CN107179303A - Droplet fluorescence detection method, device, system, storage medium and computer equipment - Google Patents

Abstract

The present invention relates to a microdroplet fluorescence detection method, device, system, storage medium and computer equipment. The microdroplet fluorescence detection method includes: acquiring fluorescence waveform signals and scattered light waveform signals respectively corresponding to the fluorescence emitted by the microdroplets and scattered light; The optical signal collected under the same acquisition frequency and the same acquisition start time as the scattered light; extract the peak position of the effective band in the scattered light waveform signal; extract the peak value at the corresponding peak position from the fluorescence waveform signal according to the peak position to obtain the fluorescence peak value ; Store the extracted fluorescence peak value as the detection data of the droplet corresponding to the peak position in the fluorescence waveform signal. According to the peak position of the scattered light waveform signal, the peak value can be extracted at the corresponding position in the fluorescence waveform signal to obtain the fluorescence peak value. It is not necessary to analyze and extract all the bands, which can reduce the amount of data analysis of the fluorescence waveform signal. Thereby improving the efficiency of fluorescence detection.

Description

Droplet fluorescence detection method, device, system, storage medium and computer equipment

technical field

The invention relates to the technical field of measurement and control, in particular to a droplet fluorescence detection method, device, system, storage medium and computer equipment.

Background technique

High-precision instruments such as flow cytometers and digital PCR (Polymerase Chain Reaction) systems are used to reliably detect microdroplets (or microspheres) sequentially queued from the flow channel. At present, there are two common methods for detecting droplets. One is the impedance method, that is, electrodes are added to both sides of the flow channel, and the number and size of the droplets are determined according to the impedance change when the droplets pass through; the other is the impedance method. One is the fluorescence detection method. The working principle of the fluorescence detection method is: taking the digital PCR system as an example, the probe in the water-in-oil microdroplet is specifically hydrolyzed after amplification, and emits fluorescence within a specific wavelength range under excitation light of a certain wavelength, such as FAM The peak absorption wavelength of the dye is 495nm, and the peak emission wavelength is 520nm; the peak absorption wavelength of the VIC dye is 538nm, and the peak emission wavelength is 554nm; the fluorescence detection method uses a light source to excite microdroplets with fluorescent substances, and collects the corresponding fluorescence when the microdroplets pass through the flow channel Waveform signal and extract peak value, pulse width and other data for storage, so as to analyze and determine whether the droplet is negative or positive.

When the size of the droplet is relatively small, and there is no significant contribution to the impedance change in the measurement of the impedance method, the fluorescence detection method is often the only choice. The traditional fluorescence detection method usually extracts and stores the peak value directly according to the waveform signal corresponding to the collected fluorescence, and needs to extract the entire waveform signal, which requires a large amount of data analysis and low detection efficiency.

Contents of the invention

Based on this, it is necessary to solve the problem of low detection efficiency of the traditional fluorescence detection method, and provide a droplet fluorescence detection method, device, system, storage medium and computer equipment that can improve the detection efficiency.

A microdroplet fluorescence detection method, comprising:

Obtaining the fluorescence waveform signal and the scattered light waveform signal respectively corresponding to the fluorescence and scattered light emitted by the droplet, the fluorescence and scattered light being optical signals collected under the same acquisition frequency and the same acquisition start time;

extracting the peak position of the effective band in the scattered light waveform signal;

extracting a peak value corresponding to the peak position from the fluorescence waveform signal according to the peak position to obtain a fluorescence peak value;

The extracted fluorescence peak value is stored as the detection data of the droplet corresponding to the peak position in the fluorescence waveform signal.

A microdroplet fluorescence detection device, comprising:

The waveform signal acquisition module is used to acquire the fluorescence waveform signal and the scattered light waveform signal respectively corresponding to the fluorescence and the scattered light emitted by the droplet, and the fluorescence and the scattered light are optical signals collected under the same acquisition frequency and the same acquisition start time ;

a peak position extraction module, configured to extract the peak position of the effective band in the scattered light waveform signal;

A fluorescence peak extraction module, configured to extract a peak corresponding to the peak position from the fluorescence waveform signal according to the peak position to obtain a fluorescence peak;

The detection data storage module is used to store the extracted fluorescence peak value as the detection data of the droplet corresponding to the peak position in the fluorescence waveform signal.

