WO2020038235A1

WO2020038235A1 - High-flux biological, chemical and environmental detection system and method based on planar waveguide technology

Info

Publication number: WO2020038235A1
Application number: PCT/CN2019/099718
Authority: WO
Inventors: 潘涛; 柴贇
Original assignee: 上海攀颂生物科技有限公司
Priority date: 2018-08-20
Filing date: 2019-08-08
Publication date: 2020-02-27
Also published as: CN109085156A

Abstract

Provided is a high-flux biological, chemical and environmental detection system based on planar waveguide technology. The system comprises a planar optical waveguide sensor (109), comprising a first side (1091) and a second side (1092) which are opposite to each other, wherein a probe molecule or a substrate molecule (112) containing fluorophores is fixed on a surface of the first side (1091); a linear excitation light source (101), which can generate excitation light entering the planar optical waveguide sensor (109) and generate evanescent waves on the surface of the first side (1091); a chip container (107), wherein a sealed cavity for a solution to be detected (1071) is enclosed between the chip container and the surface of the first side (1091); and an imaging detection apparatus (111), which is arranged on the second side (1092) of the planar optical waveguide sensor (109). Also provided is a method for detecting a sample by using the system, wherein a sample solution is detected in a sealed manner, so that a detection result is accurate. Various kinds of probe or substrate molecules (112) can be fixed on the surface of the planar optical waveguide sensor (109), and substances in a solution can react with the various kinds of molecules on the surface and generate fluorescence changes in a specific probe area respectively, so that parallel high-flux detection of multiple indexes is completed.

Description

High-throughput biological, chemical, and environmental detection systems and methods based on planar waveguide technology

Technical field

The present application relates to a high-throughput biological, chemical, and environmental detection system and method based on planar waveguide technology, which can be used in the technical fields of biology, medicine, chemistry, and the environment to simultaneously detect multiple indicators to be measured in parallel in one detection.

Background technique

The 21st century is an era of rapid development of science and technology, especially the integration of multiple disciplines such as chemistry, biology, environmental science, medicine and electronic information will get vigorous development. Sensing technology is an important research area in the discipline of electronic information and an important means of information acquisition. And the development of technical fields such as biology, medicine, chemistry, and environment cannot be separated from the improvement and improvement of corresponding detection technology. Therefore, linking the sensing technology with the actuality of these disciplines to develop new efficient, high-throughput detection systems is also an important work in these fields.

Waveguide technology is a new detection method developed based on optical technology. It uses light waves to transmit in the waveguide in a total reflection manner to generate an evanescent wave at the interface of the sensor. This evanescent wave can excite the probe connected to the sensor surface. The fluorophore on the target or the fluorophore of the target molecule captured by the probe is combined with the change in fluorescence to realize the quantitative detection of the target molecule. The infiltration depth of the evanescent wave on the surface of the sensor is only tens to hundreds of nanometers, and the free fluorescent molecules in the solution body have little effect on the detection result, which can reduce the effect of impurities in the solution on the detection result. It has strong characteristics, high sensitivity, and fast detection speed. It has a strong application prospect in biomedicine, environmental detection, and chemical detection.

The waveguide technology platform has two directions in development. The first is a fiber-optic waveguide detection platform, which is characterized by simple use and low price, but its detection flux is not high, and it can only detect one target at a time. In order to overcome the flux problem, several planar waveguide detection platforms have been developed for synchronous high-throughput detection, that is, detecting multiple targets simultaneously. However, these systems are developed based on point light sources or line light sources. In order to realize the imaging of flat areas during the specific use process, it is necessary to use moving parts to scan the excitation light of the light source inside the waveguide material, which increases the system's The complexity, cost and difficulty of synchronous detection cannot meet the requirements of low cost and high speed detection. At the same time, most of the detection platforms of the platform waveguide are open systems, that is, the sample solution is directly added to the sensor, and then the evanescent wave is used for excitation detection. The advantage of this is that the sensor is simple in design and low in cost, but because it is in contact with air during use, not only the accuracy of the experimental results is difficult to guarantee, but also the mutual interference of the test is very serious. At the same time, in actual use, biological and chemical sample solutions require some pretreatment or reaction before fluorescent detection can be used, and waveguide detection after processing is not necessary.

Summary of the Invention

The object of the present invention is to provide a high-throughput biological, chemical, and environmental detection system and method based on planar waveguide technology. The technology that overcomes existing waveguide detection platforms (optical fiber and planar waveguide existing platforms) is not satisfactory for biology and medicine. , Chemical, environmental and other technical fields of high-throughput, low-cost, fast detection requirements.

