value and temperature allowable deviation boundary in the temperature correction benchmark, and simultaneously obtain the real-time temperature value corresponding to each sampling timestamp within the dwell period; when the real-time temperature value is within the temperature allowable deviation boundary, the corresponding sampling point is determined as a temperature consistent sampling point; when the real-time temperature value exceeds the temperature allowable deviation boundary, the corresponding sampling point is marked as a temperature abnormal sampling point; the temperature abnormal sampling point is not used to generate the corrected fluorescence intensity sequence, nor is it used to determine the candidate reaction fragment and the effective reaction window.

[0022] As a further aspect of the present invention, the process of determining the arrival time of the reaction liquid front and the number of micropump propulsion steps includes: acquiring the fluorescence intensity value at the entrance of the detection zone, the sampling timestamp corresponding to the fluorescence intensity value at the entrance of the detection zone, and the cumulative number of micropump propulsion steps; subtracting the entrance background from the fluorescence intensity value at the entrance of the detection zone based on the entrance background benchmark to obtain the net fluorescence intensity value at the entrance; comparing the increasing state of the net fluorescence intensity value at the entrance within a continuous sampling period; when the number of consecutive increases reaches the preset number of front-end recognitions and the last net fluorescence intensity value at the entrance is higher than the front-end recognition threshold, determining the corresponding sampling timestamp as the arrival time of the reaction liquid front, and determining the corresponding cumulative number of micropump propulsion steps as the number of micropump propulsion steps.

[0023] As a further aspect of the present invention, the process of determining the dwell time includes: obtaining the detection zone length, channel cross-sectional area, and single-step propulsion volume; determining the detection zone volume based on the detection zone length and channel cross-sectional area; determining the number of steps through the detection zone based on the volume correspondence between the detection zone volume and the single-step propulsion volume; using the number of micro-pump propulsion steps corresponding to the arrival time of the reaction liquid front as the inlet reference number of steps; determining the corresponding number of steps at the outlet based on the inlet reference number of steps and the number of steps through the detection zone; and searching for the sampling timestamp corresponding to the number of steps at the outlet in the micro-pump drive control record to generate the dwell time.

[0024] As a further aspect of the present invention, the process of generating the modified fluorescence intensity sequence includes:

[0025] The fluorescence intensity values ​​of the detection area center arranged according to the sampling timestamp during the dwell time period are obtained, and the dark current reference is called to perform subtraction processing on the fluorescence intensity value of the detection area center corresponding to each sampling timestamp to obtain the dark current subtracted intensity value.

[0026] Based on the central background reference, the dark current subtraction intensity value is subjected to central background subtraction to obtain the background subtraction intensity value, and the background subtraction intensity value is proportionally corrected according to the intensity change relationship of the reference optical path corresponding to the light source drift reference.

[0027] After removing sampling points with abnormal temperature and abnormal light sources, the corrected fluorescence intensity sequence is generated according to the remaining sampling timestamps.

[0028] As a further aspect of the present invention, the ratio correction specifically includes: obtaining the center value of the blank reference optical path in the light source drift reference, and simultaneously obtaining the real-time reference optical path intensity value corresponding to each sampling timestamp during the dwell time period, and comparing the deviation of the real-time reference optical path intensity value relative to the center value of the blank reference optical path.

[0029] When the deviation is within the preset allowable drift range, the background subtraction intensity value is reverse-compensated according to the ratio between the real-time reference light path intensity value and the blank reference light path center value. When the deviation exceeds the preset allowable drift range, the corresponding sampling timestamp is marked as an abnormal sampling point of the light source and removed from the corrected fluorescence intensity sequence.

[0030] As a further aspect of the present invention, the process of determining the candidate reaction fragment includes: obtaining the corrected fluorescence intensity values ​​arranged in adjacent sampling timestamps in the corrected fluorescence intensity sequence, calculating the difference between the corrected fluorescence intensity value of the later sampling timestamp and the corrected fluorescence intensity value of the earlier sampling timestamp, and obtaining the fluorescence growth amount.

[0031] The channel pressure value corresponding to the adjacent sampling interval is obtained, and the channel pressure value is compared with the pressure center value and the pressure allowable fluctuation boundary corresponding to the flow stability benchmark. When the channel pressure value falls into the pressure allowable fluctuation boundary and the fluorescence growth is not less than the preset minimum growth boundary, the adjacent sampling interval is determined as the candidate reaction fragment.

[0032] As a further aspect of the present invention, the process of determining the preset minimum growth boundary specifically includes: obtaining the blank correction intensity fluctuation amount formed by subtracting dark current, background subtraction and light source drift ratio from the fluorescence intensity value of the detection area center during the blank advancement stage, and determining the blank growth upper limit of the blank correction intensity fluctuation amount.

[0033] The upper limit of blank growth and the reaction recognition margin, which have the same dimensions as the fluorescence growth amount and are determined by the sensor resolution or the standard deviation of blank noise, are superimposed to generate the preset minimum growth boundary. When the fluorescence growth amount is lower than the preset minimum growth boundary, the corresponding adjacent sampling interval is marked as a non-reaction fluctuation interval.

[0034] As a further aspect of the present invention, the process for determining the effective reaction window and the fluorescence growth amount within the window includes:

[0035] Obtain the corrected fluorescence intensity values, fluorescence growth amount, and flow stability markers within the candidate reaction fragments, arranged sequentially according to the sampling timestamps;

[0036] A preset effective growth boundary is generated based on the preset minimum growth boundary and the preset window confirmation margin, wherein the preset effective growth boundary is not less than the preset minimum growth boundary;

[0037] The sampling intervals in which the fluorescence growth is continuously greater than the preset effective growth boundary and the corrected fluorescence intensity value is lower than the saturation upper limit are selected;

[0038] When there is no flow instability marker within the sampling interval, the sampling interval is determined as the effective reaction window, and the window fluorescence growth is generated based on the difference between the corrected fluorescence intensity values ​​corresponding to the start and end points of the effective reaction window.

[0039] As a further aspect of the present invention, the process of outputting the polycyclic aromatic hydrocarbon content value or range status includes:

[0040] The window fluorescence growth amount, the low-concentration reference fluorescence growth amount, and the high-concentration reference fluorescence growth amount are obtained, and the interval position of the window fluorescence growth amount relative to the low-concentration reference fluorescence growth amount and the high-concentration reference fluorescence growth amount is compared.