The above microdroplet fluorescence detection method and device extract the peak position of the effective band in the scattered light waveform signal after obtaining the fluorescence waveform signal and the scattered light waveform signal respectively corresponding to the fluorescence emitted by the microdroplet and the scattered light, and then according to the peak position from Extract the peak value at the corresponding peak position from the fluorescence waveform signal to obtain the fluorescence peak value, and finally store the extracted fluorescence peak value as the detection data of the droplet corresponding to the peak position in the fluorescence waveform signal. By using the scattered light waveform signal to assist the peak extraction of the fluorescence waveform signal, the fluorescence and scattered light are collected at the same acquisition frequency and the same acquisition start time, so the fluorescence waveform signal and the scattered light waveform signal are synchronized, and the peak position of the fluorescence waveform signal is the same as The peak position of the scattered light waveform signal is the same, so that according to the peak position of the scattered light waveform signal, the peak value can be extracted at the corresponding position in the fluorescence waveform signal to obtain the fluorescence peak value. It is not necessary to analyze and extract all the bands, which can reduce the The amount of data analysis on the fluorescence waveform signal, thereby improving the efficiency of fluorescence detection.

A storage medium stores a computer program, and when the stored computer program is executed by a processor, the steps of the above microdroplet fluorescence detection method are realized.

A computer device comprising a memory, a processor, and a computer program stored on the memory and operable on the processor, when the processor executes the computer program, the steps of the above-mentioned droplet fluorescence detection method are realized.

The above-mentioned storage medium and computer equipment can realize the above-mentioned droplet fluorescence detection method, and can also reduce the data analysis amount of the fluorescence waveform signal, thereby improving the fluorescence detection efficiency.

A microdroplet fluorescence detection system, comprising a scattered light collection device, a fluorescence collection device and a data processing device, the scattered light collection device and the fluorescence collection device are respectively located on both sides of the flow channel for the droplet to flow through, and both connecting said data processing means;

The scattered light collection device and the fluorescence collection device collect the scattered light and fluorescence of the droplet at the same collection frequency and the same collection start time, respectively obtain the scattered light waveform signal and the fluorescence waveform signal and send them to the data processing device;

The data processing device extracts the peak position of the effective band in the scattered light waveform signal, extracts the peak value corresponding to the peak position from the fluorescence waveform signal according to the peak position to obtain the fluorescence peak value, and converts the extracted fluorescence peak value Stored as the detection data of the droplet corresponding to the corresponding peak position in the fluorescent waveform signal.

The above-mentioned droplet fluorescence detection system collects the scattered light and fluorescence of the droplet respectively by using the scattered light collection device and the fluorescence collection device to obtain the scattered light waveform signal and the fluorescence waveform signal and send them to the data processing device, and the data processing device extracts the scattered light waveform signal The peak position of the effective band in the middle, and then extract the peak value at the corresponding peak position from the fluorescence waveform signal according to the peak position to obtain the fluorescence peak value, and finally store the extracted fluorescence peak value as the detection of the droplet corresponding to the corresponding peak position in the fluorescence waveform signal data. By using the scattered light waveform signal to assist the peak extraction of the fluorescence waveform signal, the fluorescence and scattered light are collected at the same acquisition frequency and the same acquisition start time, so the fluorescence waveform signal and the scattered light waveform signal are synchronized, and the peak position of the fluorescence waveform signal is the same as The peak position of the scattered light waveform signal is the same, so that according to the peak position of the scattered light waveform signal, the peak value can be extracted at the corresponding position in the fluorescence waveform signal to obtain the fluorescence peak value. It is not necessary to analyze and extract all the bands, which can reduce the The amount of data analysis on the fluorescence waveform signal, thereby improving the efficiency of fluorescence detection.

Description of drawings

Fig. 1 is the flowchart of droplet fluorescence detection method in an embodiment;

Fig. 2 is the schematic diagram of scattered light waveform signal and fluorescence waveform signal in the first case;

Fig. 3 is the schematic diagram of scattered light waveform signal and fluorescent waveform signal in the second case;

Fig. 4 is the schematic diagram of scattered light waveform signal and fluorescence waveform signal in the third case;

Fig. 5 is the schematic diagram of scattered light waveform signal and fluorescence waveform signal in the fourth case;

Fig. 6 is the flow chart of droplet fluorescence detection method in another embodiment;

Fig. 7 is a block diagram of a droplet fluorescence detection device in an embodiment;

Fig. 8 is a structural diagram of a droplet fluorescence detection system in an embodiment;

Fig. 9 is a schematic diagram of the working position of the droplet fluorescence detection system in an embodiment.

detailed description

Referring to FIG. 1 , the droplet fluorescence detection method in one embodiment includes the following steps.

S110: Acquire fluorescence waveform signals and scattered light waveform signals respectively corresponding to the fluorescence and scattered light emitted by the microdroplet.

Wherein, the fluorescence and the scattered light are optical signals collected under the same collection frequency and the same collection start time.

The micro-droplets carry fluorescent dyes, and the light source is used to excite the micro-droplets passing sequentially in the flow channel to excite fluorescence and generate scattered light. The fluorescence waveform signal corresponds to the fluorescence of one or more microdroplets, and the scattered light waveform signal corresponds to the scattered light of one or more microdroplets. Since the acquisition frequency of the fluorescence and the scattered light is the same as the acquisition start time, sampling synchronization can be guaranteed, and the fluorescence waveform signals corresponding to the fluorescence and the scattered light are synchronized with the scattered light waveform signals. Specifically, the fluorescence and scattered light can be collected respectively by the fluorescence collection device and the scattered light collection device at the same collection frequency and the same collection start time, and the fluorescence waveform signal and the scattered light waveform signal can be obtained through the conversion of the photoelectric signal.

