An online microbial counting sensor based on microscopic imaging

Abstract

The present invention discloses an online microbial counting sensor based on microscopic imaging, comprising a trace solution flow module, a Y-shaped optical fiber module, a lens group module, an image sensing module and a computing module, wherein the trace solution flow module comprises an SPR chip, a stainless steel bottom layer, a transparent window and an electromagnetic component, wherein the SPR chip is mounted on the stainless steel bottom layer, the transparent window covers the side of the SPR chip away from the stainless steel bottom layer and the electromagnetic component is located on the outer circle of the transparent window. The present invention discloses an online microbial counting sensor based on microscopic imaging, which realizes real-time monitoring of the number of microorganisms in the fermentation process by integrating microfluidics technology and surface plasmon resonance technology. Compared with the prior art, the present invention can provide faster and more accurate microbial counting, and is easy to operate and low in cost.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial counting, and in particular relates to an online microbial counting sensor realized based on a microscopic imaging method. Background Art

[0002] In the field of biological fermentation, the number and activity of microorganisms are key factors affecting the fermentation process and product quality. Existing microbial counting methods, such as plate counting, are cumbersome and time-consuming, and cannot meet the needs of real-time monitoring.

[0003] Therefore, further improvements are made to the above problems. Summary of the invention

[0004] The main purpose of the present invention is to provide an online microbial counting sensor based on microscopic imaging, which realizes real-time monitoring of the number of microorganisms in the fermentation process by integrating microfluidics technology and surface plasmon resonance (SPR) technology. Compared with the prior art, the present invention can provide faster and more accurate microbial counting, and is easy to operate and low in cost; it can provide real-time microbial counting data, and improve the monitoring efficiency and accuracy of the fermentation process.

[0005] To achieve the above objectives, the present invention provides an online microbial counting sensor based on microscopic imaging, comprising a trace solution flow module, a Y-shaped optical fiber module, a lens group module, an image sensing module and a computing module, wherein:

[0006] The micro-solution flow module comprises an SPR chip, a stainless steel bottom layer (designed as a slidable structure), a transparent window and an electromagnetic component, wherein the SPR chip is mounted on the stainless steel bottom layer, the transparent window covers a side of the SPR chip away from the stainless steel bottom layer and the electromagnetic component is located on the outer circle of the transparent window, wherein:

[0007] When detection and counting are required, the electromagnetic component is powered on and the stainless steel bottom layer is attracted, so that the stainless steel bottom layer brings the SPR chip closer, thereby forming a trace solution flow layer of preset thickness between the SPR chip and the transparent window, so that the solution of the number of microorganisms to be detected flows in the trace solution flow layer (the main purpose of forming a liquid cavity with a trace solution flow layer of 1 μm thickness is to keep the liquid cavity thin enough to avoid the overlap of cells in imaging as much as possible, and the thickness of 1 μm is a relatively thin height that can be achieved in engineering);

[0008] The SPR chip includes, from bottom to top, a substrate layer (PET, preferably more than ten μm), a nanogold layer (preferably 6 nm), and a molecular modification layer modified with ligands, wherein the substrate layer is mounted on the stainless steel bottom layer, and the molecular modification layer contacts the flowing solution and specifically captures specific protein structures, so that the cell bodies with specific protein structures remain on the SPR chip at a certain density for a certain period of time (so as to be imaged);

[0009] The Y-shaped optical fiber module includes an imaging optical fiber and an illumination optical fiber (emitting light of 600nm wavelength to optimize image quality), the front end of the imaging optical fiber and the front end of the illumination optical fiber are both installed on the transparent window, the illumination optical fiber is used to illuminate an area in the trace solution flow layer, the imaging optical fiber is used to obtain the image information of the SPR chip imaging (a picture per unit area) in real time, and transmit the unamplified image to the lens group module located at the rear end of the imaging optical fiber (the unamplified image is equivalent to 1x imaging, and the imaging area actually obtained is a circular area with a diameter of 0.1-0.2mm);

[0010] The lens group module magnifies the obtained image (preferably 500 times) and projects it onto the effective imaging area of ​​the image sensing module (CMOS image sensor), so that the image sensing module outputs an image of a preset size (preferably a 1920*1080 image) to the calculation module, and the calculation module calculates the number of microorganisms in the image and outputs the required results after statistical analysis (for example, the average value within a certain period of time, the calculation of the number of microorganisms per milliliter of solution (CFU / mL)).

