CN1588005A - Biological chip time division complex multiple fluorescent simultaneously detecting method and device - Google Patents

Abstract

The invention discloses a biological chip time-division multiplexing multi-fluorescent simultaneous detection method and device, the method steps are: a plurality of lasers for exciting different fluorescent substances and corresponding reflection holographic band-stop filters are arranged in the scanner; Modulation, the modulated light pulses are collimated and enter the same measurement optical path through their respective filters, and the biochips of various fluorescent substances are excited in turn to generate fluorescent signals of different phases; the fluorescent signals are collected, focused, and passed through the pinhole After entering the photomultiplier tube, it is converted into a pulsed electrical signal corresponding to different phase fluorescent signals, that is, a multiplexed fluorescent signal; the signal is passed through a multiplier to obtain the respectively excited fluorescent signal to realize its demultiplexing; each fluorescent signal is converted by A/D , input to the computer, and the computer scans the biochip two-dimensionally. The invention adopts a biochip laser confocal scanner and a time-division multiplexing method. The system only needs to prepare a set of receiving systems, and one scan can realize the excitation of various fluorescent substances and the detection of fluorescent signals.

Description

Method and device for time-division multiplexing multiple fluorescence simultaneous detection of biochip

                      

The invention belongs to the field of biological optical detection, in particular to a detection method and device of a biological chip.

Background technique

The existing biochip detection methods mainly include CCD imaging method and laser confocal scanning method.

The CCD imaging method uses an excitation light source to irradiate the biochip at the same time, filters out the excitation light at the detection end, and only allows the fluorescence to pass through, and images on the CCD through the imaging system, and the two-dimensional fluorescence signal is recorded by the CCD. The CCD imaging method can obtain the information of one area on the biochip in one measurement, so it is characterized by fast detection speed. The disadvantage is that the lateral resolution is relatively low; in order to improve the lateral resolution, the magnification of the imaging system needs to be increased, so the field of view is small, that is, the chip area for one measurement is small. When the chip area to be measured is large, only Measure multiple times in blocks and then stitch them together. Block scanning actually makes the chip and the imaging system move relative to each other through mechanical movement, but due to mechanical positioning errors, it will cause splicing errors in scanned images. So this method is not suitable for high-density biochip detection. On the other hand, the sensitivity of CCD is much lower than that of photomultiplier tube, so the detection sensitivity is relatively low. As disclosed on August 13, 2003, the application number is the Chinese patent of 02112626.7, biochip fluorescence detection scanning device, which mainly consists of laser excitation light path, fluorescence receiving light path, X-Y platform and support for placing biochip. The two laser beams in the laser excitation optical path reach the same optical path through the light-combining prism, and after passing through the through hole in the center of the hollow total reflection mirror, they are focused on a point on the biochip by the laser focusing lens, and the laser excitation optical path and the fluorescence receiving optical path pass through The hollow total reflection mirror and the fluorescence dichroic mirror are separated, and two-dimensional fluorescent images on multiple biochips are obtained through the two-dimensional movement of the X-Y platform.

In the laser confocal scanning method, the laser is used as the excitation light, the laser is converged through the optical system, and the biochip is placed on the focal plane; then the biochip is scanned two-dimensionally, and when the laser is irradiated on the fluorescent substance, it will generate fluorescence, which is determined by The optical system collects the fluorescence, and finally converts the fluorescence signal into an electrical signal through a photomultiplier tube. In the laser confocal scanning method, the biochip is scanned point by point. Since the biochip is placed on the focal plane, the spot size of the excitation light is very small, so the lateral resolution is relatively high. Moreover, by setting a conjugate pinhole at the receiving end, the interference of stray light outside the focal plane can be eliminated, and the detection sensitivity is relatively high. Laser confocal scanning has become the main detection method for high-density biochips.

In practical applications, different probes need to be labeled with different fluorescent substances. At present, the widely used fluorescent substances mainly include CY3 and CY5 fluorescent dyes. Their excitation light wavelengths and generated fluorescence wavelengths are different. Therefore, detection instruments It should be able to excite different fluorescent substances and detect corresponding fluorescent signals. In addition, in order to improve the repeatability and reliability of chip detection, a biochip is marked with multiple fluorescent substances at the same time, which requires the detection instrument to detect multiple fluorescent signals at the same time. The current laser confocal biochip scanner has two methods for the detection of various fluorescence. One is to use a set of receiving systems (including conjugated pinholes, photomultiplier tubes, etc.), but multiple scans are required, and each scan can only excite one fluorescent substance and detect the corresponding fluorescent signal. It takes more time. Another method is that the instrument is equipped with multiple sets of receiving systems, which can simultaneously excite various fluorescent substances and detect the corresponding fluorescent signals. The disadvantage is that multiple sets of receiving systems need to be prepared, and the instrument debugging workload is heavy, the cost is high, and maintenance is difficult.