S130: Extract the peak position of the effective band in the scattered light waveform signal.

The scattered light waveform signal includes a plurality of continuous bands in time, and the effective band refers to a band that satisfies a preset condition. The peak position refers to the position where the peak appears in the effective band. The effective wave band is determined by analyzing the scattered light waveform signal, so as to extract the peak position of the effective wave band.

S150: Extracting the peak value at the corresponding peak position from the fluorescence waveform signal according to the peak position to obtain the fluorescence peak value.

Since the scattered light waveform signal and the fluorescent waveform signal are synchronized, the peak position of the fluorescent waveform signal is the same as the peak position of the scattered light waveform signal, so it can be determined that the peak value needs to be extracted from the fluorescent waveform signal according to the peak position in the scattered light waveform signal s position.

S170: Store the extracted fluorescence peak as detection data of the droplet corresponding to the peak position in the fluorescence waveform signal.

The corresponding relationship between the peak position and the droplet can be determined according to the pre-stored corresponding relationship, for example, the corresponding relationship between the sequence number of each droplet and the band in the scattering peak waveform signal can be stored in advance, that is, which band corresponds to which serial number of the droplet ; By determining which band the peak position belongs to, the droplet corresponding to the serial number of the peak position can be obtained, so that the fluorescence peak extracted according to the peak position is used as the detection data of the corresponding droplet in the fluorescence detection channel where the fluorescence peak is located.

Specifically, there may be multiple fluorescence waveform signals. That is, at the same time, there are multiple fluorescence collection devices that collect fluorescence from the microdroplets, and thus multiple synchronized fluorescence waveform signals are obtained. For example, a scattered light collection device is installed on one side of the flow channel where droplets flow, and two fluorescence collection devices are installed on the other side. The scattered light collection device and the two fluorescence collection devices collect simultaneously, and a synchronous scattered light waveform signal and two fluorescent waveform signals. At this time, step S150 is: extract the peak value at the corresponding position from each fluorescence waveform signal according to the peak position, and obtain the fluorescence peak value corresponding to the peak position of each fluorescence waveform signal; step S170 is: store the extracted fluorescence peak value as the corresponding peak position The detection data of the corresponding microdroplet in the fluorescence waveform signal where the fluorescence peak is located.

For example, for the peak position X, the corresponding droplet is the first droplet, the fluorescence peak value extracted from the first fluorescence waveform signal is a1, and the fluorescence peak value extracted from the second fluorescence waveform signal is b1, The information to be stored includes: the detection data of the first droplet in the first fluorescence waveform signal is a1, and the detection data in the second fluorescence waveform signal is b1.

The above microdroplet fluorescence detection method, after obtaining the fluorescence waveform signal and the scattered light waveform signal respectively corresponding to the fluorescence emitted by the microdroplet and the scattered light, extracts the peak position of the effective band in the scattered light waveform signal, and then extracts the peak position from the fluorescence waveform according to the peak position The peak value at the corresponding peak position is extracted from the signal to obtain the fluorescence peak value, and finally the extracted fluorescence peak value is stored as the detection data of the droplet corresponding to the corresponding peak position in the fluorescence waveform signal. By using the scattered light waveform signal to assist the peak extraction of the fluorescence waveform signal, the fluorescence and scattered light are collected at the same acquisition frequency and the same acquisition start time, so the fluorescence waveform signal and the scattered light waveform signal are synchronized, and the peak position of the fluorescence waveform signal is the same as The peak position of the scattered light waveform signal is the same, so that according to the peak position of the scattered light waveform signal, the peak value can be extracted at the corresponding position in the fluorescence waveform signal to obtain the fluorescence peak value. It is not necessary to analyze and extract all the bands, which can reduce the The amount of data analysis on the fluorescence waveform signal, thereby improving the efficiency of fluorescence detection.

In addition, the above microdroplet fluorescence detection method stores the extracted fluorescence peak value as the detection data of the corresponding microdroplet in the fluorescence waveform signal through the corresponding relationship between the peak position and the microdroplet, so that the fluorescence peak value and the microdroplet can be in one-to-one correspondence, Clarify the corresponding relationship between fluorescence peaks and microdroplets, avoid the phenomenon of detection data confusion between different microdroplets, and improve the accuracy of data acquisition.

