Device for observing target sample in fluid, method for generating droplets of target sample in fluid, and method for determining anaerobic sludge activity

Abstract

An apparatus for observing a target sample in a fluid, a method for generating droplets of a target sample in a fluid, and a method for determining anaerobic sludge activity. The device comprises: a microfluidic channel of a preset length connecting an input end and an output end, the microfluidic channel defining an observation area for observing the target sample; wherein the input comprises a plurality of input ports, each for supplying a fluid of a different phase to the microfluidic channel at the input, thereby producing a plurality of droplets containing the target sample, the plurality of droplets being substantially uniformly separated by a separation medium therebetween.

Description

Technical Field

[0001] This disclosure relates to an apparatus for observing a target sample in a fluid, a method for generating a target sample droplet, and a method for determining the activity of anaerobic sludge, particularly (but not limited to) a microfluidic device for determining the activity of anaerobic sludge. Background Technology

[0002] The global food waste problem has been exacerbated by economic growth and population expansion. According to statistics from the Food and Agriculture Organization of the United Nations, approximately 1.3 billion tons of food that should have been consumed by humans are wasted or discarded globally each year.

[0003] In Hong Kong, the daily production of municipal solid waste may exceed 10,000 tons annually, with 30% attributed to food waste. Most of this food waste likely ends up in landfills. However, due to limited landfill capacity, many landfills have been closed and are no longer operational. Given this situation, an anaerobic co-digestion technology for food waste / sludge has been proposed as a relatively cost-effective approach for both renewable energy production and food waste treatment. Summary of the Invention

[0004] According to a first aspect of this disclosure, an apparatus for observing a target sample in a fluid is provided. The apparatus includes: a microfluidic channel of a predetermined length connecting an input and an output, wherein the microfluidic channel defines an observation area for observing the target sample; wherein the input includes a plurality of input ports, each input port for supplying a fluid of a different phase to the microfluidic channel at the input, thereby generating a plurality of droplets containing the target sample, the plurality of droplets being substantially uniformly separated therebetween by a separation medium.

[0005] According to the first aspect, the plurality of droplets comprises an aqueous phase solution, and the separation medium comprises an oil phase solution.

[0006] According to the first aspect, the aqueous solution includes a phosphate solution.

[0007] According to the first aspect, the plurality of droplets comprise sodium pyrophosphate.

[0008] According to the first aspect, the aqueous solution includes an alginate solution.

[0009] According to the first aspect, the plurality of droplets includes sodium alginate.

[0010] According to the first aspect, the microfluidic channel includes a serpentine channel defining the observation area.

[0011] According to the first aspect, the plurality of input ports includes a first input port connected to the serpentine channel and a second input port connected to the serpentine channel through the first input port.

[0012] According to the first aspect, the second input port and the first input port are separated by a preset distance.

[0013] According to the first aspect, the first input port is used to supply the aqueous phase solution, and the second input port is used to supply the oil phase solution.

[0014] According to the first aspect, the first input port is also connected to a microfluidic channel input combiner for supplying the aqueous solution mixed with the target sample and at least one aqueous reagent.

[0015] According to the first aspect, the microfluidic channel input merger has a Y-shaped structure, the Y-shaped structure defining two inlets connected to the merging inlet.

[0016] According to the first aspect, the microfluidic channel and / or the microfluidic channel input combiner are disposed in the PDMS microfluidic device.

[0017] According to the first aspect, the at least one aqueous reagent includes a trace amount of active indicator.

[0018] According to the first aspect, the trace active indicator includes a fluorescent reagent.

[0019] According to the first aspect, the trace active indicator includes resazurin, which is used to react with anaerobic sludge to generate resorufin.

[0020] According to the first aspect, the target sample includes anaerobic sludge.

[0021] According to a second aspect of the invention, a method for generating droplets of a target sample in a fluid is provided. The method includes: providing a microfluidic channel of a predetermined length connecting an input end and an output end, wherein the microfluidic channel defines an observation area for observing the target sample; and supplying fluids of different phases to the microfluidic channel through a plurality of input ports of the input end, thereby generating a plurality of droplets containing the target sample, the plurality of droplets being substantially uniformly separated therebetween by a separation medium.