Contents of the invention

The present invention aims to solve the technical problem of simultaneously detecting the presence of multiple types of fluorescence in existing biochip laser confocal scanners, and provides a method and device capable of simultaneously exciting multiple types of fluorescent substances and detecting corresponding fluorescence signals. Its characteristic is that only one set of receiving system needs to be prepared, and only one scan is needed to realize the excitation of various fluorescent substances and the detection of fluorescent signals. The detection takes less time, the cost of the instrument is low, and the maintenance of the instrument is simple.

In order to achieve the above object, the technical scheme adopted in the present invention is as follows: a biochip time-division multiplexed multi-fluorescent simultaneous detection method, based on including a pulse signal generator, a laser, a reflective holographic band-stop filter, a microscope objective lens, and a carrier film , a two-dimensional scanning platform, a focusing lens, a pinhole, and a photomultiplier tube laser confocal scanner for biochips, characterized in that the method steps are:

1. Multiple lasers for exciting different fluorescent substances and corresponding reflection holographic band-stop filters are set in the device;

2. Perform pulse modulation on the light emitted by the above-mentioned multiple lasers, so that each laser output is an optical pulse with the same repetition frequency, pulse width and different phase differences;

3. The pulse-modulated light pulses emitted by each laser with the same repetition frequency, pulse width and different phase differences are collimated and then enter the same measurement optical path with their own reflection holographic filters. The biological chip of the substance is excited in turn to generate a fluorescent signal;

4. Fluorescence signals of different phases are collected by the microscope objective lens and converted into parallel light. After being filtered by the filter to remove the scattered light, they are focused by the focusing lens and pass through the pinhole at the focal point to eliminate the light on the non-focal plane. Stray light enters the photomultiplier tube and converts it into a series of pulsed electrical signals corresponding to different phases of fluorescent signals, that is, multiplexed fluorescent signals;

5. The multiplexed fluorescent signals are multiplied by the corresponding modulation pulses of the lasers through the multiplier to obtain the respective excited fluorescent signals, so as to realize the demultiplexing of the multiplexed fluorescent signals;

6. Each fluorescent signal is input to the multi-channel A/D converter, and the collected data is read into the computer; during the whole process of time-division multiplexed multi-fluorescent simultaneous detection, the biochip is controlled by the computer to perform two-dimensional scanning.

The described method of pulse modulating the emitted light of a plurality of lasers uses a semiconductor laser as an excitation light source, and the modulation of the laser is realized by directly modulating the laser drive current. The excitation light source adopts other types of lasers including He-Ne lasers. Modulation is achieved through a chopper.

The biological chip time-division multiplexing multi-fluorescent simultaneous detection device adopting the method includes a pulse signal generator, a laser, a reflection holographic band-stop filter, a microscopic objective lens, a carrier film, a two-dimensional scanning platform, a focusing lens, a pinhole, The photomultiplier tube is characterized in that the laser is a plurality of lasers, one of which is directly connected to the pulse signal generator, and the rest are connected to the pulse signal generator through a corresponding delayer, and the plurality of lasers The output of the reflective holographic band-stop filter enters the same measurement optical path composed of a microscope objective lens, a carrier film, a two-dimensional scanning platform, a focusing lens, a pinhole, and a photomultiplier tube. The multipliers correspondingly configured according to the number of fluorescent substances are connected, and the input end of one of the multipliers is directly connected to the pulse signal generator, and the input ends of the remaining multipliers are connected to the output ends of the correspondingly configured delayers. The multiple multipliers The output end of the device is connected to the A/D acquisition card, the A/D acquisition card is connected to the computer, and the computer is connected to the two-dimensional scanning platform.

The present invention aims at the problem that the existing biochip laser confocal scanner simultaneously detects the existence of multiple kinds of fluorescence, and provides a method and device that can simultaneously excite multiple kinds of fluorescent substances and detect corresponding fluorescent signals, and is characterized in that it only needs to be prepared A set of receiving system can realize the excitation of various fluorescent substances and the detection of fluorescent signals with only one scan, the detection time is less, the efficiency is high, the instrument cost is low, the instrument maintenance is simple, meets the actual needs, and is beneficial to biochips popularization of technology.