[0041] When the fluorescence growth in the window is between the low-concentration reference fluorescence growth and the high-concentration reference fluorescence growth, the reference concentration mapping relationship is invoked to determine the polycyclic aromatic hydrocarbon content value; based on the monotonic direction of the reference concentration mapping relationship, it is determined whether the fluorescence growth in the window is within the effective reference interval defined by the low-concentration reference fluorescence growth and the high-concentration reference fluorescence growth; if it is outside the interval, the state below or above the range is output according to the monotonic direction.

[0042] Compared with the prior art, the advantages and positive effects of the present invention are as follows:

[0043] In this invention, dark current, background, light source drift, flow stability, and temperature reference are acquired during the blank propagation stage. The continuous rise of the inlet fluorescence after correction identifies the reaction liquid front and the micropump propagation state. The actual residence time of the sample is defined by combining the channel size and the single-step propagation volume, so that the calculation is limited to the effective reaction process. The central fluorescence is subtracted by dark current, background subtraction, and light source drift ratio correction to form comparable growth data. Candidate fragments are screened based on conditions such as pressure stability, continuous rise, unsaturation, and no instability. The window growth amount is established with the low and high reference growth amount to establish a positional relationship. Transmission disturbances, environmental drift, and saturation anomalies are eliminated. The quantitative output and range judgment are more consistent with the immune reaction itself, improving repeatability, stability, and reliability. Attached Figure Description

[0044] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0045] Figure 1 This is the main flowchart of the microfluidic immunofluorescence quantitative detection method for polycyclic aromatic hydrocarbons of the present invention;

[0046] Figure 2 This is a schematic diagram of the sampling objects and synchronous data flow in the detection area of ​​this invention;

[0047] Figure 3 This is a schematic diagram illustrating the signal change effect before and after fluorescence correction in this invention;

[0048] Figure 4 This is a schematic diagram illustrating the screening effect of candidate reaction fragments and effective reaction windows in this invention;

[0049] Figure 5 This is a schematic diagram showing the relationship between the fluorescence growth amount in the window and the reference range in this invention. Detailed Implementation

[0050] The technical solution of the present invention will now be described with reference to the accompanying drawings.

[0051] Example

[0052] Please see Figures 1 to 5This embodiment provides a microfluidic immunofluorescence quantitative detection method for polycyclic aromatic hydrocarbons (PAHs) based on intelligent sensors. During the continuous detection process where the PAH sample enters the microfluidic channel with the reaction solution and undergoes an immunofluorescence reaction in the detection area, the fluorescence intensity, reference light path intensity, channel pressure, and temperature obtained from the sampling change synchronously with the advancement state. This method is carried out in sequence around blank advancement, front identification, residence time determination, fluorescence correction, reaction fragment screening, effective window determination, and content output.

[0053] During this continuous detection process, blank signals, channel flow states, and light source drift can affect the interpretation of fluorescence growth. If the fluorescence intensity at the center of the detection zone is directly used as the basis for the polycyclic aromatic hydrocarbon (PAH) content, dark current, background fluorescence, flow disturbances, or light source fluctuations can easily be mistaken for reaction signals. In this embodiment, multiple benchmarks are first established during the blank propagation phase. Then, the fluorescence change at the inlet of the detection zone is used to identify the reaction front. Subsequently, the time period during which the sample actually remains in the detection zone is limited, and fluorescence growth is extracted only within the reaction window where the flow is stable, fluorescence continuously rises, and saturation has not been reached. This ensures that the output PAH content value or range corresponds to the effective immunofluorescence reaction process.

[0054] S1: Obtain the dark current reference, background correction reference, light source drift reference, flow stability reference, and temperature correction reference for the blank propulsion phase.

[0055] The blank advance stage refers to the advancement phase before the reaction liquid or blank liquid forms an effective reaction signal for the test sample. This stage is used to obtain the reference state required for subsequent correction and screening. The dark current reference refers to the inherent sampling offset state formed by the central fluorescence intensity value of the detection zone within the non-advancement range of the micropump. It is used to subtract the baseline offset unrelated to the reaction from the central fluorescence intensity value of the detection zone during subsequent dwell periods. The background correction reference refers to the difference state formed between the fluorescence intensity value at the entrance of the detection zone and the fluorescence intensity value at the center of the detection zone in the blank state. It is used to distinguish between channel background, non-specific fluorescence, and the actual reaction signal entering the detection zone. The light source drift reference refers to the stable reference state of the reference optical path intensity value during the blank advance stage. It is used to determine whether changes in the light source during subsequent sampling are still compensable. The flow stability reference refers to the pressure center state and allowable fluctuation boundary of the channel pressure value during the blank advance stage. It is used to exclude unstable sampling intervals caused by bubbles, blockage, backflow, or sudden advance abrupt changes. The temperature correction reference refers to the environmental reference state formed by the temperature value during the blank advance stage. It is used to confirm whether the blank reference and subsequent detection processes are in a usable temperature state.

[0056] S101: During the blank advance phase, the following values ​​are synchronously collected according to the sampling timestamp: central fluorescence intensity of the detection zone, inlet fluorescence intensity of the detection zone, reference optical path intensity, channel pressure, temperature, and cumulative micropump advance steps. These values ​​from the same sampling timestamp are considered as the same set of blank sampling records. The central fluorescence intensity of the detection zone refers to the fluorescence reading corresponding to the reaction observation position in the detection zone; the inlet fluorescence intensity refers to the fluorescence reading corresponding to the inlet position before the reaction liquid enters the detection zone; the reference optical path intensity refers to the light source reference reading obtained synchronously with the fluorescence reading of the detection zone; the channel pressure refers to the pressure reading corresponding to the flow state within the channel during micropump advancement; and the temperature refers to the ambient or channel temperature reading at the same sampling time. These sampling records are arranged according to the sampling timestamp, providing a data source for the generation of the preceding benchmark and alignment of subsequent time periods.

[0057] S102: Statistically analyze and organize the fluorescence intensity values ​​at the center of the detection area within the micropump non-propelling interval, removing readings with missing samples, duplicate timestamps, abnormal sampling order, and those significantly deviating from the blank stable state, to form a dark current reference. The micropump non-propelling interval refers to the time interval in the micropump propulsion record where there is no propulsion action and no propulsion disturbance in the channel pressure. If the sampling records within the micropump non-propelling interval are insufficient to form a stable reference, the most recent valid blank stable record from the same blank propulsion will be used, and this continued state will be marked as a reference pending verification state. It will only be used again when the pressure and temperature do not deviate from the corresponding reference boundary.

[0058] S103: Statistically analyze and organize the blank difference between the fluorescence intensity values ​​at the entrance and center of the detection area to generate a background correction baseline. The blank difference refers to the fluorescence difference between the entrance and center positions at the same sampling time stamp when no effective reaction signal has been formed. When generating the background correction baseline, the sampling time stamps of the entrance and center positions are aligned; sampling records whose time stamps cannot be aligned are not included in the background correction baseline. The background correction baseline is used in subsequent frontal identification and center fluorescence correction, ensuring that both the net fluorescence intensity value at the entrance and the background subtracted intensity value are referenced to the blank background state.