To achieve the above objective, the present invention provides the following technical solutions:

An embodiment of the present application discloses a high-throughput biological, chemical, and environmental detection system based on a planar waveguide technology, including:

A planar optical waveguide sensor includes a first side and a second side opposite to each other, and a probe molecule or a substrate molecule containing a fluorescent group is fixed on a surface of the first side;

The linear excitation light source can generate the excitation light that enters the planar optical waveguide sensor and generates an evanescent wave on the surface of the first side;

A chip container is enclosed with a surface of the first side surface to form a sealed test solution cavity;

The imaging detection device is disposed on the second side of the planar optical waveguide sensor.

Preferably, in the above-mentioned high-throughput biological, chemical, and environmental detection system based on the planar waveguide technology, the imaging detection device and the chip container are respectively disposed on the upper and lower sides of the planar optical waveguide sensor.

Preferably, the above-mentioned high-throughput biological, chemical, and environmental detection system based on the planar waveguide technology further includes an optical baffle disposed between the linear excitation light source and the planar optical waveguide sensor, and the optical baffle is provided with a slit. .

Preferably, in the above-mentioned high-throughput biological, chemical, and environmental detection system based on the planar waveguide technology, the size of the slit satisfies: the generation of an evanescent wave on the surface of the first side, which can cover the probe molecules or the fluorescent group-containing All regions of the substrate molecule.

Preferably, the above-mentioned high-throughput biological, chemical, and environmental detection system based on the planar waveguide technology further includes a heating device for controlling the temperature of the chip container.

Preferably, in the above-mentioned high-throughput biological, chemical, and environmental detection system based on the planar waveguide technology, the probe molecule or the substrate molecule containing a fluorescent group is fixed to the substrate by a chemical bond, Van der Waals force, hydrogen bond, or hydrophobic interaction. A first side surface of a planar optical waveguide sensor.

Preferably, in the above-mentioned high-throughput biological, chemical, and environmental detection system based on the planar waveguide technology, a plurality of nucleic acid probes for different nucleic acid sequences to be tested are fixed on the surface of the planar optical waveguide sensor.

Preferably, in the above-mentioned high-throughput biological, chemical, and environmental detection system based on the planar waveguide technology, the target biological and chemical molecules (groups) to be measured are fixed on the surface of the planar optical waveguide sensor;

Or a molecule (group) capable of effectively competing with a test molecule (group) for binding to an antibody or a nucleic acid aptamer in a solution;

Or an antibody or aptamer molecule that directly binds to the test molecule;

Or it can be degraded or cleaved by the test molecules to lose fluorescently labeled substrate molecules.

Preferably, in the above-mentioned high-throughput biological, chemical, and environmental detection system based on the planar waveguide technology, the imaging detection device is a PMT, a CCD camera, or a CMOS camera.

Accordingly, this application also discloses a detection method, including:

A reaction solution containing a sample to be tested is contained in a chamber of the solution to be tested;

Activate the linear excitation light source to generate an evanescent wave on the surface of the first side of the planar optical waveguide sensor;

The chemical or biological reaction between the reaction solution and the test sample produces a change in fluorescence in the interval of the evanescent wave;

Collect images containing fluorescent signals to determine the concentration information of the sample to be measured.

Compared with the prior art, the advantages of the present invention are:

1. Various biological and chemical reactions can be completed in the sample solution, and changes in fluorescence intensity can be generated for detection.

2. The excitation light source is a linear area light source, which can be scanned without using mechanical moving parts, which is more stable and reliable.

3. A variety of probes or substrate molecules can be fixed on the sensor surface. The substances in the solution can interact with a variety of molecules on the surface and generate fluorescence changes in specific probe regions, respectively. Flux detection.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to explain the technical solutions in the embodiments of the present application or the prior art more clearly, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are merely These are some of the embodiments described in this application. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without paying creative labor.

FIG. 1 is a schematic structural diagram of a detection system in a specific embodiment of the present invention.

FIG. 2 is a schematic diagram showing a working principle of a planar optical waveguide sensor in a specific embodiment of the present invention; FIG.

