A microfluidic chip, a manufacturing method and an online absorbance detection system

Abstract

The present application relates to a kind of microfluidic chip, including first layer, second layer and third layer in turn superimposed and connected together, first layer is provided with sample inlet, droplet formation flow channel and incident optical fiber channel, second layer is provided with detection flow channel, third layer is provided with sample outlet, sample flow channel and collection optical fiber channel, sample inlet is communicated with the input end of droplet formation flow channel, the output end of droplet formation flow channel is communicated with one end of detection flow channel, the input end of sample flow channel is communicated with the other end of detection flow channel, sample outlet is communicated with the output end of sample flow channel, incident optical fiber channel is used to install incident optical fiber, collection optical fiber channel is used to install collection optical fiber.The present application also proposes a kind of manufacturing method.The present application also proposes a kind of online absorbance detection system.The present application helps to reduce the manufacturing cost and manufacturing difficulty of microfluidic chip, helps to improve the manufacturing efficiency of microfluidic chip, helps to detect trace droplet quickly, can improve detection sensitivity.

Description

Technical Field

[0001] This invention relates to the field of online absorbance detection technology, specifically to a microfluidic chip, a manufacturing method, and an online absorbance detection system. Background Technology

[0002] Many bacterial metabolites are low-molecular-weight compounds, and gas chromatography-mass spectrometry (MS), liquid chromatography-MS, and their derivatives are commonly used for their detection. While these traditional methods offer advantages such as accuracy and practicality, they are complex, time-consuming, and expensive, making them unsuitable for rapid and real-time detection. Some researchers have developed colorimetric and electrochemical methods to achieve accurate in-situ metabolite detection. These newer detection methods are often designed for directional sensing, which may limit their ability to detect unknown chemical species in complex mixtures of metabolites. Therefore, developing new methods for detecting bacterial metabolites is crucial. Consequently, some researchers have applied droplet microfluidic systems and absorbance detection to the detection of bacterial metabolites.

[0003] Compared to continuous flow microfluidic systems, droplet microfluidic systems for single cells offer several advantages: Droplets are immiscible, allowing each droplet to function as an independent microreactor, enabling the individual encapsulation of cell samples for single-cell and extracellular secretory metabolite analysis. Droplet microfluidic systems can generate large numbers of droplets in a short time, reaching rates of thousands of droplets per second, making them suitable for high-throughput biological and chemical analysis. The droplet diameter is uniform and controllable, with droplet volumes ranging from picoliters to nanoliters, reducing sample and reagent consumption. Droplet microfluidic systems provide a method for studying individual bacteria in dynamic environments, allowing for precise control of the more sensitive bacterial microenvironment, facilitating further exploration of bacterial life at smaller scales. Furthermore, droplet microfluidic systems achieve miniaturization, enabling the processing of picoliter-sized droplets.

[0004] Absorbance detection is a method for analyzing the light absorption characteristics of substances, and it is an effective and commonly used method for evaluating cell dynamics in microfluidic analysis. Since analytes and products affect optical absorption spectra, absorption detection can be used to estimate various cell behaviors. When a light beam illuminates a sample solution, different compounds exhibit different absorption spectral characteristics, which can be used to identify and distinguish different compounds. By measuring the absorption intensity of the sample solution, the concentration of compounds can be quantitatively analyzed. Absorbance detection has strong detection capabilities for trace amounts of target compounds and low concentrations of samples. Using absorbance detection to analyze the absorption characteristics of bacterial metabolites allows for the monitoring and study of bacterial growth status and metabolic activity. Absorbance detection is based on Lambert-Beer's law, which states that the thickness of the absorbing medium affects the absorption response, and Beer's law states that the concentration of the absorbing medium also affects the absorption response. The combination of these two laws expresses a quantitative relationship between the light absorbed by a substance. Absorbance detection is a widely used general optical detection method with advantages such as the ability to determine a wide variety of substances and simple structures. It is also one of the earliest detection methods used in microfluidic analysis systems.

[0005] The following technical challenges arise when applying droplet microfluidics systems and absorbance detection to the detection of bacterial metabolites:

[0006] In droplet microfluidic systems, despite efforts to miniaturize fluid handling systems, progress in miniaturizing detection and analysis systems has been very limited. Traditional microfluidic detection and analysis systems rely on conventional free-space optics, incorporating a series of lenses, dichroscopes, and fluorescence microscopes. The complex integration and alignment of these free-space optics make the entire microfluidic detection and analysis system bulky and expensive. Rigid, fixed optics offer virtually no freedom in experimental design and require cumbersome handling and maintenance.

