An optical system for synchronous detection of full-wavelength absorbance of microplate samples

Abstract

The present invention discloses an optical system for synchronous detection of full-wavelength absorbance of microplate samples, which is composed of a light source, a transmitting optical fiber, a transmitting lens group, a microplate, a receiving lens group, a receiving optical fiber and a spectrometer. The transmitting lens group is composed of a first lens, an aperture and a first reflector, and the first reflector is arranged at a 45-degree angle with the main optical axis of the first lens. The receiving lens group is composed of a second lens, a second reflector and a third lens, and the second reflector is arranged at a 45-degree angle with the main optical axis of the second lens. The optical system of the present invention can realize online synchronous detection of full-wavelength absorbance of biological samples in microplates. When detecting absorbance, the microplate can be moved, or the transmitting lens group and the receiving lens group can be moved, and absorbance detection can be performed on biological samples in microplates with the same single hole diameter or different single hole diameters, so as to achieve high repeatability and consistency in absorbance detection of biological samples in microplates, thereby meeting the detection accuracy required by the experiment.

Description

Technical Field

[0001] The invention relates to an optical system for biological detection, in particular to an optical system for synchronous detection of full-wavelength absorbance of microplate samples. Background Art

[0002] In the current scientific research field of microorganisms, a large number of microbial cultivation experiments need to be carried out. Due to the characteristics of small volume and large throughput of microplates, more and more researchers have begun to use microplates for liquid cultivation of microorganisms. In the process of microbial cultivation, it is generally necessary to detect the absorbance of the sample solution in the microplate to observe the growth concentration of the microorganisms. Absorbance refers to the logarithm of the ratio of the intensity of the incident light and the outgoing light of the light passing through the sample to be tested, i.e., lg (incident light intensity / outgoing light intensity). Therefore, it is necessary to accurately and stably measure the incident light intensity value and the outgoing light intensity value of the sample.

[0003] However, due to the small sample capacity in a single well of a microplate, the traditional sampling and detection method cannot be used. In addition, the single wells of a microplate are generally circular holes arranged in a matrix, and the spectrophotometer cannot be used to directly detect the absorbance of the side wall. In addition, the single wells of different types of microplates have different diameters. The liquid surface of the bacterial solution in the wells of different diameters will form different concave surfaces, and the refraction of the concave surface will have a greater impact on the absorbance detection beam.

[0004] At the same time, with the development of current microbial scientific research experiments, a new demand for simultaneous detection of full-wavelength absorbance has been proposed. Full wavelength generally refers to a spectrum that includes part of the ultraviolet, all visible light, part of the infrared, etc., with a wavelength range of about 300 to 900 nanometers. Conventional microplates on the market are made of plastic materials such as polystyrene, which have a large absorption interference with ultraviolet and infrared light, and the transmittance of plastic microplates is unstable. Its transmittance will be greatly affected by changes in temperature, acid and alkali solutions and other factors, and is not suitable for full-wavelength absorbance detection. In addition, the fully transparent hole wall also has the problem of interference from stray light.

[0005] Since there are currently no devices or instruments on the market that meet the above detection requirements, in order to solve this problem, it is necessary to design an optical detection system to achieve the function of online synchronous detection of full-wavelength absorbance of multiple types of microplate biological samples. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide an optical system for synchronous detection of full-wavelength absorbance of microplate samples in view of the deficiencies of the prior art.

[0007] The technical solution adopted by the present invention to solve the above technical problems is: an optical system for synchronous detection of full-wavelength absorbance of microplate samples, which is composed of a light source, a transmitting optical fiber, a transmitting lens group, a microplate, a receiving lens group, a receiving optical fiber and a spectrometer arranged in sequence along the light path, wherein the light source is used to emit a full-wavelength continuous light source, the transmitting lens group is used to converge the divergent light beam transmitted by the transmitting optical fiber into a fine-diameter light beam, the transmitting lens group is composed of a first lens, an aperture and a first reflector arranged in sequence along the light path, the first reflector is arranged at a 45-degree angle to the main optical axis of the first lens, and the first reflector is used to The outgoing light beam of the first lens is changed to be incident vertically upward into the sample in the hole of the microplate. The receiving lens group is used to converge the outgoing light beam of the sample and transmit it to the spectrometer through the receiving optical fiber. The receiving lens group consists of a second lens, a second reflector and a third lens arranged in sequence along the light path. The second reflector is arranged at an angle of 45 degrees to the main optical axis of the second lens. The second reflector is used to change the outgoing light beam of the second lens to be incident horizontally into the third lens. The spectrometer calculates the absorbance value of the sample by analyzing the light energy difference between the incident light and the outgoing light of the sample.