FIG. 3 is a schematic diagram showing the arrangement of probes in the application example 1 of the present invention.

detailed description

The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are part of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by a person of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

In the description of the present invention, it should be noted that the terms "center", "up", "down", "left", "right", "vertical", "horizontal", "inside", "outside", etc. The indicated orientation or positional relationship is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present invention and simplified description, and does not indicate or imply that the device or element referred to must have a specific orientation, a specific orientation Construction and operation should therefore not be construed as limiting the invention. In addition, the terms "first," "second," and "third" are used for descriptive purposes only, and should not be construed to indicate or imply relative importance.

In the description of the present invention, it should be noted that the terms "installation", "connected", and "connected" should be understood in a broad sense unless otherwise specified and limited. Connected or integrated; it can be mechanical or electrical; it can be directly connected, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood on a case-by-case basis.

With reference to FIG. 1, an embodiment of the present application provides a high-throughput biological, chemical, and environmental detection system based on a planar waveguide technology, including a linear excitation light source 101, a chip container 107, a planar optical waveguide sensor 109, and an imaging detection device 111.

As shown in FIG. 2, the planar optical waveguide sensor 109 uses a light-tight medium, including glass, quartz, and polymer materials. The planar optical waveguide sensor 109 includes a first side 1091 and a second side 1092 opposite to each other. Probe molecules or substrate molecules 112 containing fluorescent groups are immobilized on the surface (see FIG. 3).

The linear excitation light source 101 is a laser or LED light source. The linear excitation light source 101 can generate excitation light that enters the planar optical waveguide sensor 109 and generates an evanescent wave on the surface of the first side 1092.

A sealed test solution cavity 1071 is enclosed between the chip container 107 and the surface on the first side.

In a preferred embodiment, the planar optical waveguide sensor is horizontally covered on the top of the chip container 107.

The imaging detection device 111 is a PMT, a CCD camera, or a CMOS camera, and is disposed on the second side of the planar optical waveguide sensor 109. An image of the planar optical waveguide sensor can be obtained through the imaging detection device 111, and concentration information of a substance to be measured or a reaction caused by the substance to be measured can be obtained from a fluorescent signal in the image.

In an embodiment, the front end of the imaging detection device 111 is further provided with a lens group 110. The lens group 110 includes an optical filter, and the optical filter is used to filter the interference of the excitation light.

In one embodiment, a lens or a lens group 102 disposed at the front end of the linear excitation light source 101 is further included.

In one embodiment, an optical baffle 103 is further provided between the linear excitation light source 101 and the planar optical waveguide sensor 109. The optical baffle 103 is provided with a slit 104.

A slit on the baffle is used to generate a linear transmission of a specific long width (such as a rectangle).

In one embodiment, it further includes a base 106, and the chip container 107 and the optical baffle 103 are supported on the base 106.

Further, the base 106 is supported on the sample platform 105, and the sample platform 105 may be provided with a heating device for controlling the temperature of the chip container.

In one embodiment, the sample platform 105 can also realize one-axis or multi-axis movement.

In operation, firstly, a probe molecule or a substrate molecule 112 containing a fluorescent group is fixed on the first side surface (preferably the lower surface) of the planar optical waveguide sensor 109. The fixing method may be chemical bonding, van der Waals force, hydrogen bonding, hydrophobic Role and many other ways.

Then, the planar optical waveguide sensor 109 and the chip container 107 constitute a closed detection chip, which is used to contain the reaction solution 108 containing the sample to be measured.

Next, the chip container 107 carrying the reaction solution is placed on the sample platform 105, and a temperature control program is started, and a chemical or biological reaction occurs between the reaction solution and the test sample in the detection chip through time temperature control, including but not limited to:

(1) Amplification, cleavage and modification of nucleic acid (DNA or RNA);

(2) Hybridization binding between nucleic acid and probe;

(3) Immunity between antibody and antigen;

(4) the interaction between the aptamer and the substrate;

(5) Indirect competitive immune response between the probe fixed on the lower surface of the chip, the substance to be tested, and the same fluorescently labeled antibody;

(6) Cutting reaction of the substance to be tested directly or indirectly to the fluorescent substrate containing the lower surface of the chip.

These reactions will cause a change in fluorescence in the interval of the evanescent wave on the lower surface of the planar optical waveguide sensor 109. Target molecules that are not in the evanescent wave region do not generate fluorescence without being excited by the excitation light.

Next, at the appropriate timing of the reaction, or after the reaction is completed, turn on the linear excitation light source 101 and let the excitation light pass through the slit to illuminate the side of the planar optical waveguide sensor 109 with a critical angle that produces an evanescent wave. The size is designed so that after the incident light enters the waveguide material, a total reflection (evanescent wave) on the bottom surface can cover all areas where a probe is present on the lower surface of the waveguide material.