[0022] According to the second aspect, the plurality of droplets comprises an aqueous phase solution, and the separation medium comprises an oil phase solution.

[0023] According to the second aspect, the aqueous solution includes a phosphate solution.

[0024] According to the second aspect, the plurality of droplets includes sodium pyrophosphate.

[0025] According to the first aspect, the aqueous solution includes an alginate solution.

[0026] According to the second aspect, the plurality of droplets includes sodium alginate.

[0027] According to the second aspect, the microfluidic channel includes a serpentine channel defining the observation area.

[0028] According to the second aspect, the plurality of input ports includes a first input port connected to the serpentine channel and a second input port connected to the serpentine channel through the first input port.

[0029] According to the second aspect, the second input port and the first input port are separated by a preset distance.

[0030] According to the second aspect, supplying fluids of different phases to the microfluidic channel through the plurality of input ports of the input terminal includes the following steps: continuously supplying the oil phase solution through the second input port at a first preset flow rate to fill the serpentine channel with the oil phase solution; and continuously supplying the aqueous phase solution through the first input port at a second preset flow rate.

[0031] According to the second aspect, the first preset flow rate is ten times the second preset flow rate.

[0032] According to the second aspect, the first preset flow rate is 10 μL / min, and the second preset flow rate is 1 μL / min.

[0033] According to the second aspect, the method further includes the step of mixing the target sample with at least one aqueous reagent to form an aqueous solution, the aqueous solution being supplied to the serpentine channel through the second input port.

[0034] According to the second aspect, the at least one aqueous reagent includes a trace amount of active indicator.

[0035] According to the second aspect, the trace activity indicator includes a fluorescent reagent.

[0036] According to the second aspect, the trace active indicator includes resazurin, which is used to react with anaerobic sludge to generate resorufin.

[0037] According to the second aspect, the target sample includes anaerobic sludge.

[0038] According to a third aspect of the present invention, a method for determining the activity of anaerobic sludge is provided. The method includes the following steps: generating a plurality of sludge droplets in a serpentine channel according to the second aspect; exciting the sludge droplets by irradiating the observation area at a preset wavelength; and recording the rate of change in fluorescence intensity of the micro-activity indicator.

[0039] According to the third aspect, the trace active indicator includes resazurin, which is used to react with anaerobic sludge to generate resorufin.

[0040] According to the third aspect, the method further includes: mixing and homogenizing anaerobic sludge with sodium alginate and sodium pyrophosphate, wherein the sodium alginate and sodium pyrophosphate are used to supply the microfluidic channel through the input end.

[0041] According to the third aspect, the method further includes the step of recording changes in fluorescence intensity using a camera under darkroom conditions. Attached Figure Description

[0042] Embodiments of this disclosure will be described below by way of example with reference to the accompanying drawings.

[0043] Figure 1 This is a schematic diagram illustrating an apparatus for observing a target sample in a fluid according to an embodiment of the present disclosure.

[0044] Figure 2 To show in Figure 1 Images of dispersed droplets of the target sample formed in the device.

[0045] Figure 3 To demonstrate the image captured by the camera under darkroom conditions Figure 2 Images of dispersed droplets. Detailed Implementation

[0046] Without being constrained by theoretical limitations, reducing food waste disposal in landfills and prioritizing appropriate waste management may be more preferable. For example, anaerobic co-digestion of food waste and sludge could be employed, a natural process where microorganisms decompose organic matter under anaerobic conditions to produce biogas, a potential source of renewable energy. This approach could improve food waste treatment capacity and reduce reliance on landfills.

[0047] However, the inventors believe that food waste, if not properly managed, can negatively impact anaerobic digestion systems. For example, due to the low pH of food waste, introducing large amounts into the system may alter the acidity level, making it more difficult for the system to effectively decompose waste and produce biogas. To mitigate the potential instability in the anaerobic co-digestion process and improve system efficiency, real-time monitoring of the anaerobic co-digestion process may be crucial. This system enables early detection of potential system failures and facilitates timely adjustments by operators.