[0007] In droplet microfluidic systems, the small droplet detection volume and short absorption optical path in the microfluidic channel detection area result in low absorbance detection sensitivity, which greatly limits the combination of absorbance detection and droplet microfluidic systems for bacterial metabolite detection.

[0008] The sample flow channels of microfluidic chips in droplet microfluidic systems are usually set in PDMS layers. PDMS layers are usually made using photolithography molding. The optical path requirements for online absorbance detection are different, so it is necessary to redesign the chip layout and re-make the PDMS layer using photolithography molding, which is time-consuming, labor-intensive and costly.

[0009] The PDMS layer of microfluidic chips has low rigidity, making it prone to swelling and leakage. Summary of the Invention

[0010] The purpose of this invention is to provide a microfluidic chip, a manufacturing method, and an online absorbance detection system to alleviate or eliminate at least one of the aforementioned technical problems.

[0011] The present invention discloses a microfluidic chip comprising a first layer, a second layer, and a third layer stacked and bonded together in sequence. The first layer is provided with a sample inlet, a droplet forming channel, and an incident optical fiber channel. The second layer is provided with a detection channel, which is a straight channel penetrating the second layer. The third layer is provided with a sample outlet, a sample outlet channel, and a collection optical fiber channel. The sample inlet is connected to the input end of the droplet forming channel, the output end of the droplet forming channel is connected to one end of the detection channel, the input end of the sample outlet channel is connected to the other end of the detection channel, and the sample outlet is connected to the output end of the sample outlet channel. The incident optical fiber channel is used to install an incident optical fiber capable of emitting a detection beam to one end of the detection channel, and the collection optical fiber channel is used to install a collection optical fiber capable of collecting the detection beam emitted from the other end of the detection channel.

[0012] Optionally, the second layer is an plexiglass layer, and the first and third layers are both epoxy resin layers.

[0013] Optionally, the droplet forming channel is formed by a groove-like structure on the surface of the first layer toward the second layer and the surface of the second layer toward the first layer, and the sample discharging channel is formed by a groove-like structure on the surface of the third layer toward the second layer and the surface of the second layer toward the third layer.

[0014] Optionally, the cross-sectional area of ​​the detection channel is smaller than the cross-sectional area of ​​the droplet forming channel.

[0015] Optionally, the incident fiber channel, the collecting fiber channel, and the detection channel are on the same center line, the incident fiber channel is connected to the output end of the detection channel, and the collecting fiber channel is connected to the input end of the sample outlet channel.

[0016] Optionally, the first layer is provided with two sample inlets, namely an oil phase inlet and an aqueous phase inlet. The droplet forming channel includes a first channel segment connected to one end of the detection channel and a second channel segment perpendicular to and intersecting the first channel segment. The oil phase inlet is connected to both ends of the second channel segment, and the aqueous phase inlet is connected to the other end of the first channel segment.

[0017] Optionally, the detection channel is a through-hole that penetrates the second layer along the thickness direction of the second layer.

[0018] The present invention also proposes a manufacturing method for manufacturing microfluidic chips, comprising the following steps:

[0019] The first layer is manufactured, in which an inlet, a droplet forming channel and an incident optical fiber channel are formed, and the inlet is connected to the input end of the droplet forming channel;

[0020] A second layer is manufactured, in which a detection channel is formed. The detection channel is a straight channel that runs through the second layer.

[0021] A third layer is manufactured, in which a sample outlet, a sample flow channel and a collection fiber optic channel are formed, and the sample outlet is connected to the output end of the sample flow channel.

[0022] The first, second, and third layers are stacked and connected together in sequence, so that the output end of the droplet forming channel is connected to one end of the detection channel, and the input end of the sample outlet channel is connected to the other end of the detection channel.

[0023] The incident fiber channel is used to install an incident fiber capable of emitting a detection beam to one end of the detection channel, and the collection fiber channel is used to install a collection fiber capable of collecting the detection beam emitted from the other end of the detection channel.

[0024] Optionally, manufacturing the second layer also includes the following steps: determining the length of the detection channel based on the optical path requirements of online absorbance detection, and determining the thickness of the second layer based on the length of the detection channel.