When the linear excitation light source 101 is turned on, all regions where the probe is present on the lower surface of the planar optical waveguide sensor 109 will have an evanescent wave of excitation light, which will excite fluorescent molecules in this area, but the fluorescent molecules in the solution body will not emit light to cause interference. . At this time, the imaging detection device 111 is turned on, and an image of the upper surface of the planar optical waveguide sensor 109 is collected from the top to the bottom. The image includes the fluorescent signal of the probe or the fluorescent substrate region. By combining the original probe position from the image, the concentration information of the test substance or the reaction caused by the amplification or test substance can be obtained.

Finally, according to the temperature-time-data acquisition parameters set by the time limit, we can obtain several fluorescent pictures of the waveguide material. After analyzing these pictures, we can finally determine the concentration of the measured substance and other information (analysis method and judgment method) (The existing technology is mature, which is not described in this embodiment).

In summary, the detection system is low-cost and reliable, has convenient control, fast reading and high sensitivity, and can be used in a variety of detection scenarios. The detection system also includes temperature control, sampling synchronization and data processing storage components.

Application Example 1-Biological detection (taking nucleic acid detection as an example)

Sexually Transmitted Diseases (STD)-Diseases that are transmitted by sexually transmitted bacteria through sexual behavior, causing genitourinary tract, or even the entire system infection, are called sexually transmitted diseases, commonly known as sexually transmitted diseases in China. There are many kinds of common sexually transmitted diseases, the most important of which are gonorrhea, syphilis, mycoplasma urealyticum, chlamydia trachomatis, and capsular bacillus granulomatosis, etc. In clinical tests, it is necessary to distinguish the type and severity of the infectious disease pathogens in patients. However, common culture or PCR methods cannot meet the requirements of high-throughput detection.

With reference to FIG. 3, the multiple detection of STD in this embodiment can be summarized as the following process:

1. Design a pair of Cy5 fluorescently labeled universal primers to simultaneously amplify the DNA of different types of pathogens in the sample, and design unique probes for different pathogens for hybridization.

2. Collect the patient's genital abscess or exudate, extracts from local lymph nodes, use nucleic acid extraction equipment to extract the nucleic acids in the sample, add them to the chip container 107, and complete the primers (PCR primers are DNA fragments, and primers for DNA replication in cells Is a piece of RNA strand), enzymes, dNTPs and buffers into high-throughput biological, chemical, and environmental detection systems.

3. Control the temperature change of the waveguide chip to complete the temperature change of the classical polymerase chain reaction PCR cycle, and each cycle undergoes denaturation, annealing and extension.

At the end of the cycle extension step of the PCR reaction, the linear excitation light source 101 is turned on, and the side of the planar optical waveguide sensor 109 is illuminated according to the critical angle of the evanescent wave. An evanescent wave of excitation light is generated on the lower surface of the planar optical waveguide sensor 109. At this time, the probe on the lower surface of the planar optical waveguide sensor 109 can capture (hybridize) the amplified nucleic acid copy of the sample containing the fluorescent group by PCR. These captured nucleic acid copies are induced to emit fluorescence by the evanescent wave under the planar optical waveguide sensor 109. And collected by the imaging detection device 111.

4. Analyze the collected fluorescent signals to determine whether the target molecule corresponding to the probe is present and how strong it is.

5 Repeat the cycle of PCR and signal acquisition 35-40 times, plot the fluorescence intensity and cycle number of the target molecule corresponding to a single probe, determine the corresponding cycle number according to the Ct judgment method of real-time quantitative PCR, and calculate the start of the corresponding target molecule. concentration.

6. Analyze the concentration of the sexually transmitted disease pathogens in clinical samples corresponding to probes 1-7, and issue a test report.

It can be seen that the high-throughput biological, chemical, and environmental detection system of the present invention can test the concentration of target nucleic acid molecules in a sample in parallel, and has a strong application prospect in biomedicine.

Application Example 2-Indirect Competitive Immunoassay (Taking Aflatoxin as an Example)

We need to choose an indirect competitive immune model, that is, using a fluorescently labeled aflatoxin antibody (or aptamer) as a detection reagent, and immobilize the molecule of the species under test (here, Aspergillus flavus) on the lower surface of the planar optical waveguide sensor 109. Molecule), the fluorescently labeled antibody (or aptamer) can compete with aflatoxin in the test sample and aflatoxin immobilized on the lower surface of the planar optical waveguide sensor 109, that is, aflatoxin in solution When there is more mycin, the antibody bound to the aflatoxin molecule immobilized on the lower surface of the planar optical waveguide sensor 109 will be reduced, and the fluorescence intensity will decrease.