[0011] As a further preferred technical solution of the above technical solution, the trace solution flow module also includes a distance sensor, which detects the distance between the transparent window and the molecular modification layer in real time, thereby feeding back the obtained distance data to the calculation module, so that the calculation module adjusts the suction force of the electromagnetic component (adjusts the applied voltage) according to the current distance, so that the trace solution flow layer is maintained at a preset thickness (ensuring the thickness, thereby avoiding the overlap of cells in imaging as much as possible and improving the accuracy of subsequent counting).

[0012] As a further preferred technical solution of the above technical solution, the trace solution flow module also includes a micro peristaltic pump, which is located on one side of the trace solution flow layer and is used to control the speed of the solution coming in from the other side of the trace solution flow layer, so that the solution moves slowly in the trace solution flow layer at a preset speed (to facilitate imaging capture and subsequent counting).

[0013] As a further preferred technical solution of the above technical solution, the stainless steel bottom layer is installed with multiple SPR chips, the trace solution flow layer is provided with multiple detection channels, each detection channel corresponds to an SPR chip, and each detection channel is matched with an imaging optical fiber and an illumination optical fiber, the lens group module respectively amplifies the image obtained by each imaging optical fiber and projects it onto the effective imaging area of ​​the image sensing module, so that the image sensing module simultaneously outputs multiple images of preset sizes to the calculation module, the calculation module respectively calculates the number of microorganisms in each image and outputs the required results after statistical analysis (performs a first averaging to obtain an average value, thereby improving the accuracy of the detection).

[0014] As a further preferred technical solution of the above technical solution, for each detection channel, the calculation module averages multiple images obtained before and after to improve the counting accuracy (performs a second average to further improve the detection accuracy). BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a structural schematic diagram of an online microbial counting sensor implemented based on microscopic imaging of the present invention.

[0016] Figure 2 It is a structural schematic diagram of a trace solution flow module of an online microbial counting sensor realized based on a microscopic imaging method of the present invention.

[0017] Figure numerals: 10, trace solution flow module; 11, SPR chip; 12, stainless steel bottom layer; 13, transparent window; 21, imaging optical fiber; 22, illumination optical fiber; 30, lens group module; 40, image sensing module; 50, computing module; 60, host computer. DETAILED DESCRIPTION

[0018] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are only examples, and those skilled in the art can think of other obvious variations. The basic principles of the present invention defined in the following description can be applied to other embodiments, variations, improvements, equivalents, and other technical solutions that do not deviate from the spirit and scope of the present invention.

[0019] In the preferred embodiments of the present invention, those skilled in the art should note that the microorganisms and the like involved in the present invention may be regarded as prior art.

[0020] Preferred embodiments.

[0021] like Figure 1-2As shown, the present invention discloses an online microbial counting sensor based on microscopic imaging, including a trace solution flow module 10, a Y-shaped optical fiber module, a lens group module 30, an image sensing module 40 and a computing module 50, wherein:

[0022] The micro-solution flow module 10 includes an SPR chip 11, a stainless steel bottom layer 12 (designed as a slidable structure), a transparent window 13 and an electromagnetic component. The SPR chip 11 is installed on the stainless steel bottom layer 12, the transparent window 13 covers the side of the SPR chip 11 away from the stainless steel bottom layer 12, and the electromagnetic component is located on the outer circle of the transparent window 13, wherein:

[0023] When detection and counting are required, the electromagnetic component is powered on and the stainless steel bottom layer 12 is attracted, so that the stainless steel bottom layer 12 brings the SPR chip 11 closer, and then forms a trace solution flow layer of preset thickness between the SPR chip 11 and the transparent window 13, so that the solution of the number of microorganisms to be detected flows in the trace solution flow layer (the main purpose of forming a liquid cavity of a trace solution flow layer with a thickness of 1 μm is to keep the liquid cavity thin enough to avoid the overlap of cells in imaging as much as possible, and the thickness of 1 μm is a relatively thin height that can be achieved in engineering);

[0024] The SPR chip 11 includes a substrate layer (PET, preferably more than ten μm), a nano-gold layer (preferably 6 nm) and a molecular modification layer modified with ligands from bottom to top. The substrate layer is installed on the stainless steel bottom layer 12. The molecular modification layer contacts the flowing solution and specifically captures the specific protein structure, so that the cell bodies with the specific protein structure remain on the SPR chip 11 at a certain density for a certain period of time (so as to be imaged);

[0025] The Y-shaped optical fiber module includes an imaging optical fiber 21 and an illumination optical fiber 22 (emitting light of 600 nm wavelength to optimize image quality). The front ends of the imaging optical fiber 21 and the illumination optical fiber 22 are both mounted on the transparent window 13. The illumination optical fiber 22 is used to illuminate an area in the trace solution flow layer. The imaging optical fiber 21 is used to obtain image information (photos per unit area) of the SPR chip 11 in real time, and transmit the unamplified image to the lens group module 30 located at the rear end of the imaging optical fiber 21 (the unamplified image is equivalent to 1x imaging, and the imaging area actually obtained is a circular area with a diameter of 0.1-0.2 mm);

[0026] The lens group module 30 magnifies the obtained image (preferably 500 times) and projects it onto the effective imaging area of ​​the image sensor module 40 (CMOS image sensor), so that the image sensor module 40 outputs an image of a preset size (preferably a 1920*1080 image) to the calculation module 50. The calculation module 50 calculates the number of microorganisms in the image and outputs the required results after statistical analysis (such as the average value within a certain period of time, the calculation of the number of microorganisms per milliliter of solution (CFU / mL)), and transmits the data to the host computer 60 after calculation for statistical analysis.

[0027] Specifically, the trace solution flow module also includes a distance sensor, which detects the distance between the transparent window 13 and the molecular modification layer in real time, thereby feeding back the obtained distance data to the calculation module 50, so that the calculation module 50 adjusts the suction force of the electromagnetic component (adjusts the applied voltage) according to the current distance, so that the trace solution flow layer is maintained at a preset thickness (ensuring the thickness, thereby avoiding the overlap of cells in imaging as much as possible and improving the accuracy of subsequent counting).

[0028] More specifically, the trace solution flow module also includes a micro peristaltic pump, which is located on one side of the trace solution flow layer and is used to control the speed of the solution coming in from the other side of the trace solution flow layer, so that the solution moves slowly in the trace solution flow layer at a preset speed (to facilitate imaging capture and subsequent counting).

[0029] Furthermore, the stainless steel bottom layer 12 is installed with multiple SPR chips 11, the trace solution flow layer is provided with multiple detection channels, each detection channel corresponds to an SPR chip 11, and each detection channel is matched with an imaging optical fiber 21 and an illumination optical fiber 22, the lens group module 30 respectively amplifies the image obtained by each imaging optical fiber 21 and projects it onto the effective imaging area of ​​the image sensor module 40, so that the image sensor module 40 simultaneously outputs multiple images of preset sizes to the calculation module 50, and the calculation module 50 respectively calculates the number of microorganisms in each image and outputs the required results after statistical analysis (performing a first averaging to obtain an average value, thereby improving the accuracy of the detection).

[0030] Furthermore, for each detection channel, the calculation module 50 averages the multiple images obtained before and after to improve the counting accuracy (performing a second average to further improve the detection accuracy).