[0008] The main detection principle of the optical system of the present invention is: the emission light for absorbance detection provided by the light source is transmitted to the emission lens group through the emission optical fiber. After the emission light is converged into a thin diameter light beam by the emission lens group, it is incident vertically upward from the bottom to the sample in the hole of the microplate, and then through the receiving lens group, the outgoing light beam of the sample is converged to the receiving optical fiber, and then transmitted to the spectrometer by the receiving optical fiber. The spectrometer calculates the absorbance value of the sample by analyzing the light energy difference between the incident light and the outgoing light of the sample.

[0009] The optical system of the present invention can realize online synchronous detection of the full-wavelength absorbance of biological samples in a microplate. During absorbance detection, the microplate can be moved, or the transmitting lens group and the receiving lens group can be moved to perform absorbance detection on biological samples in microplates with the same single hole diameter or different single hole diameters, thereby achieving high repeatability and consistency in absorbance detection of biological samples in microplates, thereby meeting the detection accuracy required by the experiment. For single-hole samples with large diameters, the absorbance detection accuracy of microorganisms that are prone to agglomeration, such as algae, can also be further improved by taking the average value through multi-point detection.

[0010] Preferably, the light source is a xenon lamp, LED lamp or halogen tungsten lamp that can emit full-wavelength continuous light source.

[0011] Preferably, the transmitting optical fiber is an optical fiber with a protective shell on the outer layer, and its two ends are metal-encapsulated transmitting ports, one end is used to connect to the light source, and the other end is used to connect to the first lens; the receiving optical fiber is an optical fiber with a protective shell on the outer layer, and its two ends are metal-encapsulated receiving ports, one end is used to connect to the third lens, and the other end is used to connect to the spectrometer.

[0012] Preferably, the transmitting optical fiber and the receiving optical fiber both use quartz fiber cores.

[0013] Preferably, the first lens, the second lens and the third lens are all convex lenses, and the first reflector and the second reflector are lenses with a reflective film coated on one side surface.

[0014] Preferably, the micro-porous plate is a porous plate of a composite material with a transparent bottom, the hole bottom is made of quartz material, and the hole wall is made of black plastic material.

[0015] Compared with the prior art, the present invention has the following advantages:

[0016] 1. Realize online synchronous detection of full-wavelength absorbance of biological samples in microplates.

[0017] 2. Realize absorbance detection of biological samples in microplates with the same or different single-hole diameters.

[0018] 3. Achieve high repeatability and consistency in absorbance detection of biological samples in microplates, thereby meeting the detection accuracy required by the experiment. For single-hole samples with large diameters, the absorbance detection accuracy of algae and other microorganisms that are prone to agglomeration can be further improved by taking the average value through multi-point detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a general schematic diagram of the optical system in the embodiment;

[0020] Figure 2 Schematic diagram of the optical path for absorbance detection of microplate samples in the embodiment;

[0021] Number Description:

[0022] 1 light source, 2 transmitting optical fiber, 21 transmitting port, 3 transmitting lens group, 31 first lens, 32 aperture, 33 first reflector, 4 microplate, 41 sample, 42 hole bottom, 43 hole wall, 5 receiving lens group, 51 second lens, 52 second reflector, 53 third lens, 6 receiving optical fiber, 61 receiving port, 7 spectrometer. DETAILED DESCRIPTION

[0023] The present invention is further described in detail below with reference to the accompanying drawings.

[0024] The optical system for synchronous detection of full-wavelength absorbance of microplate samples in the embodiment is as follows: Figure 1 and Figure 2 As shown, it is composed of a light source 1, an emitting optical fiber 2, an emitting lens group 3, a microplate 4, a receiving lens group 5, a receiving optical fiber 6 and a spectrometer 7 which are sequentially arranged along the optical path. The main detection principle of the optical system is as follows: the emission light for absorbance detection provided by the light source 1 is transmitted to the emitting lens group 3 through the emitting optical fiber 2, and the emission light is converged into a thin diameter light beam through the emitting lens group 3, and then incident vertically upward from the bottom to the sample 41 in the hole of the microplate 4, and then through the receiving lens group 5, the outgoing light beam of the sample 41 is converged to the receiving optical fiber 6, and then transmitted to the spectrometer 7 by the receiving optical fiber 6, and the spectrometer 7 calculates the absorbance value of the sample 41 by analyzing the light energy difference between the incident light and the outgoing light of the sample 41.