[0059] S104: Statistical analysis of the changes in reference optical path intensity values ​​during the blanking phase is performed to form a light source drift baseline. The light source drift baseline includes the blank reference optical path center value, allowable drift states, and abnormal drift boundaries. The blank reference optical path center value represents the stable representative state of the reference optical path intensity during the blanking phase. Allowable drift states refer to states where light source changes can still be corrected through proportional compensation. Abnormal drift boundaries refer to boundary states where light source changes are no longer suitable for inclusion in effective fluorescence correction. This baseline is used in the subsequent generation of the corrected fluorescence intensity sequence to determine whether sampling points enter the compensation process or are discarded.

[0060] S105: Determine the blank fluctuation state for the channel pressure and temperature values ​​respectively, and generate the flow stability benchmark and temperature correction benchmark. When generating the flow stability benchmark, the stable flow state of the channel during blank propagation is described by the pressure center state and the pressure allowable fluctuation boundary; when generating the temperature correction benchmark, the blank temperature center value and the temperature allowable deviation boundary are used to describe whether the blank correction relationship can be used for subsequent sampling.

[0061] In particular, when only a few temperature values ​​exceed the preset temperature stability condition during the blank advance phase, and the remaining temperature values ​​still meet the preset temperature stability condition after removing the abnormal temperature sampling records, the abnormal temperature sampling records are excluded from the temperature correction benchmark, and the temperature correction benchmark is generated based on the remaining blank sampling records.

[0062] When multiple consecutive temperature values ​​exceed the preset temperature stability condition during the blank advancement phase, or when the remaining temperature values ​​after removing abnormal temperature sampling records are insufficient to form a stable temperature reference, the temperature correction benchmark will be marked as pending verification.

[0063] When the temperature correction benchmark is in a pending verification state, subsequent sampling points are only allowed to call the temperature correction benchmark if the real-time temperature value is within the temperature allowable deviation boundary and the channel pressure value is within the pressure allowable fluctuation boundary corresponding to the flow stability benchmark.

[0064] By simultaneously processing fluorescence, reference optical path, pressure, and temperature sampling records during the blank propagation phase, dark current, background, light source drift, flow stability, and temperature state are each made into callable benchmarks, thereby enabling subsequent reaction signal judgments to not directly depend on the original fluorescence readings.

[0065] S2: Based on the background correction benchmark, the fluorescence intensity value at the entrance of the detection zone is continuously increased and compared to determine the arrival time of the reaction liquid front and the number of micropump advance steps.

[0066] The arrival time of the reaction liquid front refers to the sampling timestamp when the fluorescence intensity at the entrance of the detection zone shows a continuous upward trend after background subtraction and meets the front identification conditions. The micropump advance steps refer to the cumulative advance record of the micropump corresponding to this sampling timestamp, which is used in conjunction with the detection zone steps to determine the residence time of the sample in the detection zone.

[0067] S201: Obtain the fluorescence intensity value at the entrance of the detection zone, the sampling timestamp corresponding to the fluorescence intensity value at the entrance of the detection zone, and the cumulative propulsion steps of the micropump, and establish an entrance sampling record according to the sampling timestamp. The entrance sampling record also retains the calling status of the background correction benchmark. If the entrance sampling record lacks a sampling timestamp, the cumulative propulsion steps of the micropump, or cannot be aligned with the blank benchmark, then the entrance sampling record will not enter the leading edge recognition process.

[0068] S202: Based on the background correction benchmark, background difference processing is performed on the fluorescence intensity value at the inlet of the detection area to obtain the net fluorescence intensity value at the inlet. The net fluorescence intensity value at the inlet refers to the reaction liquid entry trend data formed by subtracting the blank background difference from the fluorescence reading at the inlet position. It is used to exclude the fixed background offset that exists at the inlet position in the blank state. If the temperature value has deviated from the allowable deviation boundary of the temperature correction benchmark, the corresponding net fluorescence intensity value at the inlet is marked as a temperature inconsistency state and is not used as the basis for determining the arrival time of the front.

[0069] S203: Perform an incremental comparison of the net fluorescence intensity values ​​at the inlet within a continuous sampling period. This incremental comparison refers to confirming, according to the sampling timestamp sequence, that subsequent net fluorescence intensity values ​​at the inlet maintain an upward trend relative to previous values, excluding transient spikes caused by single-point noise or background fluctuations. The preset number of leading-edge recognitions is derived from the calibration results of the net fluorescence fluctuation state at the inlet during the blank transition phase and the leading-edge recognition rules, used to limit the number of sampling records that need to continuously meet the upward trend. The leading-edge recognition threshold is derived from the superposition rule of the upper limit of the blank background and the remaining recognition margin of the reaction liquid, used to limit the final net fluorescence intensity value at the inlet to having deviated from the blank background state.

[0070] S204: When the continuous increase in the net fluorescence intensity value at the inlet meets the preset number of frontal boundary recognitions, and the last net fluorescence intensity value at the inlet is higher than the frontal boundary recognition threshold, the corresponding sampling timestamp is determined as the arrival time of the reaction liquid frontal boundary, and the cumulative propulsion steps of the micropump corresponding to this sampling timestamp are determined as the inlet baseline steps. If, during the continuous rise, the net fluorescence intensity at the inlet drops, sampling is missing, timestamps are out of order, or pressure instability occurs, frontal boundary recognition is restarted from the next inlet sampling record that meets the continuity requirement. Through this process, the arrival time of the reaction liquid frontal boundary corresponds one-to-one with the micropump propulsion state, providing an inlet baseline for determining the dwell time period.

[0071] By comparing the continuously increasing net fluorescence intensity value at the inlet after background correction, the identification of the reaction front is simultaneously constrained by the background benchmark, continuous trend, and identification boundary, thereby reducing false triggering caused by fluctuations in the blank background.

[0072] S3: Based on the arrival time of the reaction liquid front, the number of micropump propulsion steps, the length of the detection zone, the channel cross-sectional area, and the single-step propulsion volume, determine the residence time of the sample in the detection zone.

[0073] Here, the detection zone length refers to the effective path length of the reaction liquid flowing through the detection zone along the channel; the channel cross-sectional area refers to the lateral flow area of ​​the reaction liquid when it passes through the detection zone channel; and the single-step propulsion volume refers to the liquid propulsion volume corresponding to each propulsion action of the micropump. The residence time period refers to the sampling time interval between the entry of the sample into the detection zone and its exit from the detection zone, used to limit the data range for subsequent fluorescence correction and reaction window screening.