[0025] This invention also proposes an online absorbance detection system, comprising an incident unit, a detection unit, a sample input unit, and a sample output unit, and a microfluidic chip as described above. The incident unit includes, in sequence, an LED light source, a first fiber collimator, a first filter, and an incident fiber with its output end embedded in the incident fiber channel. The LED light source provides a detection beam, the first fiber collimator collimates the detection beam provided by the LED light source, and the first filter filters out stray light from the detection beam output by the first fiber collimator. The input end of the incident fiber is connected to the output end of the first filter. The detection unit includes, in sequence, an LED light source, a first fiber collimator, a first filter, and an incident fiber with its output end embedded in the incident fiber channel. The system includes a collecting optical fiber, a second filter, a second optical fiber collimator, a detector, and an oscilloscope. The input end of the collecting optical fiber is embedded in the collecting optical fiber channel, and the output end of the collecting optical fiber is connected to the input end of the second filter. The second filter is used to filter out stray light in the detection beam collected by the collecting optical fiber. The second optical fiber collimator is used to collimate the detection beam output by the second filter. The detector is used to convert the optical signal output by the second optical fiber collimator into an electrical signal, and the oscilloscope is used to display the electrical signal. The sample input unit is used to input a sample into the sample inlet, and the sample output unit is used to collect the sample output from the sample outlet.

[0026] This invention optimizes the structure, materials, and manufacturing methods of microfluidic chips, improving their modularity and reducing manufacturing costs and complexity, thus enhancing manufacturing efficiency. It also optimizes the internal flow channels of the microfluidic chip, increasing detection sensitivity and facilitating the integration of absorbance detection and droplet microfluidic systems for bacterial metabolite detection. This invention combines absorbance detection, droplet microfluidic systems, and fiber optic micro / nanotechnology to form an online absorbance detection system, characterized by low cost, simple structure, high degree of freedom, low sample and reagent consumption, miniature flow channels, and fast analysis speed. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the microfluidic chip described in some embodiments;

[0028] Figure 2 This is a side view of the microfluidic chip described in some embodiments;

[0029] Figure 3 This is a top view of the microfluidic chip described in some embodiments;

[0030] Figure 4 This is a schematic diagram of the online absorbance detection system described in some embodiments.

[0031] Among them, 1-microfluidic chip; 2-LED light source; 3-first fiber collimator; 4-first filter; 5-incident fiber; 6-collecting fiber; 7-second filter; 8-second fiber collimator; 9-detector; 10-oscilloscope; 11-micro-injection pump; 12-oil phase injection tube; 13-aqueous phase injection tube; 14-exit tube; 15-sample container;

[0032] 101-First layer; 102-Second layer; 103-Third layer; 104-Oil phase inlet; 105-Aqueous phase inlet; 106-Droplet formation channel; 107-Incident fiber optic channel; 108-Detection channel; 109-Collection fiber optic channel; 110-Exit channel; 111-Exit port;

[0033] 1061 - First flow channel section; 1062 - Second flow channel section; 1063 - Third flow channel section; 1064 - Fourth flow channel section; 1065 - Fifth flow channel section; 1066 - Sixth flow channel section. Detailed Implementation

[0034] The embodiments of the present invention will be described below with reference to the accompanying drawings and preferred embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be understood that the preferred embodiments are only for illustrating the present invention and not for limiting the scope of protection of the present invention.

[0035] It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Therefore, the drawings only show the components related to the present invention and are not drawn according to the actual number, shape and size of the components in the actual implementation. In the actual implementation, the form, quantity and proportion of each component can be arbitrarily changed, and the layout of the components may also be more complex.

[0036] like Figures 1 to 3 The microfluidic chip 1 shown includes a first layer 101, a second layer 102, and a third layer 103 stacked and bonded together in sequence. The first layer 101 is provided with a sample inlet, a droplet forming channel 106, and an incident optical fiber channel 107. The second layer 102 is provided with a detection channel 108, which is a straight channel penetrating the second layer 102. The third layer 103 is provided with a sample outlet 111, a sample outlet channel 110, and a collection optical fiber channel 109. The sample inlet is connected to the input end of the droplet forming channel 106. The output end of 06 is connected to one end of the detection channel 108, the input end of the sample outlet channel 110 is connected to the other end of the detection channel 108, the sample outlet 111 is connected to the output end of the sample outlet channel 110, the incident fiber channel 107 is used to install the incident fiber 5 that can emit a detection beam to one end of the detection channel 108, and the collection fiber channel 109 is used to install the collection fiber 6 that can collect the detection beam emitted from the other end of the detection channel 108. The droplet forming channel 106, the detection channel 108 and the sample outlet channel 110 are all microchannels.