[0048] In a preferred embodiment of the invention, a microfluidic analysis device—a small tool with specially designed channels and chambers to help scientists study small amounts of liquid—can be used to monitor the metabolic activity of food waste and sewage sludge during anaerobic co-digestion.

[0049] like Figure 1 The illustration shows an embodiment of a device 100 for observing a target sample in a fluid. The device 100 includes a microfluidic channel 102 of a predetermined length connecting an input 104 and an output 106. The microfluidic channel 102 defines an observation area for observing the target sample. The input 104 includes multiple input ports, each for supplying a fluid of a different phase to the microfluidic channel 102, thereby generating multiple droplets 108 containing the target sample at the input end. These droplets are uniformly separated by a separating medium 110.

[0050] In this embodiment, the microfluidic channel 102 can be disposed in a microfluidic device or microfluidic chip for receiving a small amount of fluid sample containing the sample for analysis or observation. For example, the microfluidic device 100 can be used to prepare dispersed anaerobic sludge droplets 108, wherein the formation of the anaerobic sludge droplets 108 in the microfluidic channel 102 is uniform and controlled, and this is achieved by introducing a separation medium 110 between the sludge droplets 108.

[0051] Preferably, the target sample includes anaerobic sludge, which can be mixed with a "carrier solution" such as an aqueous solution. On the other hand, to ensure that the target sample does not mix with the separation medium, the separation medium is preferably an oil solution or reagent, such that the target sample—aqueous sludge droplets 108—is immiscible with the oil solution, thereby forming a heterogeneous system.

[0052] Those skilled in the art will understand that the present invention can also be applied, for example, to the preparation of heterogeneous droplets 108 containing solid particles, which can be physically or chemically separated from the separation medium.

[0053] like Figure 1As shown, a series of droplets 108 containing the target sample (i.e., anaerobic sludge) are uniformly separated from each other by a separation medium 110. The separation between adjacent droplets 108 can be regulated by precisely controlling the supply of aqueous and oil phase solutions, for example, by controlling the flow rate and time interval of different solutions injected into the microfluidic channel. The operation of the microfluidic device 100 will be further described in this disclosure.

[0054] Preferably, the microfluidic channel 102 includes a serpentine channel for defining an observation area, wherein multiple droplets 108 are formed within the serpentine channel for visual observation or capture by an imaging device (such as a camera). The serpentine channel also has a structure that facilitates the observation of multiple droplets 108, which are adequately separated by a separation medium 110 and can be observed simultaneously within the observation area. Figure 1 As shown, the serpentine channel 102, the input end 104 and the output end 106 are respectively located at both ends, which facilitates the supply and discharge of fluid into and out of the serpentine channel 102.

[0055] The inventors utilized polydimethylsiloxane (PDMS)—a material that can be used to manufacture microfluidic devices to achieve droplet formation—to construct a microfluidic analysis device.

[0056] Preferably, the microfluidic device 100 may be designed to have multiple inputs or input ports, wherein the multiple input ports include a first input port 104A connected to a serpentine channel and a second input port 104B connected to the serpentine channel 102 via the first input port 104A, wherein the second input port 104B is separated from the first input port 104A by a predetermined distance. In an example operation, the first input port 104A can be used to supply an aqueous phase solution, and the second input port 104B is used to supply an oil phase solution.

[0057] In addition, the first input port 104A is also connected to the microfluidic channel input combiner 112 for supplying an aqueous solution mixed with the target sample and at least one aqueous reagent, such as a micro-activity indicator, which can be used to facilitate the observation of micro-activity under specific conditions.

[0058] Preferably, the micro-activity indicator may include resazurin (7-Hydroxy-3H-phenoxazin-3-one 10-oxide), a benzothiazole dye with special properties, which can act as a fluorescent substance and exhibit micro-activity after reacting with the target sample.

[0059] Rezakura exhibits a blue to purple color at pH values ​​above 6.5 and an orange color at pH values ​​below 3.8. Under near-neutral pH conditions, it can be irreversibly reduced to a pink, highly fluorescent compound—haloxy-3H-phenoxazin-3-one (7-hydroxy-3H-phenoxazin-3-one). Haloxy-hal ...