[0074] S301: Obtain the detection zone length, channel cross-sectional area, and single-step propulsion volume, and confirm that these parameters have been provided by the channel parameter record or micropump propulsion parameter record before detection. The detection zone length and channel cross-sectional area are used to determine the volume state that the detection zone can hold for the reaction liquid, and the single-step propulsion volume is used to convert this volume state into the passage state corresponding to the number of micropump propulsion steps. If the detection zone length, channel cross-sectional area, or single-step propulsion volume is missing or does not match the current detection channel, no dwell time period will be generated, and the dwell time period will be output as indeterminate.

[0075] S302: The detection zone volume is determined based on the detection zone length and channel cross-sectional area, and the number of steps through the detection zone is determined based on the volume correspondence between the detection zone volume and the single-step propulsion volume. Specifically, the detection zone volume is obtained by multiplying the detection zone length by the channel cross-sectional area, and the number of steps through the detection zone is determined based on the ratio of the detection zone volume to the single-step propulsion volume. When the ratio is not an integer, the number of steps through the detection zone can be determined by rounding up to ensure that the reaction liquid front can advance from the detection zone inlet to the detection zone outlet. Alternatively, the number of steps through the detection zone can be determined based on a pre-calibrated correspondence table between the detection zone volume, single-step propulsion volume, and number of steps through the detection zone. If there is a discrepancy between the calibration version of the single-step propulsion volume and the channel parameters, the parameter record confirmed as valid before the start of the current detection shall prevail; if a valid parameter cannot be confirmed, the subsequent dwell time period will not be generated.

[0076] S303: The cumulative number of micropump advance steps corresponding to the arrival time of the reaction liquid front is used as the inlet reference step number, and the corresponding exit step number is determined based on the inlet reference step number and the number of steps passed through the detection zone. The corresponding exit step number refers to the number of steps that the micropump cumulative advance record should reach when the reaction liquid front reaches the detection zone exit. The micropump advance control record is arranged according to the order of advance action and the corresponding sampling timestamp. If there is a pause or uneven interval, the step correspondence is not changed, and only the sampling timestamp corresponding to the exit step number is found according to the actual record.

[0077] S304: Locate the sampling timestamp corresponding to the number of steps at the outlet in the micropump propulsion control record. Define the time interval between the arrival time of the reaction liquid front and the sampling timestamp corresponding to the number of steps at the outlet as the residence period. If there is no corresponding sampling timestamp for the number of steps at the outlet in the propulsion control record, mark the propulsion record after the obtained inlet reference step as an incomplete residence period and do not use this incomplete state for the final content output. If there is a backtracking, repeated steps, or sequence break in the propulsion control record, the record segment that can maintain the continuity of the step sequence is the valid search object. If continuity cannot be maintained, output the abnormal state of the flow record.

[0078] By linking the arrival time of the reaction liquid front, the number of micropump propulsion steps, the geometric parameters of the detection zone, and the single-step propulsion parameters, the subsequent fluorescence intensity processing is only applied to the sampling period when the sample is located within the detection zone, thereby reducing the influence of non-dwelling phase signals on the determination of the reaction window.

[0079] S4: For the fluorescence intensity value of the center of the detection area during the dwell time period, perform dark current subtraction, background subtraction and light source drift ratio correction in sequence to generate a corrected fluorescence intensity sequence.

[0080] The corrected fluorescence intensity sequence refers to the central fluorescence intensity data sequence formed by arranging the data according to the sampling timestamps within the residence period and correcting for dark current subtraction, background subtraction, and light source drift ratio. This sequence is used for subsequent generation of fluorescence growth and screening of candidate reaction fragments.

[0081] S401: Obtain the central fluorescence intensity values ​​of the detection area arranged according to the sampling timestamp during the dwell period, and verify the integrity, sequential continuity, and reference recall status of the sampling timestamps. If there are duplicate central fluorescence intensity values ​​at the same sampling timestamp, retain the record that can be synchronized with the reference optical path intensity value, channel pressure value, and temperature value. If the central fluorescence intensity value is missing or cannot be synchronized with the reference optical path intensity value, the corresponding sampling timestamp will not be included in the corrected fluorescence intensity sequence.

[0082] S402: Call the dark current reference and perform dark current subtraction processing on the central fluorescence intensity value of the detection area corresponding to each sampling timestamp to obtain the dark current subtracted intensity value. Dark current subtraction processing refers to excluding the part of the central fluorescence intensity value of the detection area that corresponds to the inherent sampling offset when the micropump is not advancing, so that the subsequent processed object retains the intensity state related to the channel background and reaction fluorescence. If the dark current reference is in a state of pending verification, it will only be called when the pressure and temperature of the corresponding sampling timestamp are still within the blank reference allowable state. Sampling points that do not meet this condition are marked as references that cannot be reused.

[0083] S403: Based on the background correction benchmark, perform background difference subtraction on the dark current subtracted intensity value to obtain the background subtracted intensity value. The background subtracted intensity value refers to the reaction-related intensity state formed after excluding the difference between dark current and blank background in the central fluorescence intensity. During background subtraction, the background correction benchmark corresponding to the same detection process is used first; if there is a background correction benchmark that cannot be used, the corresponding sampling point will not be included in the subsequent proportional correction.

[0084] S404: Obtain the center value of the blank reference optical path in the light source drift reference, and simultaneously obtain the real-time reference optical path intensity value corresponding to each sampling timestamp during the dwell time period. Compare the deviation direction and deviation magnitude of the real-time reference optical path intensity value relative to the center value of the blank reference optical path. When the deviation magnitude is within the preset drift allowable range, perform reverse compensation on the background subtraction intensity value according to the proportional relationship between the real-time reference optical path intensity value and the center value of the blank reference optical path to obtain the corrected fluorescence intensity value. Reverse compensation means that when the light source of the reference optical path is enhanced, the background subtraction intensity value is reduced accordingly, and when the light source of the reference optical path is weakened, the background subtraction intensity value is increased accordingly, so that the corrected fluorescence intensity returns to the light source state corresponding to the blank reference optical path.

[0085] S405: When the deviation of the real-time reference optical path intensity value from the center value of the blank reference optical path exceeds the preset drift allowable range, the corresponding sampling timestamp is marked as an abnormal sampling point of the light source and removed from the corrected fluorescence intensity sequence. The preset drift allowable range is derived from the blank fluctuation state of the light source drift reference and the drift compensation calibration rules, and is used to distinguish between compensable drift and uncompensable anomalies. After removing the abnormal sampling point of the light source, if the remaining sampling points cannot form adjacent sampling intervals, subsequent candidate reaction fragments will no longer be generated, and the effective reaction window will be output as uncertain.

