CN100414288C - Millimeter-scale miniature laser-induced fluorescence detector for biochips - Google Patents

Abstract

The invention relates to a millimeter-level miniature laser-induced fluorescence detector used in biological chips, which belongs to biological and medical detection instruments. It includes a light source, a filter system and a photoelectric detection system, characterized in that: the light source is a semiconductor laser (1) with a peak wavelength of 470-495nm and its spectrum is cut off at 500nm, and the filter system is a cut-off wavelength of 500nm peak wavelength In the 520nm interference filter film (2), the photoelectric detection part is a miniature semiconductor photodetection device (3); the semiconductor laser, the interference filter film, and the photodetection device are integrated; the total volume of the fluorescence detector is at the millimeter level, and its length, Width and height are within the range of 1mm to 10mm. The photodetection device can be located directly below the semiconductor laser, can surround it, or can be juxtaposed with it. The invention simplifies the structure of the detection technology device, is easy to carry, eliminates the error and distortion caused, and makes the detection result closer to the true value.

Description

Millimeter-scale miniature laser-induced fluorescence detector for biochips

technical field

The invention is mainly aimed at the detection of fluorescent substances with excitation wavelength peaks at 470-495nm and emission wavelengths at 510-530nm, and belongs to biological and medical detection instruments.

Background technique

Biochip is a high-tech that has developed rapidly in the field of life sciences in recent years. Because biochips can analyze a large number of biomolecules in a short period of time, people can quickly and accurately obtain biological information in samples. The efficiency is the traditional detection method. Hundreds of thousands of times, and thus present a peak of development in a short period of time.

There are four basic points in biochip technology: chip preparation, sample preparation, biomolecular hybridization reaction and signal detection. Signal detection is an important part of biochip technology, which mainly includes three parts: hybridization signal generation, signal collection and transmission, signal processing and identification. Among the biochip signal detection methods, the fluorescence detection method has good repeatability and strong selectivity, and is currently one of the most widely used methods.

When certain substances are irradiated by light of a certain wavelength (excitation light), within a very short period of time, they can immediately emit light with different colors and intensities and longer wavelengths than the excitation light (also called absorption light), and then When the excitation light disappears, it disappears immediately, and this light is fluorescence.

The basic principle of fluorescence detection method is: the sample to be detected is labeled with fluorescein, and fully hybridized with the known gene (probe) on the chip, and the result is displayed by image after washing, and then the sample to be tested is detected by computer processing The expressed biological information. The purpose of labeling is to make detectable tracer marks on the sample to be tested.

The fluorescent substance (fluorescein) releases a certain intensity of fluorescence after being excited by the excitation light, and produces the highest release intensity at a certain wavelength, and has its own excitation absorption value. FluorX, a commonly used fluorescent substance, has an absorption peak of 480nm and an emission peak wave of 520nm. After the labeled sample to be tested is hybridized with the probe array on the biochip, the fluorescently labeled sample is bound to a specific position on the chip, and the unhybridized molecules are removed. At this time, it is necessary to use a detection device to convert the measurement results of the chip into image data that can be analyzed and processed, so as to realize the detection of the fluorescence signal of the biochip.

At present, the real-time detection technologies of biochip signals based on fluorescent labels are mainly divided into two categories.

1. Laser scanning fluorescence microscope detection, laser confocal scanning microscope detection, etc. Its disadvantages are: the scanning accuracy is mainly affected by the mechanical accuracy, repeatability and environmental conditions of the X and Y mobile platforms.

2. Signal detection technology using a cooled CCD and other weak light detection devices. Since the light field irradiated by the excitation light is the entire chip area, and the Gaussian distribution of the laser beam intensity will make the light intensity distribution of the light field uneven, and the signal intensity of the fluorescent marker is linearly related to the intensity of the excitation light, the collected signal cannot be accurately linear response. CCD detection is done based on image processing, so it is easy to cause signal distortion.

Simultaneously the structure of the device of these detection techniques is all more complicated, is not easy to carry.

Contents of the invention

The object of the present invention is to provide a millimeter-scale laser-induced fluorescence detector for detecting fluorescent substances with an excitation wavelength of 470-495nm and an emission wavelength of 510-530nm. This millimeter-level independent detection unit can not only avoid the scanning accuracy of the detection system being mainly affected by the mechanical accuracy, repeatability, and environmental conditions of the X and Y moving platforms, but also ensure the shimming of the excitation light. More importantly, it can improve The fluorescent signal intensity of the biochip fluorescent label, the scanning accuracy and repeatability, simplify the structure of the detection equipment.