[0060] In cell viability applications, resamaritan can serve as an indicator of cell viability and can also be used to quantify bacterial content in milk. In mammalian cell culture, it can detect the presence of live cells. Resamaritan can also be used to detect L-glutamate and measure the aerobic biodegradation of organic matter in wastewater. Its fluorescence color change and non-invasive properties help assess cell viability and proliferation in bacteria, yeast, and mammalian cells.

[0061] Furthermore, resamaritan-based detection methods offer the advantages of being rapid and sensitive. For samples, resamaritan can be added to cell cultures, where it is reduced to reduced resamaritan to indicate metabolic activity and cell viability. See also Figure 1 The microfluidic channel input merger 112 has a Y-shaped structure and defines two inlets 112A and 112B. These two inlets are connected to the merging inlet 112C. One inlet supplies an aqueous phase solution containing the target sample, and the other inlet supplies a micro-activity indicator solution. The aqueous phase solution containing the target sample and the micro-activity indicator solution are mixed and then supplied to the merging inlet 112C, and further enter the serpentine channel 102.

[0062] Alternatively, the microfluidic channel input merger 112 can also have different shapes or structures. It is sufficient that the merger has two inlets and one output or merge inlet for discharging the mixed solution. In some alternative examples, if the micro-activity indicator and sample solution are pre-mixed, a merger may not be necessary.

[0063] Preferably, the microfluidic channel 102 and the microfluidic channel input merger 112 can be disposed in the PDMS microfluidic device. The PDMS microfluidic device can be manufactured by imprinting or reverse imprinting manufacturing technology.

[0064] In a preferred embodiment, to manufacture a PDMS chip with Y-shaped channels, the process can begin with designing a 3D model using CAD software. After the design is complete, a negative mold template is created using a 3D printer. This template is then used in a mold-making process to manufacture the PDMS chip. Subsequently, individual PDMS layers are applied to the hermetically molded chip using plasma bonding technology. To complete the manufacturing process, the chip also requires heat treatment.

[0065] In one example embodiment, the PDMS chip may be fabricated with a Y-shaped channel 112 connected to the serpentine microchannel 102. Port 104B of the serpentine channel 102 serves as the input for the oil phase, while port 106 serves as the output for the waste liquid. The two ports of the Y-shaped channel 112 serve as input terminals 102 for the aqueous phase, with one port 112A designated as the input terminal for anaerobic sludge and the other port 112B as the input terminal for microbial activity indicators.

[0066] The oil phase injection port 104B, serving as the inlet for the oil phase, is located before and at a certain distance from the merging inlet 112C to utilize the shear force of the continuous phase to separate the sludge into droplets 108. The Y-shaped channel 112 facilitates subsequent detection of the anaerobic sludge activity. Sludge is injected through injection port 112A, while the sludge activity indicator—rezarcina—is introduced through injection port 112B. At the merging inlet 112C, the two components will merge under the shear force of the oil phase, forming droplets 108.

[0067] In this example, the serpentine channel 102 is substantially filled with oil-phase separation medium or aqueous-phase sludge droplets 108. The following steps can be used to fill the microfluidic channel:

[0068] First, an oil phase solution is continuously injected into the serpentine channel 102 through the second input port 104B at a first preset flow rate; second, an aqueous phase solution is continuously injected through the first input port 104A at a second preset flow rate.

[0069] For example, the first preset flow rate is ten times the second preset flow rate; for instance, the first preset flow rate is 10 μL / min and the second preset flow rate is 1 μL / min. In practice, the expected length of the separation medium is ten times the length of the droplet 108 in the serpentine channel. Skilled technicians will understand that the flow rate, the size / length of the droplet 108, and the length of the separation medium can be adjusted to other values ​​as needed. The input flow rate at each input port can be adjusted in the syringe pump's settings interface (not shown).

[0070] To further improve the formation of sludge droplets 108, the aqueous phase solution may preferably include a phosphate solution and / or an alginate solution, such as sodium pyrophosphate and sodium alginate, which help to “stabilize” each sludge droplet 108 formed in the serpentine channel.