[0086] S406: Temperature status screening of sampling points within the dwell time period is performed based on a temperature correction benchmark. Specifically, the blank temperature center value and temperature allowable deviation boundary in the temperature correction benchmark are obtained, and the real-time temperature value corresponding to each sampling timestamp within the dwell time period is simultaneously obtained. When the real-time temperature value is within the temperature allowable deviation boundary, the corresponding sampling point is determined as a temperature-consistent sampling point and allowed to enter the corrected fluorescence intensity sequence. When the real-time temperature value exceeds the temperature allowable deviation boundary, the corresponding sampling point is marked as a temperature anomalous sampling point, and this temperature anomalous sampling point is not included in the generation of the corrected fluorescence intensity sequence.

[0087] S407: Arrange the corrected fluorescence intensity values, after dark current subtraction, background subtraction, light source drift ratio correction, and temperature state screening, in the order of sampling timestamps. After removing abnormal sampling points from the light source and temperature states, a corrected fluorescence intensity sequence is generated. These abnormal sampling points are not used for subsequent fluorescence growth calculations, candidate reaction fragment determination, or effective reaction window determination. Each item in this sequence retains its corresponding sampling timestamp, pressure state marker, light source correction state marker, and temperature state marker for subsequent identification of fluorescence growth and screening of candidate reaction fragments. If the removal of an abnormal temperature sampling point results in discontinuities between adjacent sampling timestamps, the corresponding interval is marked as a discontinuous interval and is not considered a candidate reaction fragment.

[0088] By sequentially subtracting dark current, background, and correcting the light source drift ratio for the fluorescence intensity value at the center of the detection area, the corrected fluorescence intensity sequence corresponds to the fluorescence change of the sample in the detection area, thus providing a consistent signal range for subsequent growth determination.

[0089] S5: Calculate the fluorescence growth of adjacent sampling intervals based on the modified fluorescence intensity sequence, and determine the sampling intervals where the channel pressure value falls within the flow stability benchmark as candidate reaction fragments.

[0090] Fluorescence growth refers to the increase in corrected fluorescence intensity between adjacent sampling time stamps, reflecting changes in the immunofluorescence reaction signal during the residence period. Candidate reaction fragments are sampling fragments that simultaneously meet the fluorescence growth boundary and the flow stability boundary within adjacent sampling intervals, used for further screening of effective reaction windows.

[0091] S501: Obtain the corrected fluorescence intensity values ​​arranged adjacently by sampling timestamp in the corrected fluorescence intensity sequence. Perform difference processing on the corrected fluorescence intensity value of the later sampling timestamp relative to the corrected fluorescence intensity value of the previous sampling timestamp to obtain the fluorescence growth amount. If there are rejected sampling points with abnormal light sources, unusable reference sampling points, or abnormal temperature sampling points between adjacent sampling timestamps, the adjacent sampling interval is marked as a discontinuous interval and will not be included in the candidate reaction fragment;

[0092] If the start or end point of an adjacent sampling interval is marked as a light source abnormality sampling point, a reference sampling point that cannot be used, or a temperature abnormality sampling point, then the adjacent sampling interval is uncertain as a candidate reaction segment.

[0093] S502: Acquire the channel pressure value corresponding to the adjacent sampling interval and compare the channel pressure value with the pressure center state and pressure allowable fluctuation boundary corresponding to the flow stability reference. When the channel pressure value falls within the pressure allowable fluctuation boundary, mark the adjacent sampling interval as a flow stable interval. When the channel pressure value exceeds the pressure allowable fluctuation boundary or shows a sudden change in propulsion, stagnation, or backflow, mark the adjacent sampling interval as a flow instability segment. Flow instability segments do not enter the effective response window and disrupt the continuity between adjacent candidate segments.

[0094] S503: Obtain the blank correction intensity sequence formed by subtracting dark current, background and correcting light source drift ratio of the fluorescence intensity value of the center of the detection area during the blank advancement stage, and calculate the intensity difference between adjacent blank sampling points to obtain the blank correction intensity fluctuation.

[0095] The upper limit of blank growth is determined based on the statistical center value and statistical discrete value of the blank correction intensity fluctuation, wherein the statistical center value includes the mean or median, and the statistical discrete value includes the standard deviation, absolute deviation, or quantile range.

[0096] The blank growth upper limit is superimposed with the reaction recognition margin, which has the same dimension as the fluorescence growth amount and is determined by the sensor resolution or the standard deviation of blank noise, to generate a preset minimum growth boundary.

[0097] When the fluorescence growth corresponding to an adjacent sampling interval is lower than the preset minimum growth boundary, the adjacent sampling interval is marked as a non-reactive fluctuation interval.

[0098] S504: When the channel pressure value corresponding to an adjacent sampling interval falls within the allowable pressure fluctuation boundary, the fluorescence growth is not lower than the preset minimum growth boundary, and the adjacent sampling interval does not cross the abnormal sampling points of the light source and the abnormal sampling points of the temperature, the adjacent sampling interval is identified as a candidate reaction fragment. When the fluorescence growth is lower than the preset minimum growth boundary, the corresponding adjacent sampling interval is marked as a non-reaction fluctuation interval. Non-reaction fluctuation intervals can be retained as process records, but will not be included in the effective reaction window screening. If adjacent sampling intervals simultaneously exhibit flow instability and non-reaction fluctuation states, they will be preferentially processed as flow instability fragments to avoid fluorescence changes caused by flow disturbances being mistakenly included in reaction fragments.

[0099] By using adjacent growth states of the modified fluorescence intensity sequence and channel pressure stability states together to determine candidate reaction fragments, fragments entering the next step can simultaneously meet the requirements of signal growth and flow stability, thereby reducing the impact of flow disturbances and blank fluctuations on the reaction window.

[0100] S6: For candidate reaction fragments that show continuous increases, do not reach the upper limit of saturation, and do not cross the flow instability segment, determine the effective reaction window and the amount of fluorescence growth within the window.

[0101] The effective reaction window refers to the sampling interval within the candidate reaction fragment that continuously satisfies reaction growth, has not reached saturation, and lacks flow instability markers. The fluorescence growth within the window refers to the growth state between the corrected fluorescence intensity values ​​corresponding to the start and end points of the effective reaction window, used for subsequent positional determination with the fluorescence growth of low-concentration and high-concentration reference fluorescence.