Description of drawings

Fig. 1 is the structure diagram of biochip time-division multiplexing multi-fluorescent simultaneous detection device;

Fig. 2 is the timing diagram of excitation light pulse;

Fig. 3 is a schematic diagram of demultiplexing;

Figure 4 Workflow diagram.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

This embodiment is a method and device for simultaneous detection of biochips labeled with two fluorescent substances, CY3 and CY5. The absorption peak wavelength of the CY3 fluorescent substance is about 530nm, so a semiconductor laser with a wavelength of 532nm is used to pump a frequency-doubled laser as an excitation light source, and the peak wavelength of fluorescence generated by excitation is 570nm. The absorption peak wavelength of the CY5 fluorescent substance is about 630nm, so a semiconductor laser with a wavelength of 635nm is used as the excitation light source, and the fluorescence peak wavelength generated by excitation is 670nm. Semiconductor lasers have a very high modulation frequency, which can meet the actual modulation needs. In this embodiment, biochips with two fluorescent substances are used, so two corresponding lasers are used.

As shown in FIG. 1 , the pulse signal generator 1 outputs a pulse voltage signal, and the pulse voltage signal is input to the drive circuit of the 532nm laser 3 as a modulation signal, so that the 532nm laser 3 works in pulse mode. The pulse signal generator 1 outputs a pulse voltage signal, which is input to the delayer 2 at the same time to delay the pulse signal, and then input to the driving circuit of the 635nm laser 4 as a modulation signal to make the 635nm laser 4 work in pulse mode. As shown in Figure 2, the output light pulse 20 of the 532nm laser 3 and the 30 sequence of the output light pulse 30 of the 635nm laser 4 are different in timing, and the repetition frequency f (f=1/T) of the pulse is at least greater than the fluorescence signal frequency under DC operation double to ensure no loss of information. The pulse repetition period T, the pulse width τ, and the time delay t _d of the two-pulse sequence can be adjusted according to specific conditions such as chip spotting density.

The collimated beams output by the 532nm laser 3 and the 635nm laser 4 are respectively incident on the 532nm reflective holographic band-stop filter 5 and the 635nm reflective holographic band-stop filter 6, and are reflected by the 532nm reflective holographic band-stop filter 5 and 635nm The holographic band-rejection filter 6 is reflected and enters the microscope objective lens 7, where the excitation light is converged, and the focus is located on the front surface of the slide 8, that is, the biochip, to excite fluorescence. During the measurement process, the biochip 8 is driven by the two-dimensional scanning platform 9 to perform two-dimensional scanning.

Fluorescence and scattered excitation light are collected by the microscope objective lens 7 and converted into parallel light. Two kinds of fluorescent and partially scattered excitation light with peak wavelengths of 570nm and 670nm pass through the 532nm reflective holographic band-stop filter 5, and the 635nm reflective holographic band-stop filter After the light sheet 6, the scattered excitation light is further filtered by the 532nm reflective holographic band-stop filter 10 and the 635nm reflective holographic band-stop filter 11, while the fluorescence passes through the 532nm reflective holographic band-stop filter 10 and the 635nm reflective holographic band-stop filter Filter 11. Fluorescence is converged by the focusing lens 12 and passes through the pinhole 13 placed at the focal point, while stray light cannot pass through the pinhole 13 . Two kinds of fluorescent light with peak wavelengths of 570 nm and 670 nm passing through the pinhole 13 are irradiated on the photomultiplier tube 14 and converted into electrical signals. The electrical signal is a series of pulses, and different time slots correspond to different fluorescent signals, which are called multiplexed fluorescent signals here.

The multiplexed fluorescence signal pulse sequence output by the photomultiplier tube 14 is input to two multipliers 15, 16, and multiplied by the modulation pulses of each laser respectively, as shown in Figure 3, due to the corresponding relationship of timing, the respective Demultiplexing of the multiplexed fluorescent signal is achieved by the excited fluorescent signal without containing another fluorescent signal.

Each fluorescent signal is input to the multi-channel A/D acquisition card 17 , and the acquired data is read into the computer 18 . In the above process, the computer 18 controls the operation of the two-dimensional scanning platform 9 through the two-dimensional scanning platform controller 19 to perform two-dimensional scanning on the biochip. The x-direction drive pulse output by the two-dimensional scanning platform controller 19 is input to the A/D acquisition card 17 as a trigger signal for data acquisition.