For example, referring to Fig. 2, Fig. 3, Fig. 4 and Fig. 5, taking two fluorescent waveform signals as an example, fluorescent A represents the waveform corresponding to the first fluorescent waveform signal, and fluorescent B represents the corresponding waveform of the second fluorescent waveform signal. Waveform diagram, scattered light C represents the waveform diagram of scattered light waveform signal. Possible situations during the droplet detection process include: the scattered light waveform signal and the two fluorescent waveform signals are normal, as shown in Figure 2; due to the flow channel, one of the fluorescent waveform signals is too weak and is submerged in noise, as shown in Figure 2 As shown in Figure 3 ; when there is air in the flow channel and the fluid velocity is unstable, the scattered light waveform signal and the fluorescent waveform signal are very chaotic, both of which are clutter, as shown in Figure 4; the phenomenon of splitting and fusion of droplets occurs, resulting in the possible emergence of droplets Too much larger or smaller than the normal droplet, so that the scattered light waveform signal and the fluorescence waveform signal are too large or too small, as shown in Figure 5. The traditional fluorescence detection method needs to extract and judge each band in the fluorescence waveform signal of each channel, which requires a large amount of calculation. At the same time, for the situation shown in Figure 3, at the peak point of the first fluorescent waveform signal, the second fluorescent waveform signal has no obvious peak, and the second fluorescent waveform signal is too weak to be submerged in the noise. At this time, The peak value cannot be extracted from the second fluorescence waveform signal, and droplet data storage omissions are prone to occur; at this time, the peak values of the two fluorescence waveform signals will be different in the traditional fluorescence detection method, and the same index position will be saved in the fluorescence peak array. The two fluorescence peaks below are not necessarily the extraction results of the same droplet, resulting in data confusion. By adopting the above microdroplet fluorescence detection method, the amount of calculation can be reduced and the detection efficiency can be improved, and at the same time, data misalignment and omission can be avoided by corresponding the fluorescence peak value to the microdroplet.

In one embodiment, referring to FIG. 6 , step S130 includes step S131 to step S135 .

S131: Extracting the peak value and pulse width of each band in the scattered light waveform signal and judging whether the extraction is successful.

The specific extraction operation of the peak value and the pulse width of the band can be realized by using the existing known technology. If the peak value and pulse width are extracted, it means that the extraction is successful, and step S133 is executed at this time.

S133: Determine whether the extracted peak value and pulse width are respectively within a preset peak value range and a preset pulse width range.

The peak value range and the pulse width range are used to limit the normal value range of the peak value and the pulse width respectively; Execute step S135.

Specifically, the peak value range and the pulse width range may be stored in advance according to empirical values, or may be obtained by receiving a value range input by a user in real time. For example, in one embodiment, before step S133, a range determination step is further included: receiving the input peak range and pulse width range as a preset peak range and a preset pulse width range respectively. Specifically, the step of determining the range may be performed after step S131, or may be performed after step S110 but before step S131, or may be performed before step S110.

S135: Use the extracted band with the peak value and pulse width as an effective band, and extract the peak position of the effective band.

If the extracted peak value is within the peak value range and the extracted pulse width is within the pulse width range, it means that the corresponding band satisfies the preset condition, and the corresponding band is taken as an effective band to extract the peak position.

From step S131 to step S135, by extracting the peak value and pulse width of the band and analyzing whether they are in the corresponding peak range and pulse width range, it is determined whether the corresponding band is an effective band, and the bands outside the effective band can be effectively eliminated to improve Accuracy of peak position extraction. It can be understood that in other embodiments, other parameters of the band may also be extracted to analyze whether it is a valid band.

In one embodiment, continue to refer to FIG. 6 , after step S131 , if the extraction of the peak value and pulse width is not successful, step S132 is executed.

S132: Record the droplets corresponding to the corresponding bands as clutter droplets.

If the peak value and pulse width are not extracted, it means that the extraction is unsuccessful, and it can be determined that the corresponding band is submerged by noise. For example, in the waveform shown in Figure 4, the peak value and pulse width cannot be extracted for all bands; The data corresponding to the extracted bands are discarded, and the droplets corresponding to the corresponding bands are recorded as clutter droplets.

Specifically, after step S133, if the extracted peak value and pulse width are not within the peak value range and pulse width range respectively, step S134 is executed.

S134: Record the droplet corresponding to the corresponding band as a droplet with an abnormal size.

The extracted peak value and pulse width are not within the peak range and pulse width range respectively, including the following cases: the peak value is not within the peak range and the pulse width is not within the pulse width range, the peak value is not within the peak range and the pulse width is within the pulse width Within a wide range, the peak value is within the peak value range and the pulse width is not within the peak value range; among them, the peak value is not within the peak value range, including the two cases where the peak value is smaller than the minimum value of the peak value range and the peak value is greater than the maximum value of the peak value range, and the pulse width is not within the peak value range. The pulse width range includes two cases where the pulse width is smaller than the minimum value of the pulse width range and the pulse width is greater than the maximum value of the pulse width range. When the extracted peak value and pulse width are not within the peak value range and pulse width range respectively, see the situation shown in Figure 5, it means that the waveform shape of the corresponding band is too large or too small, so that the corresponding droplet is too large or too small , and record the corresponding droplet as an abnormally sized droplet.