[0037] Using the above technical solution, the detection beam emitted from the incident optical fiber 5 illuminates the sample droplets in the detection channel 108, and the collecting optical fiber 6 collects the detection beam emitted from the detection channel 108, enabling absorbance detection of the sample droplets in the detection channel 108. The microfluidic chip 1 with a three-layer structure, by placing the detection channel 108 in the second layer 102, allows for easy adjustment of the length of the detection channel 108 according to inspection requirements. Only a preform of appropriate thickness needs to be selected and perforated to manufacture the second layer 102; the structures of the first layer 101 and the third layer 103 remain unchanged, eliminating the need for redesigning the chip layout and re-lithography. This improves the modularity of the microfluidic chip 1, helps reduce its manufacturing cost and difficulty, and improves its manufacturing efficiency.

[0038] In some embodiments, the second layer 102 is an acrylic glass layer, and the first layer 101 and the third layer 103 are both epoxy resin layers. Using the above technical solution, the second layer 102 is manufactured using acrylic glass. The detection channel 108 can be formed by drilling holes. When the length of the detection channel 108 needs to be adjusted according to inspection requirements, acrylic glass of different thicknesses can be selected to manufacture the second layer 102, eliminating the need to redesign the chip layout and re-perform photolithography.

[0039] The first layer 101 and the third layer 103 are manufactured using epoxy resin. Epoxy resin is cheaper and has higher hardness than PDMS, which can prevent swelling and leakage. Furthermore, a PDMS mold can be made using photolithography, and the first layer 101 and the third layer 103 can be repeatedly manufactured by casting epoxy resin onto the PDMS mold, which greatly reduces the manufacturing difficulty and cost of the microfluidic chip 1.

[0040] In practice, the acrylic glass layer and the epoxy resin layer can be bonded by applying acetone or ethanol. The acetone or ethanol solvent will only affect the roughness of the acrylic glass layer, but will not change the roughness of the epoxy resin layer with channels. Black or transparent epoxy resin can be selected to manufacture the first layer 101 and the third layer 103 according to different testing requirements.

[0041] In some embodiments, the droplet forming channel 106 is formed by a groove-like structure on the surface of the first layer 101 facing the second layer 102 and the surface of the second layer 102 facing the first layer 101, and the sample dispensing channel 110 is formed by a groove-like structure on the surface of the third layer 103 facing the second layer 102 and the surface of the second layer 102 facing the third layer 103. This technical solution facilitates the repeated manufacturing of the first layer 101 and the third layer 103 using epoxy resin in a PDMS mold.

[0042] In some embodiments, the cross-sectional area of ​​the detection channel 108 is smaller than that of the droplet forming channel 106. By employing the above technical solution, and by setting the detection channel 108 to have a smaller cross-sectional area, the detection channel 108 can compress the sample droplet transported from the droplet forming channel 106 to a longer length, thereby extending the optical path of the detection beam within the sample droplet. This improves the detection sensitivity of the online absorbance detection system and facilitates the integration of absorbance detection and droplet microfluidics systems for bacterial metabolite detection. A longer optical path results in a stronger signal; extending the optical path of the detection beam within the sample droplet increases the signal intensity, further enhancing the detection capability of the sample droplet.

[0043] As a preferred example, the droplet forming channel 106 has a square cross-sectional shape, and the detection channel 108 has a circular cross-sectional shape. The droplet forming channel 106 has a width of 40nm-200nm and a depth of 50nm-100nm; the detection channel 108 has an aperture of 30nm-190nm. By reducing the diameter of the detection channel 108, it can compress the sample droplets flowing from the droplet forming channel 106 into elongated droplets, thereby extending the optical path of the detection beam within the sample droplets. Experimental verification shows that the scheme adopted in the above preferred example has the best performance.