[0071] For example, sodium alginate can be added to anaerobic sludge at a concentration of 4 g / L, while sodium pyrophosphate can be added at a concentration of 2.9 g / L. The mixture is thoroughly homogenized to disperse the anaerobic sludge, prevent re-aggregation, and inhibit settling. Sludge mixing and homogenization occur before it is supplied to the serpentine channel. Alternatively, other concentrations / volumes of solution can be added depending on the material properties of the target sample.

[0072] In this example, the anaerobic sludge is first mixed with sodium pyrophosphate and sodium alginate to disperse the aggregates of anaerobic sludge and prevent the sludge particles from re-aggregating. Once the target sample enters the chip, the mixture of sodium pyrophosphate, sodium alginate, and anaerobic sludge, as the dispersed phase, is transformed into droplets 108 under the action of the continuous phase of oil.

[0073] This paper further elaborates on a method for determining the activity of anaerobic sludge using a microfluidic device. Preferably, the method includes the following steps: as described above, generating multiple sludge droplets 108 in a serpentine channel; exciting the sludge droplets 108 by irradiating the observation area at a preset wavelength; and recording the rate of change of fluorescence intensity of a micro-activity indicator. As mentioned above, the present invention preferably uses a micro-activity indicator, including resazurite, which reacts with anaerobic sludge to generate a red fluorescent substance—halogenin. This indicator is mixed with the sludge through a Y-shaped merging structure, and then the mixture is supplied to the serpentine channel.

[0074] Furthermore, the method may also include a step of mixing and homogenizing anaerobic sludge with sodium alginate and sodium pyrophosphate, and then supplying it to a microfluidic channel through an input port, thereby ensuring the stability of the droplets 108 formed within the serpentine channel. (See reference...) Figure 2 The image shows an example of sludge droplets 108 generated in a microfluidic device, where the droplets 108 are uniformly separated by an oil phase separation medium.

[0075] In this example, within the PDMS chip, the oil phase is injected into the serpentine channel 102 at a flow rate of 10 μL / min through input port 104B using an injection pump. Simultaneously, anaerobic sludge is injected through input port 112C of the Y-shaped channel 112 at a flow rate of 1 μL / min. When the anaerobic sludge encounters the oil phase within the serpentine channel, fluid shear forces begin to exert their effect, causing the anaerobic sludge to disperse (due to the immiscibility of the anaerobic sludge and the oil phase), thus forming droplets 108.

[0076] After dispersed sludge droplets 108 are formed in a serpentine channel, changes in the fluorescence intensity of the droplets 108 can be observed or recorded using a camera under darkroom conditions, thereby reflecting changes in the microactivity of the sludge droplets 108. An exemplary embodiment illustrates the observation of dispersed sludge droplets 108 under darkroom conditions, wherein the microactivity of the sludge is represented by fluorescent spots captured by a camera.

[0077] In this example, under darkroom conditions, the fluorescence intensity of sludge droplets can be detected using halogenated sludge, a strongly fluorescent compound generated from the reaction of sludge with resazurite. Prior to the observation step, sludge is injected through injection port 112A, while the sludge activity indicator resazurite is introduced through injection port 112B. At the merging inlet 112C, the two components merge and form droplets 108 through oil-phase shearing. Once droplets 108 enter the serpentine channel, the anaerobic sludge continues to react with resazurite, generating halogenated sludge. When the excitation wavelength is 530 nm, resazurite emits strong fluorescence, which is recorded by the camera. By analyzing the rate of change in fluorescence intensity, the activity of the sludge can be determined.

[0078] The inventors proposed that the device can also employ a strategy of inhibiting methanogenesis by adding Fe3+ and utilizing resazurite. By analyzing the intensity and changes in the fluorescence signal generated by resazurite reduction, the inventors further evaluated the impact of food waste on anaerobic digestion activity. They observed that co-digesting food waste with anaerobic sludge produced higher biogas yields compared to anaerobic digestion alone. Specifically, a 1:3 ratio of anaerobic sludge to food waste effectively promoted anaerobic digestion.