The millimeter-scale miniature laser-induced fluorescence detector used in the biochip of the present invention includes a light source, a filter system and a photoelectric detection system, and is characterized in that: the light source is a semiconductor with a peak wavelength of 470-495nm and a depth cutoff of its spectrum at 500nm Laser 1, the filter system is an interference filter film 2 with a cutoff wavelength of 500nm and a peak wavelength of 520nm, and the photoelectric detection part is a micro semiconductor photodetection device 3; the semiconductor laser 1, interference filter film 2, and photodetection device 3 are integrated ; The total volume of the fluorescence detector is at the millimeter level, and its length, width, and height are within the range of 1 mm to 10 mm. This kind of fluorescence detector can be infinitely close to the detected object, or enter the measured object for fluorescence detection, so the optical path of fluorescence from excitation to incident on the photodetector is very short, and the distance is about tens of microns to ten millimeters. The light source adopts a semiconductor laser device, which has good collimation and narrow spectral bandwidth, which can avoid the filter system.

The photodetection device 3 of the present invention can be located directly under the semiconductor laser 1 , can also be paralleled with the semiconductor laser 1 and face the detected object, and can also surround the semiconductor laser 1 .

The photodetection device 3 in Fig. 1 is located directly below the semiconductor laser 1, the interference filter film 2 is between the photodetection device 3 and the semiconductor laser 1, the light source and the photodetection device are located on the same side of the detection object, and the semiconductor laser device is located on the detection object. center of the device. The area of the photosensitive area of the detection device is 2 to 50 times the cross-sectional area of the semiconductor laser device. The photosensitive area of the detection device refers to the absorption layer of the detection device; the cross section here refers to the cross section parallel to the photosensitive area of the detection device. When the semiconductor laser is above the detection device, part of the fluorescence will be reflected or absorbed by the surface of the semiconductor laser and cannot be received by the detection device. Therefore, the area of the photosensitive area of the detection device is required to be slightly larger than the cross-sectional area of the semiconductor laser device.

In Fig. 2, the semiconductor laser 1 and the photodetection device face the detected object side by side. The photodetection device 2 is coated with a filter film with a cut-off wavelength of 500nm and a peak wavelength of 520nm, with a thickness of 1-10 μm. The carrier 4 of the fluorescence detector is used to fix the semiconductor laser 1 and the photodetection device 3, which may not be present.

In Fig. 3, the interference filter film 2 is located above the photodetection device 3 and together with the photodetection device 3 surrounds the semiconductor laser 1, the semiconductor laser 1 in the center is circular or rectangular, and the photodetection device 3 is circular or polygonal.

The electrodes of the photodetection device 3 are made of indium tin oxide transparent film to increase the effective light-receiving area.

The interference filter film 2 is directly coated on the detection device, or is first coated on other transparent substrates, and the transparent substrate is then attached to the photodetection device 3 .

The interference filter film 2 has a thickness of 1-10 μm and is used to filter stray light outside the fluorescence excitation band.

The fluorescent detection device can be formed into an array to form an optical addressing fast scanning system.

The working principle of the present invention is: the semiconductor laser in the micro-laser-induced fluorescence detector emits and shapes a light beam with a peak value of 470-495 nm (or not shaped, but infinitely close to the measured object), while ensuring a depth cut-off at 500 nm. Fluoresceins such as FluorX emit fluorescence with a peak value at 520nm under laser excitation, and the fluorescence is received by the photodetector device through the filter to achieve photoelectric conversion and output a corresponding electrical signal.

The length, width, and height of the device can be miniaturized, within the range of 1 mm to 10 mm; multiple micro-fluorescence instruments can be used in an array to realize multi-point real-time dynamics of fluorescence detection, optical addressing, fast scanning and other functions. In the scanning detection, there is no relative movement between the light source, the measured object and the photoelectric detection system, which eliminates the error and distortion caused by the displacement and makes the detection result closer to the true value. Replace the existing macro instruments, such as the general PCR quantitative and qualitative detection system using plastic reaction tubes.

Description of drawings

Fig. 1 device of the present invention (photodetection device is positioned at semiconductor laser just below) schematic diagram;

Fig. 2 device of the present invention (photodetection device and semiconductor laser are juxtaposed) schematic diagram;

Fig. 3 device (semiconductor laser is surrounded by photodetection device) schematic diagram of the present invention;

Preferred embodiment of the present invention of Fig. 4;

Fig. 5 is used for PCR real-time dynamic detection in preferred embodiment array combination of the present invention;

In Fig. 1-5, 1. Semiconductor laser device 2. Interference filter film 3. Photodetector device 4. Fluorescence detector carrier; 5. Biochip upper piece 6. Biochip lower piece 7. Microchannel 8. Containing fluorescein FluorX solution 9. Emission light 10. Fluorescence

Detailed ways

A preferred embodiment of the present invention will be described in detail with reference to accompanying drawings 4-5.