[0044] In some embodiments, the incident fiber channel 107, the collecting fiber channel 109, and the detection channel 108 are on the same center line. The incident fiber channel 107 is connected to the output end of the detection channel 108, and the collecting fiber channel 109 is connected to the input end of the sample outlet channel 110. By adopting the above technical solution, the incident fiber channel 107, the collecting fiber channel 109, and the detection channel 108 are on the same center line, facilitating the introduction of the detection beam into the detection channel 108 and the collection of the detection beam exiting the detection channel 108. The droplet directly contacts the incident fiber 5 and the collecting fiber 6, which can further improve the detection sensitivity and reduce interference from other stray light. In specific implementations, epoxy resin can be used to seal the gaps between the incident fiber 5 and the incident fiber channel 107, and between the collecting fiber 6 and the collecting fiber channel 109.

[0045] In some embodiments, the first layer 101 is provided with two inlets, namely an oil phase inlet 104 and an aqueous phase inlet 105. The droplet forming channel 106 includes a first channel section 1061 connected to one end of the detection channel 108 and a second channel section 1062 perpendicular to and intersecting the first channel section 1061. The oil phase inlet 104 is connected to both ends of the second channel section 1062, and the aqueous phase inlet 105 is connected to the other end of the first channel section 1061. Using the above technical solution, the first channel section 1061 and the second channel section 1062 constitute a cross-shaped channel, which can utilize the oil phase to shear the aqueous phase to generate water-in-oil sample droplets, and the size of the sample droplets can be controlled by controlling the flow rates of the oil and aqueous phases.

[0046] As a specific example, the droplet forming channel 106 further includes a third channel section 1063, a fourth channel section 1064 connected between one end of the third channel section 1063 and one end of the second channel section 1062, a fifth channel section 1065 connected between the other end of the third channel section 1063 and the other end of the second channel section 1062, and a sixth channel section 1066 connected at one end to the middle of the third channel section 1063. The second channel section 1062, the third channel section 1063, the fourth channel section 1064, and the fifth channel section 1065 constitute a rectangular frame-shaped channel structure. The oil phase inlet 104 is connected to the other end of the sixth channel section 1066. Using the above technical solution, a droplet forming channel capable of generating water-in-oil sample droplets is constructed by combining a rectangular frame-shaped channel structure with the first channel section 1061. This method is simple in structure and easy to implement.

[0047] In some embodiments, the detection channel 108 is a through-hole extending through the second layer 102 along its thickness direction. Using the above technical solution, the length of the detection channel 108 is consistent with the thickness of the second layer 102, which facilitates determining the thickness of the second layer 102 according to the optical path requirements of online absorbance detection, thus helping to reduce manufacturing difficulty. Furthermore, drilling can be used to form the detection channel 108, which helps to reduce costs and further reduce manufacturing difficulty.

[0048] Furthermore, the detection channel 108 is perpendicular to the first channel section 1061, and the detection channel 108 is perpendicular to the sample outlet channel 110. By adopting the above technical solution, stray light interference can be reduced, the alignment accuracy between the detection beam and the sample droplet can be improved, the luminous flux of the emitted light can be increased, and the intensity of the collected light signal can also be improved.

[0049] In some embodiments, the cross-sectional area of ​​the sample outlet channel 110 is larger than the cross-sectional area of ​​the detection channel 108. Setting the cross-sectional area of ​​the sample outlet channel 110 to be larger allows for the smooth discharge of sample droplets.

[0050] The present invention also proposes a manufacturing method for manufacturing a microfluidic chip 1, comprising the following steps:

[0051] A first layer 101 is manufactured, in which an inlet, a droplet forming channel 106 and an incident optical fiber channel 107 are formed, and the inlet is connected to the input end of the droplet forming channel 106.

[0052] A second layer 102 is manufactured, in which a detection channel 108 is formed. The detection channel 108 is a straight channel that penetrates the second layer 102.

[0053] A third layer 103 is manufactured, in which a sample outlet 111, a sample flow channel 110 and a collection fiber optic channel 109 are formed, and the sample outlet 111 is connected to the output end of the sample flow channel 110.

[0054] The first layer 101, the second layer 102 and the third layer 103 are stacked and bonded together in sequence, so that the output end of the droplet forming channel 106 is connected to one end of the detection channel 108, and the input end of the sample outlet channel 110 is connected to the other end of the detection channel 108.