[0079] These findings are significant, highlighting the potential of food waste as a substrate for anaerobic digestion, which can drive renewable energy generation by increasing biogas production. The monitoring capabilities of this analytical device can improve the stability of anaerobic digestion systems, especially during food waste co-digestion. The device can mitigate problems that may arise from abrupt or significant changes in feed or operating conditions, thereby improving the overall efficiency of the system.

[0080] These embodiments also have certain advantages, namely that the method uses a microfluidic chip containing a Y-shaped connector and a serpentine channel, which may be an effective way to form multiple dispersed sludge droplets for observation or image recording.

[0081] Advantageously, introducing sodium alginate and sodium pyrophosphate into anaerobic sludge as dispersants can prevent the anaerobic sludge from re-aggregating and settling. Furthermore, using an organic oil phase to fill the channels of the microfluidic chip and utilizing fluid shear force to disperse polar anaerobic sludge and generate droplets further enhances the system's performance.

[0082] Furthermore, this invention effectively addresses the challenges encountered in the prior art, particularly the problem of anaerobic sludge agglomeration and blockage of microchannels, hindering droplet formation. According to the method of embodiments of this invention, the preparation of anaerobic sludge droplets can also be achieved simply, continuously, and rapidly. Each droplet contains a relatively uniform amount of anaerobic sludge, thereby facilitating rapid detection of the activity of the anaerobic sludge.

[0083] In addition, sodium alginate can be added to the anaerobic sludge at a concentration of 4 g / L, and sodium pyrophosphate can be added at a concentration of 2.9 g / L to disperse the anaerobic sludge, prevent it from re-aggregating, and inhibit sedimentation. Subsequently, anaerobic sludge liquid droplets are prepared using the dispersed anaerobic sludge, ensuring that the content of anaerobic sludge in each droplet is relatively consistent.

[0084] Those skilled in the art will understand that various changes and / or modifications can be made to the present disclosure as illustrated in the specific embodiments without departing from the spirit or opposite scope of the present disclosure as broadly described. Therefore, the embodiments are to be considered illustrative rather than restrictive in all respects.

[0085] Unless otherwise specified, any references to prior art contained herein should not be construed as an admission that the information is common general knowledge.

Claims

1. A device for observing a target sample in a fluid, characterized in that, include: A microfluidic channel of a preset length connecting an input end and an output end, wherein the microfluidic channel defines an observation area for observing the target sample; wherein the input end includes multiple input ports, each of the input ports being used to supply fluid of a different phase to the microfluidic channel at the input end, thereby generating multiple droplets containing the target sample, the multiple droplets being substantially uniformly separated therebetween by a separation medium.

2. The apparatus according to claim 1, characterized in that, in, The plurality of droplets comprise an aqueous solution, and the separation medium comprises an oil solution.

3. The apparatus according to claim 2, characterized in that, in, The multiple droplets include sodium pyrophosphate.

4. The apparatus according to claim 2, characterized in that, The multiple droplets include sodium alginate.

5. The apparatus according to claim 2, characterized in that, in, The microfluidic channel includes a serpentine channel that defines the observation area.

6. The apparatus according to claim 5, characterized in that, in, The plurality of input ports include a first input port connected to the serpentine channel and a second input port connected to the serpentine channel through the first input port; wherein the second input port and the first input port are separated by a preset distance, the first input port is used to supply the aqueous phase solution, and the second input port is used to supply the oil phase solution.

7. The apparatus according to claim 6, characterized in that, in, The first input port is also connected to a microfluidic channel input combiner; the microfluidic channel input combiner is used to supply the aqueous solution mixed with the target sample and at least one aqueous reagent.

8. The apparatus according to claim 7, characterized in that, in, The microfluidic channel and / or the microfluidic channel input combiner are disposed in the PDMS microfluidic device.

9. The apparatus according to claim 7, characterized in that, in, The at least one aqueous reagent includes a trace amount of active indicator.

10. The apparatus according to claim 9, characterized in that, in, The target samples include anaerobic sludge.

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