[0102] S601: Obtain the corrected fluorescence intensity values, fluorescence growth, and flow stability markers within the candidate reaction fragments, arranged continuously according to the sampling timestamps, and confirm whether the candidate reaction fragments maintain continuity in timestamps and states. If there are flow-instability fragments, abnormal light source sampling points, inconsistent temperature sampling points, or discontinuous intervals between two candidate reaction fragments, the two fragments will not be merged into the same valid reaction window.

[0103] S602: Screening sampling intervals where the fluorescence growth is continuously greater than the preset effective growth boundary and the corrected fluorescence intensity value is lower than the saturation upper limit. The preset effective growth boundary is derived from the preset minimum growth boundary, blank fluctuation state, and effective reaction identification rules, and is used to further exclude intervals with insufficient growth from candidate reaction fragments. The saturation upper limit is derived from the calibration rules of the fluorescence detection linear response boundary, and is used to exclude sampling intervals where the corrected fluorescence intensity can no longer reflect the reaction growth relationship. If the corrected fluorescence intensity value reaches saturation, the sampling point and the subsequent saturation continuation interval will not enter the effective reaction window.

[0104] S603: When there are no flow instability markers, abnormal light source sampling points, or abnormal temperature sampling points within a sampling interval, and the fluorescence growth within that sampling interval maintains a continuous upward trend, the sampling interval is determined as a valid reaction window. A continuous upward trend means that the fluorescence growth of adjacent sampling intervals within the valid reaction window is not lower than a preset valid growth boundary, and there is no break caused by abnormal rejection. If multiple sampling intervals meet the conditions simultaneously, the valid reaction window is selected according to the priority rule corresponding to the stable residence state of the reaction liquid in the detection area. The priority rule is in the order of continuity, unsaturated state, and no flow instability state, without introducing any ranking criteria unrelated to the claims. When there are no flow instability markers, abnormal light source sampling points, or abnormal temperature sampling points within a sampling interval, and the fluorescence growth within that sampling interval maintains a continuous upward trend, the sampling interval is determined as a valid reaction window.

[0105] S604: Generates the fluorescence growth amount within the effective reaction window based on the difference between the corrected fluorescence intensity values ​​corresponding to the start and end points. The fluorescence growth amount is generated using only the corrected fluorescence intensity values, after dark current subtraction, background subtraction, and source drift ratio correction, rather than the original central fluorescence intensity value. If the sampling point corresponding to the start or end point of the effective reaction window is marked as abnormal, the boundary of the effective reaction window is redefined; if a complete boundary cannot be redefined, the effective reaction window is output as indeterminate and does not proceed to content mapping.

[0106] By imposing continuous, unsaturated, and non-crossing flow-instability limits on candidate reaction fragments, the fluorescence growth in the window originates from the stable immunofluorescence reaction region, thereby enabling subsequent content output to be based on a traceable and effective reaction process.

[0107] S7: Based on the positional relationship between the window fluorescence growth and the low-concentration and high-concentration reference fluorescence growth, output the polycyclic aromatic hydrocarbon content value or range status.

[0108] The low-concentration reference fluorescence growth refers to the reference growth state formed within the effective reaction window during the low-concentration reference detection process, while the high-concentration reference fluorescence growth refers to the reference growth state formed within the same effective reaction window during the high-concentration reference detection process. The reference concentration mapping relationship is a lookup table or mapping rule established by the relationship between the low-concentration and high-concentration reference fluorescence growth and the corresponding reference concentration. It is used to determine the polycyclic aromatic hydrocarbon (PAH) content value when the window fluorescence growth is within the reference range. The measurement range status refers to the output detection range status when the window fluorescence growth is lower than the low-concentration reference fluorescence growth or higher than the high-concentration reference fluorescence growth. It is used to indicate that the current result is not suitable for providing a content value based on the reference concentration mapping relationship.

[0109] S701: Acquire the fluorescence growth of the window, the low-concentration reference fluorescence growth, and the high-concentration reference fluorescence growth, and confirm that all three are determined using the same dark current subtraction, background subtraction, light source drift ratio correction, temperature state screening, and effective reaction window determination. Same correction means that all have undergone dark current subtraction, background subtraction, and light source drift ratio correction. Same effective reaction window means that all growth values ​​are obtained after excluding flow instability, saturation, and abnormal sampling points. If the correction of the reference fluorescence growth value is inconsistent with the current window fluorescence growth value, the reference concentration mapping relationship is not invoked, and the inconsistency status is output.

[0110] S702: Compare the interval position of the fluorescence growth in the comparison window relative to the fluorescence growth of the low-concentration reference and the fluorescence growth of the high-concentration reference. The interval position comparison is performed using textual rules, that is, judging whether the fluorescence growth in the window is between the low-concentration and high-concentration reference growth states, lower than the low-concentration reference growth state, or higher than the high-concentration reference growth state. If the order of the fluorescence growth of the low-concentration and high-concentration references is inconsistent with the mapping relationship of the reference concentration, the content output is paused and marked as an abnormal reference state.

[0111] S703: When the fluorescence growth within the window falls within the reference range formed by the fluorescence growth of low-concentration and high-concentration reference samples, the polycyclic aromatic hydrocarbon (PAH) content is determined using the reference concentration mapping relationship. The reference concentration mapping relationship is established using the fluorescence growth of multiple standard samples at known concentrations, and the standard samples and the sample to be tested use the same fluorescence correction aperture and effective reaction window. When determining the PAH content, the corresponding content is obtained by using table lookup, linear interpolation, piecewise fitting, or nonlinear standard curve fitting, based on the position of the fluorescence growth within the window between adjacent reference fluorescence growths. When the reference concentration mapping relationship is negatively correlated, the conversion is performed according to the direction in which the fluorescence growth decreases with increasing concentration.

[0112] S704: When the fluorescence growth in the window is lower than that of the low-concentration reference fluorescence growth, the output is in the "below range" state; when the fluorescence growth in the window is higher than that of the high-concentration reference fluorescence growth, the output is in the "above range" state. Both "below range" and "above range" states are considered range states and do not replace the polycyclic aromatic hydrocarbon (PAH) content value. If the preceding steps output an uncertain effective reaction window state, an uncertain residence time state, an abnormal continuous light source state, an abnormal flow record state, or an abnormal reference state, then the PAH content value will not be output, and the detection state corresponding to the source of the abnormality will be output, allowing the results to be traced back to the corresponding step.

[0113] By determining the interval position of the fluorescence growth rate within the window and the fluorescence growth rates of low-concentration and high-concentration references, the content value is output only within the growth range that the reference can cover, thus ensuring that the output of polycyclic aromatic hydrocarbon content is consistent with the effective reaction window and the mapping relationship between the reference.