[0055] The incident fiber channel 107 is used to install an incident fiber 5 that can emit a detection beam to one end of the detection channel 108, and the collection fiber channel 109 is used to install a collection fiber 6 that can collect the detection beam emitted from the other end of the detection channel 108.

[0056] By adopting the above technical solution, when the length of the detection channel 108 needs to be adjusted according to the inspection requirements, it is only necessary to select a blank of the appropriate thickness and make holes to manufacture the second layer 102. The structure of the first layer 101 and the third layer 103 remains unchanged. There is no need to redesign the chip layout or re-perform photolithography, which improves the modularity of the microfluidic chip 1, helps to reduce the manufacturing cost and manufacturing difficulty of the microfluidic chip 1, and helps to improve the manufacturing efficiency of the microfluidic chip 1.

[0057] In some embodiments, manufacturing the second layer 102 further includes the following steps: determining the length of the detection channel 108 according to the optical path requirements of online absorbance detection, and determining the thickness of the second layer 102 according to the length of the detection channel 108. Further, the detection channel 108 is a through-hole penetrating the second layer 102 along its thickness direction. Using the above technical solution, the length of the detection channel 108 is consistent with the thickness of the second layer 102, which facilitates determining the thickness of the second layer 102 according to the optical path requirements of online absorbance detection, helping to reduce manufacturing difficulty. Furthermore, drilling can be used to form the detection channel 108, which helps to reduce costs and further reduce manufacturing difficulty.

[0058] In some embodiments, the second layer 102 is an acrylic glass layer, and the first layer 101 and the third layer 103 are both epoxy resin layers. The manufacturing method includes the following steps: manufacturing the second layer 102 using acrylic glass; forming the detection channel 108 by drilling holes; selecting acrylic glass of appropriate thickness to manufacture the second layer 102 when the length of the detection channel 108 needs to be adjusted according to inspection requirements; manufacturing the first layer 101 and the third layer 103 using epoxy resin; fabricating a PDMS mold using photolithography; manufacturing the first layer 101 and the third layer 103 by casting epoxy resin onto the PDMS mold; bonding the first layer 101 and the second layer 102 by applying acetone or ethanol; and bonding the third layer 103 and the second layer 102 by applying acetone or ethanol. In the above technical solution, the PDMS mold can be reused, and the processing difficulty of the second layer 102 is low, which helps to reduce the manufacturing difficulty and cost of the microfluidic chip 1.

[0059] Furthermore, before the epoxy resin of the first layer 101 has fully cured, two hollow steel needles are inserted to form the oil phase inlet 104 and the aqueous phase inlet 105. Before the epoxy resin of the first layer 101 has fully cured, solid steel needles are inserted; after the epoxy resin of the first layer 101 has fully cured, the solid steel needles are removed, forming the incident fiber optic channel 107. Before the epoxy resin of the third layer 103 has fully cured, solid steel needles are inserted; after the epoxy resin of the third layer 103 has fully cured, the solid steel needles are removed, forming the collecting fiber optic channel 109. The above technical solution is easy to implement.

[0060] like Figure 4As shown, this invention also proposes an online absorbance detection system, including an incident unit, a detection unit, a sample input unit, and a sample output unit, and a microfluidic chip 1 of any of the above. The incident unit includes an LED light source 2, a first fiber collimator 3, a first filter 4, and an incident fiber 5 with its output end embedded in the incident fiber channel 107, arranged sequentially. The LED light source 2 is used to provide a detection beam, the first fiber collimator 3 is used to collimate the detection beam provided by the LED light source 2, and the first filter 4 is used to filter out stray light in the detection beam output by the first fiber collimator 3. The input end of the incident fiber 5 is connected to the output end of the first filter 4; the detection unit includes... The system includes a collecting fiber 6, a second filter 7, a second fiber collimator 8, a detector 9, and an oscilloscope 10 arranged sequentially. The input end of the collecting fiber 6 is embedded in the collecting fiber channel 109, and the output end of the collecting fiber 6 is connected to the input end of the second filter 7. The second filter 7 is used to filter out stray light in the detection beam collected by the collecting fiber 6. The second fiber collimator 8 is used to collimate the detection beam output by the second filter 7. The detector 9 is used to convert the optical signal output by the second fiber collimator 8 into an electrical signal. The oscilloscope 10 is used to display the electrical signal. The sample input unit is used to input the sample into the sample inlet, and the sample output unit is used to collect the sample output from the sample outlet 111.