[0025] The light source 1 is used to emit a full-wavelength continuous light source, the emitting lens group 3 is used to converge the divergent light beam transmitted by the emitting optical fiber 2 into a thin diameter light beam, the emitting lens group 3 is composed of a first lens 31, an aperture 32 and a first reflector 33 arranged in sequence along the light path, the first reflector 33 is arranged at a 45-degree angle to the main optical axis of the first lens 31, the first reflector 33 is used to change the outgoing light beam of the first lens 31 to be vertically incident upward into the sample 41 in the hole of the microplate 4, the receiving lens group 5 is used to converge the outgoing light beam of the sample 41 and transmit it to the spectrometer 7 through the receiving optical fiber 6, the receiving lens group 5 is composed of a second lens 51, a second reflector 52 and a third lens 53 arranged in sequence along the light path, the second reflector 52 is arranged at a 45-degree angle to the main optical axis of the second lens 51, the second reflector 52 is used to change the outgoing light beam of the second lens 51 to be horizontally incident into the third lens 53, and the spectrometer 7 calculates the absorbance value of the sample 41 by analyzing the light energy difference between the incident light and the outgoing light of the sample 41.

[0026] In this embodiment, the light source 1 is a xenon lamp that emits a full-wavelength continuous light source. The xenon lamp can emit a full-wavelength continuous light source of 190 to 2000 nanometers, and has the characteristics of no need for preheating, stable luminous intensity, and long life.

[0027] The transmitting optical fiber 2 is a multimode optical fiber with a protective shell on the outer layer, and the two ends are metal-encapsulated transmitting ports 21, one end is used to connect to the light source 1, and the other end is used to emit a conical light beam with a certain divergence angle and connect to the first lens 31; the receiving optical fiber 6 is a multimode optical fiber with a protective shell on the outer layer, and the two ends are metal-encapsulated receiving ports 61, one end is used to connect to the third lens 53, and the other end is used to connect to the spectrometer 7. The transmitting optical fiber 2 and the receiving optical fiber 6 in this embodiment both use a quartz core with high purity, and the wavelength range of light allowed to pass is 250 to 950 nanometers, the optical fiber numerical aperture is preferably 0.12 to 0.26, and the optical fiber core diameter is preferably 50 to 400 microns.

[0028] The first lens 31 is arranged at the rear end of the emission port 21, and is used to initially converge and collimate the outgoing light of the emission port 21. The first lens 31 is designed as a convex lens with a diameter of 3 to 10 mm and a focal length of 5 to 12 mm. The material is preferably fused quartz glass, and the light transmission wavelength range is 185 to 1100 nanometers, with good transmittance.

[0029] The aperture 32 is a light shielding plate with a small circular hole in the center, arranged at the rear end of the first lens 31, for further limiting the diameter of the output beam of the first lens 31 and for eliminating excess stray light. The diameter of the small circular hole of the aperture 32 is preferably 0.5 to 5 mm.

[0030] The first reflector 33 is a lens with a reflective film coated on one side surface, and is used to change the direction of the outgoing light beam of the first lens 31 , and is also helpful to compress the vertical occupied space of the emitting lens group 3 .

[0031] The microplate 4 is designed as a multi-well plate with a transparent bottom made of composite material. The positions of the holes are arranged in a matrix, and each single hole can hold a certain volume of sample 41. The bottom 42 of the hole of the microplate 4 is made of quartz material to improve the light transmittance. The plane thickness of the bottom 42 of the hole is designed to be 0.3-0.6mm, and the parallelism requirement is 0.02-0.05mm to reduce the refraction effect of the bottom 42 of the hole on the detection light path. The hole wall 43 of the microplate 4 is made of black heat-resistant and chemically resistant plastic material to eliminate the interference of multiple reflections of scattered light in the hole. The microplate 4 is designed with a variety of hole number specifications, such as 12-hole microplate, 24-hole microplate, 48-hole microplate, 96-hole microplate, 384-hole microplate, etc. The diameters of single holes of microplates with different numbers of holes are different. Single holes of different diameters are suitable for culturing different types of microorganisms. Figure 2As shown, sample 41 is a solution to be tested. In biological experiments, sample 41 is generally a bacterial solution mixed with bacterial species and culture solution in a certain proportion. When sample 41 is added to a single hole of microplate 4, due to the adsorption effect between the liquid and the single hole wall 43, the liquid surface of sample 41 tends to form a liquid surface that is concave in the direction of gravity. At this time, sample 41 has the effect of a concave lens, which will produce a refraction and dispersion effect on the incident light beam. The full-wavelength incident light beam of the emitting lens group 3 diverges after passing through sample 41, and samples 41 in single holes of different diameters will produce different concave radii, thereby producing different divergence angles.

[0032] The second lens 51 is arranged at the rear end of the light beam emitted from the sample 41, and is used to initially converge the divergent light beam emitted from the sample 41. The second lens 51 is designed as a convex lens with a diameter of 8 to 20 mm and a focal length of 6 to 18 mm. The material is preferably fused quartz glass, and the light transmission wavelength range is 185 to 1100 nanometers, with good transmittance.