By classifying and recording the droplets corresponding to the bands other than the effective band, and recording the droplets corresponding to the bands that cannot extract the peak value and pulse width as clutter droplets, the extracted peak value and pulse width are not in the peak range The droplet corresponding to the corresponding band within the pulse width range is recorded as an abnormally sized droplet, which is convenient for the user to check to understand the detailed situation.

In an embodiment, both the fluorescence waveform signal and the scattered light waveform signal include waveform data corresponding to each moment within a preset time period. That is, one microdroplet can correspond to multiple fluorescence waveform data at different times, and the fluorescence waveform data of one or more microdroplets constitute the fluorescence waveform signal.

Continuing to refer to FIG. 6 , after step S110 , step S120 is also included before step S150 .

S120: Buffer the waveform data at each time point corresponding to the fluorescence waveform signal.

By caching waveform data, it is convenient for subsequent search and use. Specifically, after step S120, the waveform data corresponding to the scattered light waveform signal at each time point may also be buffered.

Specifically, step S150 includes step S151 to step S154.

S151: Obtain the time corresponding to the peak position, and search for the band within the preset time range where the time corresponding to the peak position in the fluorescence waveform signal is located. Wherein, the preset time range takes the time corresponding to the peak position as the middle value.

The scattering peak waveform signal is composed of waveform data arranged in chronological order, and different times correspond to different positions, so the time corresponding to the peak position can be specifically used. The preset time range with the time corresponding to the peak position as an intermediate value is a time period including the time corresponding to the peak position.

S152: Extract the peak value of the found band and judge whether the extraction is successful. If yes, execute step S153; otherwise, execute step S154.

Since the fluorescent waveform signal and the scattered light waveform signal are synchronized, the waveform data corresponding to the peak position in the scattered light waveform signal and the waveform data at the time corresponding to the peak position in the fluorescent waveform signal correspond to the same droplet at the same position Detection data; because there may be errors in the collection process of fluorescence and scattered light, therefore, by searching for the band in the preset time range with the moment of the peak position as the intermediate value in the fluorescence waveform signal, and extracting the peak value of the searched band, it can improve Get peak accuracy.

S153: Use the extracted peak as the fluorescence peak at the corresponding peak position.

S154: Find the waveform data corresponding to the moment corresponding to the peak position from the buffered waveform data as the fluorescence peak at the corresponding peak position.

For the wave band that has been extracted from the scattered light waveform signal to the peak position, it means that the corresponding droplet is a normal droplet, and there are two situations in which the fluorescent waveform signal exists: the fluorescent waveform signal is normal, and the peak value can be extracted, refer to Figure 2; The fluorescent waveform signal is submerged by noise, and the peak value cannot be extracted. Refer to the second fluorescent waveform signal submerged by noise in Figure 3. If the peak value is successfully extracted from the searched band, the extracted peak value will be used as the fluorescence peak value with high accuracy; if the peak value cannot be extracted, the corresponding waveform data will be searched from the cached data as the fluorescence peak value; Extracting the peak can ensure that the corresponding fluorescence peak can be obtained, which can avoid the data omission phenomenon in the traditional fluorescence detection method, and the data acquisition accuracy is high.

Specifically, there are multiple peak positions. Therefore, each peak position corresponds to a respective fluorescence peak. Please continue to refer to FIG. 6 , after step S170 , step S180 and step S190 are also included.

S180: Determine whether all peak positions correspond to fluorescence peaks.

If not, it means that there are still peaks in the fluorescent signal that have not been extracted, and step S190 is executed at this time.

S190: Obtain the next peak position to obtain a new peak position, and return to step S150.

Specifically, step S190 returns to step S151. In this embodiment, step S150 and step S170 are performed sequentially according to the order of peak positions, that is, step S150 obtains the fluorescence peak value according to the previous peak position, and step S170 stores the fluorescence peak value as the detection data of the corresponding droplet, and then returns to step S150 The fluorescence peak is obtained according to the next peak position until the fluorescence peak is obtained according to all peak positions. In this way, it can be ensured that all peak positions correspond to a fluorescence peak, avoiding missing data.

A storage medium stores a computer program, and when the stored computer program is executed by a processor, the steps of the above microdroplet fluorescence detection method are realized.

A computer device includes a memory, a processor, and a computer program stored in the memory and operable on the processor. When the processor executes the computer program, the steps of the above microdroplet fluorescence detection method are realized.

The above-mentioned storage medium and computer equipment include the above-mentioned droplet fluorescence detection method, similarly, the amount of data analysis on the fluorescence waveform signal can be reduced, thereby improving the efficiency of fluorescence detection; and the accuracy of data acquisition is high.