[0061] In some embodiments, the sample introduction unit includes a micro-injection pump 11, an oil phase injection tube 12, and an aqueous phase injection tube 13, with the oil phase injection tube 12 and the aqueous phase injection tube 13 respectively connected between the micro-injection pump 11 and the microfluidic chip 1. Using the above technical solution, the aqueous and oil phases can be delivered separately, allowing for the testing and quantification of analytes (aqueous phase) at different concentrations. In a specific implementation, the output end of the oil phase injection tube 12 is connected to the oil phase inlet 104, and the output end of the aqueous phase injection tube 13 is connected to the aqueous phase inlet 105.

[0062] In some embodiments, the sample dispensing unit includes a sample container 15 and a sample dispensing tube 14 connected between the sample container 15 and the sample dispensing port 111. The sample container 15 is used to collect sample droplets output from the sample dispensing port 111. In specific implementations, the sample container 15 may be an Erlenmeyer flask.

[0063] Using the above-mentioned online absorbance detection system, a single-wavelength absorbance optical detection platform was built. It features low overall cost and simplicity. The light source is an LED light source 2. After passing through fiber alignment and filtering, the detection beam passes through the microfluidic chip 1. After passing through the sample droplet, the detection beam is finally received by the detector 9 after filtering. The detector 9 is connected to an oscilloscope 10 to observe the changes in peak shape.

[0064] The single wavelength is chosen because each substance absorbs light at different wavelengths. This detection system uses a single wavelength to accurately measure a specific substance. Different narrow-band light sources can be used for different substances. Compared with a wide light source optical path, the wavelengths covered are greatly reduced, which greatly improves the accuracy of detection. The use of LED light source 2 provides convenience for realizing a single-wavelength absorbance optical detection platform.

[0065] Fiber-optic in-situ absorbance detection systems offer significant advantages over traditional free-space light detection systems. Because light passing through the fiber optic cable occupies a smaller volume, it generates a larger light field intensity. Furthermore, in these in-situ absorbance detection systems, the combination of fiber optics and microfluidics allows for precise focusing of light onto the sample droplet, as light can be directly emitted into the fiber. Therefore, it is unnecessary to expand the detection beam before focusing the sample droplet to achieve optimal light field intensity. These advantages enable a lower sample detection limit, making such detection systems suitable for analyzing large numbers of samples.

[0066] The effective optical path of absorbance affects the overall detection sensitivity. This application improves the detection sensitivity by changing the cross-section of the detection channel 108 and compressing the sample droplet. The combination of absorbance with microfluidic technology and fiber micro-nano technology has the advantages of low sample and reagent consumption, small channel size and fast analysis speed.

[0067] In practical implementation, a suitable light source can be selected based on the analyte, and the final optical path system can be integrated and optimized. Water-in-oil microdroplets can be used for detection, with the analyte as the aqueous phase and the oil phase as silicone oil or fluorinated oil. Concentration quantification can be achieved by testing analytes at different concentrations.

[0068] The principle of absorbance detection is as follows: absorbance is the degree to which a substance absorbs light. Common liquids absorb light to a certain extent; the magnitude of absorption depends on the substance's inherent properties and concentration. According to Lambert-Beer's Law, the degree of light absorption by a solution is directly proportional to the product of the solution's concentration and the thickness of the liquid layer. Calculations based on this law can reveal the concentration and other relevant characteristics of the substance being detected. The mathematical expression of Lambert-Beer's Law is:

[0069]

[0070] The meanings of each parameter in the formula are as follows: Absorbance: Incident light intensity: : represents the intensity of transmitted light; : represents the transmittance; The absorption constant of a substance; : The thickness (cm) of the liquid layer through which the light beam passes; Concentration of a substance (mol / L);

[0071] According to the Lambert-Beer law, under ideal conditions, for the same substance irradiated by the same wavelength of light, its absorption coefficient can be considered fixed. Once the thickness of the liquid layer is determined, the concentration of the substance can be deduced by measuring the ratio of the intensity of the transmitted light to the intensity of the incident light.

[0072] The above embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. In the description of this specification, the reference to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., means that a specific feature, structure, material, or characteristic associated with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any suitable manner in one or more embodiments or examples. Furthermore, those skilled in the art can combine and integrate the different embodiments or examples described in this specification.