[0114] Based on the above steps, this embodiment establishes multiple benchmarks during the blank propagation phase, identifies the reaction liquid leading edge at the inlet of the detection zone, generates a corrected fluorescence intensity sequence during the residence period in the detection zone, and determines the effective reaction window from candidate reaction fragments with stable flow and continuous growth. Through this method, the fluorescence growth amount within the window can eliminate the influence of dark current, background, light source drift, flow instability, temperature inconsistency, and saturation state, and can be used for the output of polycyclic aromatic hydrocarbon content values ​​or range status.

[0115] In one embodiment, the polycyclic aromatic hydrocarbon (PAH) sample to be tested forms a competitive immune reaction with a fluorescently labeled antibody, an immobilized PAH hapten, or a PAH antigen conjugate within a microfluidic channel. The fluorescence intensity value at the center of the detection zone is used to characterize the change in the binding state of PAHs and the fluorescently labeled antibody in the sample to be tested. The PAHs can be one or more of benzo[a]pyrene, naphthalene, anthracene, phenanthrene, and pyrene, or can be the total PAH content expressed as an equivalent concentration of benzo[a]pyrene.

[0116] Therefore, in this embodiment, the arrival time of the reaction liquid front is determined not only by the continuous rise of the inlet fluorescence, but also by the cumulative propulsion steps of the corresponding micropump to form the inlet baseline step count; the residence time of the sample to be tested is not only obtained by time judgment, but also by the detection zone being jointly limited by the step count and the micropump propulsion record; the candidate reaction fragments and effective reaction windows are determined not only based on the fluorescence growth and pressure stability, but also by excluding sampling points with abnormal light sources and abnormal temperatures. Through the above processing, the fluorescence growth in the window originates from the continuous reaction interval where the sample to be tested is actually located within the detection zone and the sampling state is stable.

[0117] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A microfluidic immunofluorescence quantitative detection method for polycyclic aromatic hydrocarbons based on intelligent sensors, characterized in that, Includes the following steps: Obtain the dark current reference, background correction reference, light source drift reference, flow stability reference, and temperature correction reference for the blank propagation stage. Each reference is generated by synchronously acquiring the fluorescence intensity values ​​at the center of the detection area, the fluorescence intensity values ​​at the entrance of the detection area, the intensity values ​​of the reference optical path, the channel pressure values, and the temperature values ​​within the same blank propagation stage. Based on the background correction benchmark, the fluorescence intensity value at the entrance of the detection area is continuously compared to determine the arrival time of the reaction liquid front, and the cumulative number of micro-pump advance steps corresponding to the arrival time of the reaction liquid front is determined as the entrance benchmark step number. Based on the arrival time of the reaction liquid front, the inlet reference number of steps, the length of the detection zone, the channel cross-sectional area, and the single-step propulsion volume, the number of steps through the detection zone is determined, and the dwell time of the sample to be tested in the detection zone is determined according to the inlet reference number of steps and the number of steps through the detection zone. For the fluorescence intensity value of the center of the detection area during the dwell time period, dark current subtraction, background subtraction and light source drift ratio correction are performed in sequence. Based on the temperature correction benchmark, the sampling points are screened for temperature status. After removing abnormal light source sampling points and abnormal temperature sampling points, a corrected fluorescence intensity sequence is generated. The fluorescence growth of adjacent sampling intervals is calculated based on the modified fluorescence intensity sequence, and the sampling intervals whose fluorescence growth meets the preset minimum growth boundary, whose channel pressure value falls within the flow stability reference, and which do not cross abnormal sampling points are determined as candidate reaction fragments. For candidate reaction fragments that continuously increase, do not reach the saturation upper limit, and do not cross the flow instability fragment, abnormal light source sampling point, and abnormal temperature sampling point, determine the effective reaction window and the window fluorescence growth amount; Based on the positional relationship between the fluorescence growth in the window and the fluorescence growth in the low-concentration and high-concentration references, the polycyclic aromatic hydrocarbon content value or range status is output.

2. The microfluidic immunofluorescence quantitative detection method for polycyclic aromatic hydrocarbons based on intelligent sensors according to claim 1, characterized in that, The process of obtaining the dark current reference, the background correction reference, the light source drift reference, the flow stability reference, and the temperature correction reference includes: During the blank propulsion phase, the fluorescence intensity values ​​at the center of the detection area, the fluorescence intensity value at the entrance of the detection area, the intensity value of the reference optical path, the channel pressure value, and the temperature value are collected. The fluorescence intensity values ​​at the center of the detection area where the sampling timestamp is in the non-propelled range of the micropump are statistically analyzed to generate the dark current reference. The fluorescence intensity values ​​at the entrance of the detection area during the blank advancement phase are statistically analyzed to generate an entrance background reference. The fluorescence intensity values ​​of the detection area center after subtraction from the dark current reference during the blank advancement phase are statistically analyzed to generate a center background reference. Based on the difference between the entrance background reference and the center background reference, an entrance-center blank difference reference is generated; The background correction benchmark includes the entrance background benchmark, the center background benchmark, and the entrance-center blank difference benchmark. The blank fluctuation range is determined for the reference optical path intensity value, channel pressure value, and temperature value, respectively, and the light source drift reference, the flow stability reference, and the temperature correction reference are generated. The process of screening sampling points for temperature status based on the temperature correction benchmark includes: Obtain the blank temperature center value and temperature allowable deviation boundary in the temperature correction benchmark, and simultaneously obtain the real-time temperature value corresponding to each sampling timestamp within the dwell period; when the real-time temperature value is within the temperature allowable deviation boundary, the corresponding sampling point is determined as a temperature consistent sampling point; when the real-time temperature value exceeds the temperature allowable deviation boundary, the corresponding sampling point is marked as a temperature abnormal sampling point; the temperature abnormal sampling point is not used to generate the corrected fluorescence intensity sequence, nor is it used to determine the candidate reaction fragment and the effective reaction window.

3. The microfluidic immunofluorescence quantitative detection method for polycyclic aromatic hydrocarbons based on intelligent sensors according to claim 1, characterized in that, The process of determining the arrival time of the reaction liquid front and the inlet reference step number includes: The fluorescence intensity value at the entrance of the detection zone, the sampling timestamp corresponding to the fluorescence intensity value at the entrance of the detection zone, and the cumulative propulsion steps of the micropump are obtained. Based on the entrance background benchmark, the fluorescence intensity value at the entrance of the detection zone is subtracted from the entrance background to obtain the net fluorescence intensity value at the entrance. The net fluorescence intensity value at the entrance within a continuous sampling period is compared in an incremental manner. When the number of consecutive increments reaches the preset number of front-end recognitions and the net fluorescence intensity value at the entrance at the last time is higher than the front-end recognition threshold, the corresponding sampling timestamp is determined as the arrival time of the front-end of the reaction liquid, and the corresponding cumulative propulsion steps of the micropump are determined as the entrance benchmark steps.