[0031] For the calculation module 50, the imaging volume is the bottom area S1 = π* (0.1 / 2) 2 =0.000785mm3 , the height is 0.1mm, then 1mL (1000000mm 3 ) has 1,000,000 / 0.000785=1,270,000 such volumes. Then CFU / mL=calculated number of image microorganisms*1,270,000.

[0032] It is worth mentioning that the technical features such as microorganisms involved in the patent application of this invention should be regarded as prior art. The specific structure, working principle and possible control method and spatial layout method of these technical features can be selected by conventional methods in the field, and should not be regarded as the inventive point of this patent. This patent will not be further elaborated.

[0033] For those skilled in the art, it is still possible to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An online microbial counting sensor based on microscopic imaging, characterized in that: It includes a trace solution flow module, a Y-type optical fiber module, a lens group module, an image sensing module and a computing module, wherein: The micro-solution flow module comprises an SPR chip, a stainless steel bottom layer, a transparent window and an electromagnetic component, wherein the SPR chip is mounted on the stainless steel bottom layer, the transparent window covers a side of the SPR chip away from the stainless steel bottom layer and the electromagnetic component is located on the outer circle of the transparent window, wherein: When detection and counting are required, the electromagnetic component is powered on and attracts the stainless steel bottom layer, so that the stainless steel bottom layer brings the SPR chip closer, thereby forming a trace solution flow layer of a preset thickness between the SPR chip and the transparent window, so that the solution of the number of microorganisms to be detected flows in the trace solution flow layer; The SPR chip includes a substrate layer, a nanogold layer and a molecular modification layer modified with ligands from bottom to top, wherein the substrate layer is installed on the stainless steel bottom layer, and the molecular modification layer contacts the flowing solution and specifically captures the specific protein structure, so that the cell bodies with the specific protein structure remain on the SPR chip at a certain density for a certain period of time; The Y-shaped optical fiber module includes an imaging optical fiber and an illumination optical fiber, the front end of the imaging optical fiber and the front end of the illumination optical fiber are both installed on the transparent window, the illumination optical fiber is used to illuminate an area in the trace solution flow layer, and the imaging optical fiber is used to obtain the image information of the SPR chip imaging in real time, and transmit the unmagnified image to the lens group module located at the rear end of the imaging optical fiber; The lens group module magnifies the acquired image and projects it onto the effective imaging area of ​​the image sensor module, so that the image sensor module outputs an image of a preset size to the calculation module, and the calculation module calculates the number of microorganisms in the image and outputs the required results after statistical analysis.

2. The online microbial counting sensor based on microscopic imaging according to claim 1 is characterized in that: The trace solution flow module also includes a distance sensor, which detects the distance between the transparent window and the molecular modification layer in real time, thereby feeding back the obtained distance data to the calculation module, so that the calculation module adjusts the suction force of the electromagnetic component according to the current distance, thereby maintaining the trace solution flow layer at a preset thickness.

3. The online microbial counting sensor based on microscopic imaging according to claim 2 is characterized in that: The trace solution flow module also includes a micro peristaltic pump, which is located on one side of the trace solution flow layer and is used to control the speed of the solution coming from the other side of the trace solution flow layer so that the solution moves slowly in the trace solution flow layer at a preset speed.

4. The online microbial counting sensor based on microscopic imaging according to claim 3 is characterized in that: The stainless steel bottom layer is installed with multiple SPR chips, the trace solution flow layer is provided with multiple detection channels, each detection channel corresponds to an SPR chip, and each detection channel is matched with an imaging optical fiber and an illumination optical fiber. The lens group module respectively amplifies the image obtained by each imaging optical fiber and projects it onto the effective imaging area of ​​the image sensing module, so that the image sensing module simultaneously outputs multiple images of preset sizes to the calculation module, and the calculation module respectively calculates the number of microorganisms in each image and outputs the required results after statistical analysis.

5. The online microbial counting sensor based on microscopic imaging according to claim 4 is characterized in that: For each detection channel, the calculation module averages multiple images obtained before and after to improve the counting accuracy.

Images