Referring to FIG. 7 , a droplet fluorescence detection device includes a waveform signal acquisition module 110 , a peak position extraction module 130 , a fluorescence peak extraction module 150 and a detection data storage module 170 .

The waveform signal acquiring module 110 is used to acquire the fluorescence waveform signal and the scattered light waveform signal respectively corresponding to the fluorescence and scattered light emitted by the droplet. The fluorescence and scattered light are optical signals acquired at the same acquisition frequency and the same acquisition start time.

The peak position extraction module 130 is used to extract the peak position of the effective band in the scattered light waveform signal.

The fluorescence peak extraction module 150 is used to extract the peak value at the corresponding peak position from the fluorescence waveform signal according to the peak position to obtain the fluorescence peak value.

The detection data storage module 170 is used for storing the extracted fluorescence peak as detection data of the droplet corresponding to the peak position in the fluorescence waveform signal.

In the above-mentioned microdroplet fluorescence detection device, after the waveform signal acquisition module 110 acquires the fluorescence waveform signal and the scattered light waveform signal respectively corresponding to the fluorescence emitted by the microdroplet and the scattered light, the peak position extraction module 130 extracts the part of the effective band in the scattered light waveform signal peak position, then the fluorescence peak extraction module 150 extracts the peak value at the corresponding peak position from the fluorescence waveform signal according to the peak position to obtain the fluorescence peak value, and finally the detection data storage module 170 stores the extracted fluorescence peak value as the droplet corresponding to the corresponding peak position Detection data in the fluorescence waveform signal. By using the scattered light waveform signal to assist the peak extraction of the fluorescence waveform signal, the fluorescence and scattered light are collected at the same acquisition frequency and the same acquisition start time, so the fluorescence waveform signal and the scattered light waveform signal are synchronized, and the peak position of the fluorescence waveform signal is the same as The peak position of the scattered light waveform signal is the same, so that according to the peak position of the scattered light waveform signal, the peak value can be extracted at the corresponding position in the fluorescence waveform signal to obtain the fluorescence peak value. It is not necessary to analyze and extract all the bands, which can reduce the The amount of data analysis on the fluorescence waveform signal, thereby improving the efficiency of fluorescence detection.

In addition, through the corresponding relationship between the peak position and the droplet, the extracted fluorescence peak is stored as the detection data of the corresponding droplet in the fluorescence waveform signal, so that the fluorescence peak and the droplet can be one-to-one corresponding, and the fluorescence peak and the droplet can be clearly defined. The corresponding relationship between them can avoid the phenomenon of detection data confusion between different droplets, and improve the accuracy of data acquisition.

Specifically, the peak position extraction module 130 can specifically extract the peak position of the effective band in the scattered light waveform signal by using the method including steps S131 to S135 in the droplet fluorescence detection method, which will not be described in detail here.

Specifically, both the fluorescence waveform signal and the scattered light waveform signal include waveform data corresponding to each moment within a preset time period. The droplet fluorescence detection device further includes a buffering module, configured to buffer the waveform data corresponding to each time point of the fluorescence waveform signal after the waveform signal acquisition module 110 acquires the fluorescence waveform signal and the scattered light waveform signal. Specifically, the fluorescence peak extraction module 150 can obtain the fluorescence peak value according to the peak position by using the method from step S151 to step S154 in the droplet fluorescence detection method, which will not be described in detail here.

Specifically, the above-mentioned droplet fluorescence detection device also includes a cycle detection module (not shown in the figure), which is used to judge whether all peak positions correspond to fluorescence peaks, if not, obtain the next peak position to obtain a new peak position, and control the fluorescence peak position. The peak extraction module 150 performs corresponding functions.

Referring to FIG. 8 , a droplet fluorescence detection system includes a scattered light collection device 210, a fluorescence collection device 220, and a data processing device 230. The scattered light collection device 210 and the fluorescence collection device 220 are respectively located in the flow channel for the droplet to flow through. 300 (refer to FIG. 9 ), both sides are connected to the data processing device 230 .

The scattered light collection device 210 and the fluorescence collection device 220 collect the scattered light and fluorescence of the droplet at the same collection frequency and the same collection start time, respectively obtain the scattered light waveform signal and the fluorescence waveform signal and send them to the data processing device 230 .

The data processing device 230 extracts the peak position of the effective wave band in the scattered light waveform signal, extracts the peak value at the corresponding peak position from the fluorescence waveform signal according to the peak position to obtain the fluorescence peak value, and stores the extracted fluorescence peak value as the corresponding peak position. Detection data of droplets in fluorescent waveform signals.