Referring to accompanying drawing 4: the upper and lower pieces 5 and 6 of the biochip are made of PMMA plexiglass material, and the thickness of each piece of plexiglass is 1mm. Excimer laser processing technology is used to process microchannels on it. The upper and lower pieces 5 and 6 of the biochip are permanently sealed by thermocompression bonding. The light source is a blue light semiconductor laser device 1 with a peak wavelength of 473nm. Its emitted light 9 irradiates the FluorX solution 8 containing fluorescein in the microchannel 7 of the biochip, and the excited fluorescence 10 is filtered by the interference filter film 2 and then photoelectrically detected. Received by the device 3, the optical signal is converted into a corresponding electrical signal by the photodetector device, so as to identify the intensity of the fluorescent signal.

Referring to accompanying drawing 5: Multiple miniature fluorescent instruments can be used in an array to realize real-time dynamics of PCR fluorescence detection at multiple points. Real-time quantitative RT-PCR (Real-time reverse transcriptionquantitative polymerase chain reaction, Real-time RT-PCR), the so-called real-time quantitative PCR refers to the real-time determination of specific products by continuously detecting the strength of fluorescent signals during the exponential amplification of PCR. amount, and based on this, the initial amount of the target gene is deduced. The channel on the PCR chip goes through three temperature zones of denaturation, annealing and extension each cycle. Every time a DNA strand is amplified, a fluorescent molecule is formed, which can realize the complete synchronization of the accumulation of fluorescent signals and the formation of PCR products. The optical properties of each fluorescent signal can be identified by the microfluorometer.

Each miniature fluorometer corresponds to the area to be detected on each cycle of PCR respectively, the microchannel 7 of the biochip is filled with the FluorX solution 8 of fluorescein, and the emitted light 9 of the semiconductor laser device 1 is irradiated onto the microchannel 7 of the biochip In the fluorescein solution 8, the excited fluorescence 10 is received by the photodetection device 3 after being filtered by the interference filter film 2, and is converted from an optical signal to a corresponding electrical signal by the photodetection device. According to the different output of the electrical signal The values allow to infer the characteristics of the products of each cycle of PCR.

Claims (9)

1. the millimeter level miniature laser induced fluorescence detector that uses of biochip, include light source, filter system and photodetector system, it is characterized in that: light source is the semiconductor laser (1) that peak wavelength ends in the 500nm place degree of depth at 470～495nm, its spectrum, filter system is that cutoff wavelength is the interference light filtering film (2) of 500nm peak wavelength at 520nm, and photoelectric detection part is micro semiconductor photoelectric detector (3); Semiconductor laser (1), interference light filtering film (2), photoelectric detector (3) are integrated in one; The fluorescence detector cumulative volume is in the millimeter rank, and its length is in 1mm～10mm scope.

2. the millimeter level miniature laser induced fluorescence detector that biochip according to claim 1 uses, it is characterized in that: photoelectric detector (3) be positioned at semiconductor laser (1) under, between photoelectric detector (3) and the semiconductor laser 1 interference light filtering film (2), semiconductor laser (1) and photoelectric detector (3) are positioned at the same side of detected object, semiconductor laser device is positioned at the centre of sensitive detection parts, and the photosensitive area area of photoelectric detector (3) is 2～50 times of semiconductor laser (1) cross-sectional area.

3. the millimeter level miniature laser induced fluorescence detector that biochip according to claim 1 uses is characterized in that: semiconductor laser (1) be arranged on interference light filtering film (2) above the photoelectric detector and photoelectric detector (3) side by side towards detected object.

4. the millimeter level miniature laser induced fluorescence detector that biochip according to claim 1 uses, it is characterized in that: interference light filtering film (2) is positioned at photoelectric detector (3) top, surround semiconductor laser (1) jointly with photoelectric detector (3), the semiconductor laser (1) that is positioned at central authorities is circle or rectangle, and photoelectric detector (3) is circle or polygon.

5. according to the millimeter level miniature laser induced fluorescence detector of claim 1 or 2 or 3 or 4 described biochips uses, it is characterized in that: the electrode of photoelectric detector (3) adopts the indium tin oxide transparent film, increases effective light-receiving area.

6. the millimeter level miniature laser induced fluorescence detector that uses according to claim 1 or 2 or 3 or 4 described biochips, it is characterized in that: interference light filtering film (2) directly is plated in the top of sensitive detection parts, perhaps be plated in earlier on other transparent substrate, transparent substrate is attached to above the photoelectric detector (3) again.

7. according to the millimeter level miniature laser induced fluorescence detector of claim 1 or 2 or 3 or 4 described biochips uses, it is characterized in that: interference light filtering film (2) thickness is at 1～10 μ m.

8. according to the millimeter level miniature laser induced fluorescence detector of claim 1 or 2 or 3 or 4 described biochips uses, it is characterized in that: a described millimeter level miniature laser induced fluorescence detector can be formed array and form the quick scanning system of light addressing.

9. according to the millimeter level miniature laser induced fluorescence detector of claim 1 or 2 or 3 or 4 described biochips uses, it is characterized in that: adopt fluorescence detector carrier (4) to be used for fixing semiconductor laser (1) and photoelectric detector (3).

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