The workflow of the present invention, as shown in Figure 4, before detection, at first set the number of scanning lines N, scanning step distance, scanning range, scanning speed; The controller sends the scanning end information; after the computer reads the end information, it moves a scanning step in the Y direction, and then starts another line of scanning; after all scanning is completed, the data temporarily stored in the computer memory is saved to the computer hard disk . During the scanning process, forward scan and reverse scan in the X direction are both effective row scans. When converting a data file into an image file, it is necessary to reverse the reverse scan data to ensure the corresponding relationship between the chip position and the data.

The method of the invention can also realize time-division multiplexed simultaneous detection of more than two kinds of fluorescence. In addition, the laser adds a DC working mode, and by switching the laser, only one kind of fluorescence can be detected. When the excitation light source is other types of lasers, a chopper can be used to realize pulse modulation, as long as the timing relationship of the light pulses of each laser is guaranteed.

Claims (3)

1. A biochip time-division multiplexing multi-fluorescent simultaneous detection method, based on a pulse signal generator, a laser, a reflection holographic band-stop filter, a microscope objective lens, a carrier film, a two-dimensional scanning platform, a focusing lens, and a pinhole , the biological chip laser confocal scanner of photomultiplier tube, it is characterized in that, method step is: (1) Multiple lasers for exciting different fluorescent substances and corresponding reflection holographic band-stop filters are arranged in the instrument; (2) Pulse modulation is performed on the emitted light of the above-mentioned plurality of lasers, so that each laser is output as an optical pulse with the same repetition frequency, pulse width and different phase differences; (3) The pulse-modulated light pulses emitted by each laser with the same repetition frequency, pulse width and different phase differences are collimated and then enter the same measurement optical path using their own reflection holographic filters. The biochips of fluorescent substances are excited in turn to generate fluorescent signals; (4) Fluorescent signals of different phases are collected by the microscope objective lens and converted into parallel light. After filtering through the filter to remove the scattered light, they are focused by the focusing lens and pass through the pinhole at the focal point to eliminate the non-focal plane. The stray light enters the photomultiplier tube and converts it into a series of pulsed electrical signals corresponding to different phase fluorescent signals, that is, multiplexed fluorescent signals; (5) The multiplexed fluorescent signals are multiplied by the multipliers respectively with the modulation pulses of the corresponding lasers, respectively to obtain the respectively excited fluorescent signals, so as to realize the demultiplexing of the multiplexed fluorescent signals; (6) Each fluorescent signal is input to the multi-channel A/D converter, and the collected data is read into the computer; during the whole process of time-division multiplexed multi-fluorescent simultaneous detection, the biochip is controlled by the computer to perform two-dimensional scanning. 2. The biochip time-division multiplexing multi-fluorescent simultaneous detection method according to claim 1, characterized in that, in the method of pulse modulation of the emitted light of a plurality of lasers, a semiconductor laser is used as an excitation light source, and the laser The modulation is realized by directly modulating the driving current of the laser, the excitation light source adopts other types of lasers including He-Ne laser, and the modulation of the laser is realized by a chopper. 3. A biochip time-division multiplexing multi-fluorescent simultaneous detection device implementing the method of claim 1, which includes a pulse signal generator, a laser, a reflective holographic band-stop filter, a microscope objective lens, a slide, and a two-dimensional scanning Platform, focusing lens, pinhole, photomultiplier tube, characterized in that the laser is a plurality of lasers, one of the lasers is directly connected to the pulse signal generator, and the rest of the lasers are generated through correspondingly configured delayers and pulse signals The output of the multiple lasers enters the same measurement system consisting of a microscope objective lens, a carrier film, a two-dimensional scanning platform, a focusing lens, a pinhole, and a photomultiplier tube after passing through a correspondingly configured reflection holographic band-stop filter. In the optical path, the output terminals of the photomultiplier tubes are respectively connected to multipliers configured according to the number of fluorescent substances, and the input terminals of one of the multipliers are directly connected to the pulse signal generator, and the input terminals of the other multipliers are output with correspondingly configured delayers. The terminals are connected, the output terminals of the multiple multipliers are connected to the A/D acquisition card, the A/D acquisition card is connected to the computer, and the computer is connected to the two-dimensional scanning platform.

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