[0033] The second reflector 52 is a lens with a reflective film coated on one side surface, which is used to change the direction of the outgoing light beam of the second lens 51 and is helpful to compress the vertical occupied space of the receiving lens group 5.

[0034] The third lens 53 is arranged at the rear end of the light beam emitted by the second reflector 52, and is used to converge the light beam emitted by the second lens 51 again. The third lens 53 is designed as a convex lens with a diameter of 4 to 8 mm and a focal length of 6 to 10 mm. The material is preferably fused quartz glass, and the light transmission wavelength range is 185 to 1100 nanometers, with good transmittance.

[0035] Through the cooperation of the transmitting lens group 33 and the receiving lens group 5, the light beam incident from the light source 1 to the sample 41 can be completely transmitted to the spectrometer 7, thereby greatly reducing the light refraction and dispersion errors caused by the differences in the concentrations of different samples 41 and the liquid surface depressions. The spectrometer 7 is used to analyze the light transmitted back by the receiving optical fiber 6. It has the ability to split the full-wavelength light beam with a resolution of 1 nanometer. It can perform separate light energy calculations for different wavelengths, and then calculate the absorbance values ​​of different wavelengths within the range of 300 to 900 nanometers by analyzing the difference between the incident light and the outgoing light of the sample 41.

[0036] In summary, the present invention has better solved the problem of detecting multi-wavelength absorbance of biological samples in multi-type microplates. Through optical simulation software, light path dispersion simulation is performed on multiple wavelengths such as 300 nanometers, 600 nanometers, and 900 nanometers, and light path refraction simulation is performed on the concave surface of samples with different diameters of single holes. In combination with actual prototype testing, it is confirmed that the present invention achieves a high repeatability in the absorbance detection of single-hole samples in microplates, and at the same time achieves a high consistency in the absorbance detection between each single hole, effectively solving the problem of online synchronous detection of absorbance at all wavelengths in high-throughput culture experiments on microplates. In addition, through practical research, it is found that for microorganisms that are easy to agglomerate, such as algae, it is necessary to use a microplate with a single hole diameter of more than 12 mm for cultivation. It is also possible to move the transmitting lens group and the receiving lens group or the microplate to detect the absorbance of multiple liquid surface positions in the single hole, and then calculate the absorbance average value, which can further reduce the detection error of biological samples on the microplate.

Claims

1. An optical system for synchronous detection of full-wavelength absorbance of microplate samples, It is characterized in that The invention is composed of a light source, a transmitting optical fiber, a transmitting lens group, a micro-well plate, a receiving lens group, a receiving optical fiber and a spectrometer which are arranged in sequence along the light path. The light source is used to transmit a full-wavelength continuous light source. The transmitting lens group is used to converge the divergent light beam transmitted by the transmitting optical fiber into a thin-diameter light beam. The transmitting lens group is composed of a first lens, an aperture and a first reflector which are arranged in sequence along the light path. The first reflector is arranged at an angle of 45 degrees to the main optical axis of the first lens. The first reflector is used to change the outgoing light beam of the first lens to be incident vertically upward into the sample in the hole of the micro-well plate. The receiving lens group is used to converge the outgoing light beam of the sample and transmit it to the spectrometer through the receiving optical fiber. The receiving lens group is composed of a second lens, a second reflector and a third lens which are arranged in sequence along the light path. The two reflectors are arranged at an angle of 45 degrees to the main optical axis of the second lens. The second reflector is used to change the outgoing light beam of the second lens to be horizontally incident on the third lens. The spectrometer calculates the absorbance value of the sample by analyzing the light energy difference between the incident light and the outgoing light of the sample; the transmitting optical fiber is an optical fiber with a protective shell on the outer layer, and both ends are metal-encapsulated transmitting ports, one end is used to connect to the light source, and the other end is used to connect to the first lens; the receiving optical fiber is an optical fiber with a protective shell on the outer layer, and both ends are metal-encapsulated receiving ports, one end is used to connect to the third lens, and the other end is used to connect to the spectrometer; the first lens, the second lens, and the third lens are all convex lenses, and the first reflector and the second reflector are both lenses with a reflective film coated on one side surface.

2. An optical system for synchronous detection of full-wavelength absorbance of microplate samples according to claim 1, It is characterized in that The light source is a xenon lamp, LED lamp or halogen tungsten lamp which can emit full-wavelength continuous light source.

3. An optical system for synchronous detection of full-wavelength absorbance of microplate samples according to claim 1, It is characterized in that The transmitting optical fiber and the receiving optical fiber both adopt quartz fiber core.

4. An optical system for synchronous detection of full-wavelength absorbance of microplate samples according to claim 1, It is characterized in that The micro-porous plate is a multi-porous plate with a transparent bottom made of a composite material, the hole bottom is made of quartz material, and the hole wall is made of black plastic material.

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