Claims

1. A microfluidic chip, characterized in that, The device comprises a first layer, a second layer, and a third layer stacked and bonded together from top to bottom. The first layer has a sample inlet, a droplet forming channel, and an incident fiber channel. The second layer has a detection channel, which is a straight channel penetrating the second layer. The third layer has a sample outlet, a sample outlet channel, and a collection fiber channel. The sample inlet is connected to the input end of the droplet forming channel, the output end of the droplet forming channel is connected to one end of the detection channel, the input end of the sample outlet channel is connected to the other end of the detection channel, and the sample outlet is connected to the output end of the sample outlet channel. The incident fiber channel is used to install an incident fiber capable of emitting a detection beam to one end of the detection channel, and the collection fiber channel is used to install a collection fiber capable of collecting the detection beam emitted from the other end of the detection channel. The cross-sectional area of ​​the detection channel is smaller than the cross-sectional area of ​​the droplet forming channel; The cross-sectional area of ​​the sample outlet channel is larger than the cross-sectional area of ​​the detection channel; The detection channel is a through-hole that penetrates the second layer along the thickness direction of the second layer; The droplet forming channel has a square cross-sectional shape, and the detection channel has a circular cross-sectional shape; the second layer is an plexiglass layer, and the first and third layers are both epoxy resin layers; The incident fiber channel, the collecting fiber channel, and the detection channel are on the same center line. The incident fiber channel is connected to the output end of the detection channel, and the collecting fiber channel is connected to the input end of the sample outlet channel.

2. The microfluidic chip according to claim 1, characterized in that, The droplet forming channel is formed by a groove-like structure on the surface of the first layer toward the second layer and the surface of the second layer toward the first layer, and the sample discharging channel is formed by a groove-like structure on the surface of the third layer toward the second layer and the surface of the second layer toward the third layer.

3. The microfluidic chip according to claim 1, characterized in that, The first layer is provided with two sample inlets, namely an oil phase inlet and an aqueous phase inlet. The droplet forming channel includes a first channel section with one end connected to one end of the detection channel and a second channel section perpendicular to and intersecting the first channel section. The oil phase inlet is connected to both ends of the second channel section, and the aqueous phase inlet is connected to the other end of the first channel section.

4. A manufacturing method for manufacturing a microfluidic chip as described in any one of claims 1-3, characterized in that, Includes the following steps: The first layer is manufactured, in which an inlet, a droplet forming channel and an incident optical fiber channel are formed, and the inlet is connected to the input end of the droplet forming channel; A second layer is manufactured, in which a detection channel is formed. The detection channel is a straight channel that runs through the second layer. A third layer is manufactured, in which a sample outlet, a sample flow channel and a collection fiber optic channel are formed, and the sample outlet is connected to the output end of the sample flow channel. The first, second, and third layers are stacked and bonded together from top to bottom, so that the output end of the droplet forming channel is connected to one end of the detection channel, and the input end of the sample outlet channel is connected to the other end of the detection channel.

5. The manufacturing method according to claim 4, characterized in that, Manufacturing the second layer also includes the following steps: determining the length of the detection channel based on the optical path requirements of online absorbance detection, and determining the thickness of the second layer based on the length of the detection channel.

6. An online absorbance detection system, characterized in that, The device includes an incident unit, a detection unit, a sample input unit, and a sample output unit, and a microfluidic chip as described in any one of claims 1-3. The incident unit comprises, in sequence, an LED light source, a first fiber collimator, a first filter, and an incident fiber with its output end embedded in the incident fiber channel. The LED light source provides a detection beam, the first fiber collimator collimates the detection beam provided by the LED light source, and the first filter filters out stray light from the detection beam output by the first fiber collimator. The input end of the incident fiber is connected to the output end of the first filter. The detection unit includes, in sequence, a collection fiber. The system comprises a second filter, a second fiber collimator, a detector, and an oscilloscope. The input end of the collecting fiber is embedded in the collecting fiber channel, and the output end of the collecting fiber is connected to the input end of the second filter. The second filter is used to filter out stray light in the detection beam collected by the collecting fiber. The second fiber collimator is used to collimate the detection beam output by the second filter. The detector is used to convert the optical signal output by the second fiber collimator into an electrical signal. The oscilloscope is used to display the electrical signal. The sample input unit is used to input a sample into the sample inlet, and the sample output unit is used to collect the sample output from the sample outlet.

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