4. The microfluidic immunofluorescence quantitative detection method for polycyclic aromatic hydrocarbons based on intelligent sensors according to claim 1, characterized in that, The process of determining the duration of stay includes: The length of the detection zone, the cross-sectional area of ​​the channel, and the single-step propulsion volume are obtained. The volume of the detection zone is determined based on the length of the detection zone and the cross-sectional area of ​​the channel. The number of steps through the detection zone is determined based on the volume correspondence between the volume of the detection zone and the single-step propulsion volume. The inlet reference step number corresponding to the arrival time of the reaction liquid front is used as the starting step number for calculating the dwell time period. The corresponding step number at the outlet is determined based on the inlet reference step number and the number of steps through the detection zone. The sampling timestamp corresponding to the number of steps at the outlet is found in the micro-pump drive control record to generate the dwell time period.

5. The microfluidic immunofluorescence quantitative detection method for polycyclic aromatic hydrocarbons based on intelligent sensors according to claim 1, characterized in that, The process of generating the modified fluorescence intensity sequence includes: The fluorescence intensity values ​​of the detection area center arranged according to the sampling timestamp during the dwell time period are obtained, and the dark current reference is called to perform subtraction processing on the fluorescence intensity value of the detection area center corresponding to each sampling timestamp to obtain the dark current subtracted intensity value. Based on the central background reference, the dark current subtraction intensity value is subjected to central background subtraction to obtain the background subtraction intensity value, and the background subtraction intensity value is proportionally corrected according to the intensity change relationship of the reference optical path corresponding to the light source drift reference. After removing sampling points with abnormal temperature and abnormal light sources, the corrected fluorescence intensity sequence is generated according to the remaining sampling timestamps.

6. The microfluidic immunofluorescence quantitative detection method for polycyclic aromatic hydrocarbons based on intelligent sensors according to claim 5, characterized in that, The ratio correction specifically includes: Obtain the center value of the blank reference optical path in the light source drift reference, and simultaneously obtain the real-time reference optical path intensity value corresponding to each sampling timestamp during the dwell time period, and compare the deviation of the real-time reference optical path intensity value from the center value of the blank reference optical path. When the deviation is within the preset allowable drift range, the background subtraction intensity value is reverse-compensated based on the ratio between the real-time reference light path intensity value and the blank reference light path center value. When the deviation exceeds the preset allowable drift range, the corresponding sampling timestamp is marked as an abnormal sampling point of the light source and removed from the corrected fluorescence intensity sequence. The abnormal sampling point of the light source is not used to determine the candidate reaction fragment and the effective reaction window.

7. The microfluidic immunofluorescence quantitative detection method for polycyclic aromatic hydrocarbons based on intelligent sensors according to claim 1, characterized in that, The process of determining the candidate reaction fragment includes: Obtain the corrected fluorescence intensity values ​​arranged in adjacent sampling timestamps in the corrected fluorescence intensity sequence, and calculate the difference between the corrected fluorescence intensity value of the later sampling timestamp and the corrected fluorescence intensity value of the earlier sampling timestamp to obtain the fluorescence growth amount. The channel pressure value corresponding to the adjacent sampling interval is obtained, and the channel pressure value is compared with the pressure center value and the pressure allowable fluctuation boundary corresponding to the flow stability benchmark. When the channel pressure value falls into the pressure allowable fluctuation boundary, the fluorescence growth is not less than the preset minimum growth boundary, and the adjacent sampling interval does not cross the light source abnormal sampling point and temperature abnormal sampling point, the adjacent sampling interval is determined as the candidate reaction fragment.

8. The microfluidic immunofluorescence quantitative detection method for polycyclic aromatic hydrocarbons based on intelligent sensors according to claim 7, characterized in that, The process of determining the preset minimum growth boundary specifically includes: The blank correction intensity fluctuation is obtained by subtracting dark current, background and light source drift ratio from the fluorescence intensity value of the center of the detection area during the blank advancement phase, and the blank growth limit of the blank correction intensity fluctuation is determined. The upper limit of blank growth and the reaction recognition margin, which have the same dimensions as the fluorescence growth amount and are determined by the sensor resolution or the standard deviation of blank noise, are superimposed to generate the preset minimum growth boundary. When the fluorescence growth amount is lower than the preset minimum growth boundary, the corresponding adjacent sampling interval is marked as a non-reaction fluctuation interval.

9. The microfluidic immunofluorescence quantitative detection method for polycyclic aromatic hydrocarbons based on intelligent sensors according to claim 1, characterized in that, The process for determining the effective reaction window and the fluorescence growth amount within the window includes: Obtain the corrected fluorescence intensity values, fluorescence growth amount, and flow stability markers within the candidate reaction fragments, arranged sequentially according to the sampling timestamps; A preset effective growth boundary is generated based on the preset minimum growth boundary and the preset window confirmation margin, wherein the preset effective growth boundary is not less than the preset minimum growth boundary; The sampling intervals in which the fluorescence growth is continuously greater than the preset effective growth boundary and the corrected fluorescence intensity value is lower than the saturation upper limit are selected; When there are no flow instability markers, abnormal light source sampling points, or abnormal temperature sampling points within the sampling interval, the sampling interval is determined as the effective reaction window, and the window fluorescence growth amount is generated based on the difference between the corrected fluorescence intensity values ​​corresponding to the start and end points of the effective reaction window.

10. The microfluidic immunofluorescence quantitative detection method for polycyclic aromatic hydrocarbons based on intelligent sensors according to claim 1, characterized in that, The output process for polycyclic aromatic hydrocarbon (PAH) content values ​​or range status includes: The window fluorescence growth amount, the low-concentration reference fluorescence growth amount, and the high-concentration reference fluorescence growth amount are obtained, and the interval position of the window fluorescence growth amount relative to the low-concentration reference fluorescence growth amount and the high-concentration reference fluorescence growth amount is compared. When the fluorescence growth in the window is between the low-concentration reference fluorescence growth and the high-concentration reference fluorescence growth, the reference concentration mapping relationship is invoked to determine the polycyclic aromatic hydrocarbon content value; based on the monotonic direction of the reference concentration mapping relationship, it is determined whether the fluorescence growth in the window is within the effective reference interval defined by the low-concentration reference fluorescence growth and the high-concentration reference fluorescence growth; if it is outside the interval, the state below or above the range is output according to the monotonic direction.

Images