The above-mentioned droplet fluorescence detection system collects the scattered light and fluorescence of the droplet respectively by using the scattered light collection device 210 and the fluorescence collection device 220 to obtain the scattered light waveform signal and the fluorescence waveform signal and send them to the data processing device 230, and the data processing device 230 extracts The peak position of the effective band in the scattered light waveform signal, and then extract the peak value at the corresponding peak position from the fluorescence waveform signal according to the peak position to obtain the fluorescence peak value, and finally store the extracted fluorescence peak value as the droplet corresponding to the corresponding peak position in the fluorescence waveform Detection data in the signal. By using the scattered light waveform signal to assist the peak extraction of the fluorescence waveform signal, the fluorescence and scattered light are collected at the same acquisition frequency and the same acquisition start time, so the fluorescence waveform signal and the scattered light waveform signal are synchronized, and the peak position of the fluorescence waveform signal is the same as The peak position of the scattered light waveform signal is the same, so that according to the peak position of the scattered light waveform signal, the peak value can be extracted at the corresponding position in the fluorescence waveform signal to obtain the fluorescence peak value. It is not necessary to analyze and extract all the bands, which can reduce the The amount of data analysis on the fluorescence waveform signal, thereby improving the efficiency of fluorescence detection.

In addition, through the corresponding relationship between the peak position and the droplet, the extracted fluorescence peak is stored as the detection data of the corresponding droplet in the fluorescence waveform signal, so that the fluorescence peak and the droplet can be one-to-one corresponding, and the fluorescence peak and the droplet can be clearly defined. The corresponding relationship between them can avoid the phenomenon of detection data confusion between different droplets, and improve the accuracy of data acquisition.

In one embodiment, referring to FIG. 9 , there are multiple fluorescence collection devices 220 , and the data processing device 230 extracts the peak value at the corresponding peak position from the fluorescence waveform signal corresponding to each fluorescence collection device 220 according to the peak position, and obtains each The fluorescent waveform signal corresponds to the fluorescent peak at the peak position, and the extracted fluorescent peak is stored as detection data of the droplet corresponding to the peak position in the fluorescent waveform signal where the fluorescent peak is located.

Specifically, the scattered light collection device 210 includes a scattered light channel optical system and a first ADC (Analog-to-Digital Converter), and the first ADC is connected to the scattered light channel optical system and a data processing device 230; the fluorescence collection device 220 It includes a fluorescence channel optical system and a second ADC, and the second ADC is connected to the fluorescence channel optical system and a data processing device 230 . The acquisition frequency and acquisition start time of the first ADC and the second ADC are the same.

The scattered light channel optical system collects the scattered light of the droplet and performs photoelectric conversion to obtain the corresponding electrical signal and sends it to the first ADC. The first ADC collects the electrical signal corresponding to the scattered light according to the preset collection frequency and collection start time to obtain the scattered light The waveform signal is sent to the data processing device 230; the fluorescence channel optical system collects the fluorescence of the droplet and performs photoelectric conversion to obtain a corresponding electrical signal and sends it to the second ADC, and the second ADC collects the fluorescence according to the preset collection frequency and collection start time The corresponding electrical signal is obtained as a fluorescence waveform signal and sent to the data processing device 230 . The accuracy of data processing can be improved by setting corresponding ADCs for different channel optical systems and performing classified collection.

Referring to Fig. 9, D is the light source, E is the light source optical system; A and B are the photodetectors in the optical systems of the two fluorescent channels respectively, and C is the photodetector in the scattered light channel optical system, and the photodetectors are used to convert the light The signal is converted into an electrical signal; F is the collection channel of the scattered light channel optical system; G and H are the collection channels of the two fluorescence channel optical systems respectively. The light source excites the microdroplets passing sequentially in the flow channel 300 to excite fluorescence and generate scattered light; the fluorescence is collected by the optical system of the fluorescence channel, and received by detector A and detector B respectively; the scattered light is collected by the optical system of the scattered light channel, Received by the scattered light detector C, the detector C is placed on the spherical surface with the center of the microdroplet under test, θ is its included angle, and the size of the θ angle can be adjusted according to the signal-to-noise ratio of the signal.

The technical features of the above-mentioned embodiments can be combined arbitrarily. To make the description concise, all possible combinations of the technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, should be considered as within the scope of this specification.