Next, the sample to be tested is added to the chip container 107, and the temperature control program is started. The excitation light source is turned on to a certain extent, and an evanescent wave is generated on the lower surface of the planar optical waveguide sensor 109. The evanescent wave excites the aflatoxin captured in the area. The antibody produces fluorescence. At this time, the imaging detection device 111 is turned on to obtain a detection picture, and the fluorescence intensity of the probe region is analyzed in the picture, so that the concentration of the aflatoxin to be measured in the sample can be analyzed. If we simultaneously fix other mycin molecules on the lower surface of the planar optical waveguide sensor 109, and add antibodies (fluorescent labels) to other mycins in the solution, we can simultaneously detect the concentrations of multiple mycins in the sample to be tested, and complete Qualcomm.量测。 Quantity detection.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, but not limited thereto. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: It is still possible to modify the technical solutions described in the foregoing embodiments, or to replace some or all of the technical features equivalently; and these modifications or replacements do not depart from the essence of the corresponding technical solutions of the technical solutions of the embodiments of the present invention. range.

Claims

A high-throughput biological, chemical, and environmental detection system based on planar waveguide technology, which includes:

A planar optical waveguide sensor includes a first side and a second side opposite to each other, and a probe molecule or a substrate molecule containing a fluorescent group is fixed on a surface of the first side;

The linear excitation light source can generate the excitation light that enters the planar optical waveguide sensor and generates an evanescent wave on the surface of the first side;

A chip container is enclosed with a surface of the first side surface to form a sealed test solution cavity;

The imaging detection device is disposed on the second side of the planar optical waveguide sensor.
The high-throughput biological, chemical, and environmental detection system based on the planar waveguide technology according to claim 1, wherein the imaging detection device and the chip container are respectively disposed on the upper and lower sides of the planar optical waveguide sensor.
The high-throughput biological, chemical, and environmental detection system based on the planar waveguide technology according to claim 1, further comprising an optical baffle disposed between the linear excitation light source and the planar optical waveguide sensor, and the optical baffle Slit.
The high-throughput biological, chemical, and environmental detection system based on the planar waveguide technology according to claim 3, wherein the size of the slit is such that an evanescent wave generated on the surface of the first side can cover the probe molecules or the All regions of the substrate molecule of the fluorophore.
The high-throughput biological, chemical, and environmental detection system based on the planar waveguide technology according to claim 1, further comprising a heating device for controlling the temperature of the chip container.
The high-throughput biological, chemical, and environmental detection system based on the planar waveguide technology according to claim 1, wherein the probe molecule or the substrate molecule containing a fluorescent group passes a chemical bond, Van der Waals force, hydrogen bond, or The hydrophobic effect is fixed on the first side surface of the planar optical waveguide sensor.
The high-throughput biological, chemical, and environmental detection system based on the planar waveguide technology according to claim 1, wherein the surface of the planar optical waveguide sensor is fixed with a plurality of nucleic acid probes for different nucleic acid sequences to be tested.
The high-throughput biological, chemical, and environmental detection system based on the planar waveguide technology according to claim 1, wherein the surface of the planar optical waveguide sensor is fixed with the target biological and chemical molecules (groups) to be measured;

Or a molecule (group) capable of effectively competing with a test molecule (group) for binding to an antibody or a nucleic acid aptamer in a solution;

Or an antibody or aptamer molecule that directly binds to the test molecule;

Or it can be degraded or cleaved by the test molecules to lose fluorescently labeled substrate molecules.
The high-throughput biological, chemical, and environmental detection system based on the planar waveguide technology according to claim 1, wherein the imaging detection device is a PMT, a CCD camera, or a CMOS camera.
The detection method of the system according to any one of claims 1 to 9, further comprising:

A reaction solution containing a sample to be tested is contained in a chamber of the solution to be tested;

Activate the linear excitation light source to generate an evanescent wave on the surface of the first side of the planar optical waveguide sensor;

The chemical or biological reaction between the reaction solution and the test sample produces a change in fluorescence in the interval of the evanescent wave;

Collect images containing fluorescent signals to determine the concentration information of the sample to be measured.