The above-mentioned embodiments only express several implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but should not be construed as limiting the patent scope of the invention. It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims (10)

1. A microdroplet fluorescence detection method, characterized in that, comprising: Obtaining fluorescence waveform signals and scattered light waveform signals respectively corresponding to the fluorescence and scattered light emitted by the microdroplet, where the fluorescence and scattered light are optical signals collected at the same acquisition frequency and at the same acquisition start time; extracting the peak position of the effective band in the scattered light waveform signal; extracting a peak value corresponding to the peak position from the fluorescence waveform signal according to the peak position to obtain a fluorescence peak value; The extracted fluorescence peak value is stored as the detection data of the droplet corresponding to the peak position in the fluorescence waveform signal. 2. droplet fluorescence detection method according to claim 1, is characterized in that, described extracting the peak position of effective band in described scattered light waveform signal, comprises: Extracting the peak value and pulse width of each band in the scattered light waveform signal and judging whether the extraction is successful; If the extraction is successful, it is judged whether the extracted peak value and pulse width are respectively within a preset peak value range and a preset pulse width range; If yes, the band with the extracted peak value and pulse width is taken as an effective band, and the peak position of the effective band is extracted. 3. droplet fluorescence detection method according to claim 2, is characterized in that, after the peak value and the pulse width of each band in the described scattered light waveform signal of described extraction and judging whether to extract successfully, also comprise: If the extraction is unsuccessful, the droplet corresponding to the corresponding band is recorded as a clutter droplet; After determining whether the extracted peak value and pulse width are respectively within the preset peak value range and the preset pulse width range, it also includes: If not, the droplet corresponding to the corresponding band is recorded as a droplet with abnormal size. 4. The microdroplet fluorescence detection method according to claim 1, characterized in that, both the fluorescence waveform signal and the scattered light waveform signal include waveform data corresponding to each moment within a preset time length, and the acquired microdroplet emits After the fluorescence waveform signal and the scattered light waveform signal respectively corresponding to the fluorescence and scattered light, before extracting the peak value corresponding to the peak position from the fluorescence waveform signal according to the peak position to obtain the fluorescence peak value, it also includes buffering the The step of describing the waveform data at each moment corresponding to the fluorescence waveform signal; The extraction of the peak value corresponding to the peak position from the fluorescence waveform signal according to the peak position to obtain the fluorescence peak value includes: Obtain the time corresponding to the peak position, and search for the wave band within the preset time range where the time corresponding to the peak position in the fluorescent waveform signal is located, and the preset time range takes the time corresponding to the peak position as an intermediate value ; Extract the peak value of the found band and judge whether the extraction is successful; If so, then use the extracted peak as the fluorescence peak at the corresponding peak position; If not, the waveform data corresponding to the time corresponding to the peak position is searched from the buffered waveform data as the fluorescence peak at the corresponding peak position. 5. The microdroplet fluorescence detection method according to claim 1, wherein there are multiple peak positions, and the extracted fluorescence peak value is stored as the microdroplet corresponding to the effective band in the fluorescence peak position. After the detection data in the waveform signal, it also includes: Determine whether all peak positions correspond to fluorescence peaks; If not, acquire the next peak position to obtain a new peak position, and return to the step of extracting the peak value corresponding to the peak position from the fluorescence waveform signal according to the peak position to obtain the fluorescence peak value. 6. A storage medium storing a computer program, characterized in that, when the stored computer program is executed by a processor, the steps of the method according to any one of claims 1-5 are implemented. 7. A computer device, comprising a memory, a processor, and a computer program stored on the memory and operable on the processor, characterized in that, when the processor executes the computer program, the computer program according to claims 1-5 is implemented. The steps of any one of the described methods. 8. A microdroplet fluorescence detection device, characterized in that it comprises: The waveform signal acquisition module is used to acquire the fluorescence waveform signal and the scattered light waveform signal respectively corresponding to the fluorescence and the scattered light emitted by the droplet, and the fluorescence and the scattered light are optical signals collected under the same acquisition frequency and the same acquisition start time ; a peak position extraction module, configured to extract the peak position of the effective band in the scattered light waveform signal; A fluorescence peak extraction module, configured to extract a peak corresponding to the peak position from the fluorescence waveform signal according to the peak position to obtain a fluorescence peak; The detection data storage module is used to store the extracted fluorescence peak value as the detection data of the droplet corresponding to the peak position in the fluorescence waveform signal. 9. A droplet fluorescence detection system, characterized in that it comprises a scattered light collection device, a fluorescence collection device and a data processing device, the scattered light collection device and the fluorescence collection device are respectively located in the stream for the droplet to flow through. Both sides of the road are connected to the data processing device; The scattered light collection device and the fluorescence collection device collect the scattered light and fluorescence of the droplet at the same collection frequency and the same collection start time, respectively obtain the scattered light waveform signal and the fluorescence waveform signal and send them to the data processing device; The data processing device extracts the peak position of the effective band in the scattered light waveform signal, extracts the peak value corresponding to the peak position from the fluorescence waveform signal according to the peak position to obtain the fluorescence peak value, and converts the extracted fluorescence peak value Stored as the detection data of the droplet corresponding to the corresponding peak position in the fluorescent waveform signal. 10. The droplet fluorescence detection system according to claim 9, wherein the number of the fluorescence collection devices is multiple, and the data processing device obtains the fluorescence waveform corresponding to each fluorescence collection device according to the peak position. The peak value corresponding to the peak position is extracted from the signal to obtain the fluorescence peak value corresponding to each fluorescence waveform signal.