Integrated microfluidic chip device and method for detecting urea nitrogen

Abstract

The application belongs to the technical field of urea nitrogen detection, and particularly relates to an integrated microfluidic chip device and method for detecting urea nitrogen; wherein a cover plate is bonded to a main plate, and the main plate is bonded to a base plate; the main plate is provided with a light path channel, a micro flow channel, a reaction chamber, a sample chamber, a control chamber and a groove; a photoelectric detector is fixed in the groove, an LED light source, a lens, a spectroscope and a reflecting mirror are fixed in the light path channel, the sample chamber and the reaction chamber are communicated through the micro flow channel, and the sample chamber and the outside are communicated through the micro flow channel; the cover plate is etched with an injection port communicated with the reaction chamber and the control chamber; the application adopts the microfluidic analysis chip idea, and through microfabrication technology, functional elements such as the LED light source, the lens, the spectroscope, the reflecting mirror, the reaction chamber, the sample chamber, the control chamber, the micro flow channel, the photoelectric detector and the main control chip are integrated on the chip like integrated circuits, and the idea of transferring the functions of the whole laboratory to the chip is concentratedly embodied.

Description

Technical Field

[0001] This invention belongs to the field of urea nitrogen detection technology, specifically relating to an integrated microfluidic chip device and method for detecting urea nitrogen. Background Technology

[0002] Urea is a polar, highly water-soluble, and charge-neutral molecule with one oxygen atom and two nitrogen atoms acting as hydrogen bond acceptors, and two amino functional groups providing a total of four hydrogen bonds for energy. Urea production plays a crucial role in human protein catabolism. As an end product, approximately 90% of urea is excreted by the kidneys in urine after its formation, hence its name. Studies have also found that urea is excreted in human sweat, tears, saliva, and digestive fluids (feces).

[0003] Urea concentration is conventionally expressed as urea nitrogen. Since a urea molecule contains two nitrogen atoms, 1 mmol / L urea equals 2 mmol / L urea nitrogen. The World Health Organization recommends using urea, expressed in mmol / L, and my country's Ministry of Health has also mandated the use of urea, ceasing the use of the term urea nitrogen. However, clinical blood urea testing still uses the older term blood urea nitrogen (BUN).

[0004] In the urinary system, kidney urea nitrogen (BUN) is one of the commonly used indicators for clinicians to assess kidney function. For elderly patients, BUN is an independent predictor of urinary tract infection. BUN is also a factor in predicting whether nephrectomy is necessary in patients with kidney trauma. In the circulatory system, numerous studies strongly recommend monitoring BUN as a predictor of prognosis in both acute and chronic heart failure due to the high correlation between elevated BUN and adverse outcomes. Even after coronary artery bypass grafting, high BUN remains an independent risk factor for death, strongly recommending that cardiologists monitor patients' BUN levels throughout their hospital stay. In the respiratory system, BUN >7 mmol / L is one of the scoring indicators in the CURB-65 tool for assessing the severity and short-term mortality of community-acquired pneumonia. In multivariate analysis, elevated BUN is a variable predicting high-risk patients requiring early mechanical ventilation. In the digestive system, BUN levels >36 mg / L are a predictor of mortality from acute hepatitis A. High BUN levels can independently predict mortality in patients with purulent liver abscess. A new scoring system, incorporating BUN, hemoglobin, systolic blood pressure, and comorbidities, is used to determine risk stratification for acute upper gastrointestinal bleeding. It indicates the severity of bleeding and determines whether a patient requires clinical intervention and hospitalization. Urea also plays multiple clinical roles in the nervous system. Studies have shown that the BUN / creatinine ratio may be a novel predictor of early deterioration in stroke, and that elevated BUN following intravenous tPA treatment in patients with acute ischemic stroke is an independent predictor of adverse clinical outcomes and mortality.

[0005] Because early diagnosis and treatment of many systemic diseases require BUN (blood urea nitrogen), this biochemical test is of great clinical significance. Although the enzyme-coupled rate method and the diacetyl-oxime method are commonly used in clinical hospitals for serum urea nitrogen determination, the urease-glutamate dehydrogenase coupled rate method is faster, simpler, more specific, and provides more accurate and precise results compared to the diacetyl-oxime method, making it the preferred method for measuring serum urea nitrogen.

[0006] The principle of the urease-glutamate dehydrogenase coupling rate method for measuring serum urea is that urease catalyzes the hydrolysis of urea to produce 2 molecules of NH3 and 1 molecule of CO2, which are then coupled with glutamate dehydrogenase. For every 1 molecule of NH3 produced, 1 molecule of NADH is oxidized. The rate of decrease in absorbance at 340 nm is measured to reflect the urea content.

[0007] Although the urease-glutamate dehydrogenase coupling rate method has high specificity, it requires a spectrophotometer with continuous absorbance monitoring and a constant temperature device, making it unsuitable for hospitals without automated or semi-automated biochemical analyzers. Based on these shortcomings, we need a new device that is simple, fast, meticulously manufactured, and highly accurate, suitable for both hospital use and home use by patients to measure blood urea nitrogen levels.

[0008] Most primary hospitals still use the DAM direct colorimetric method to detect blood urea nitrogen levels, but this method has some drawbacks: because the measurement process requires boiling for color development, it is easily affected by the boiling time and the amount of liquid evaporation during boiling, which can cause errors in the results; standard reagents may become unstable if stored at room temperature for too long, turning yellow or cloudy, thus affecting subsequent colorimetric measurements; the recovery rate of diacetyl oxime colorimetric reagent is not high enough, affecting accuracy and reaction sensitivity; and the operation by hospital laboratory technicians has a certain degree of subjectivity, which may lead to errors in the test results. Summary of the Invention

[0009] To overcome the above problems, this invention provides an integrated microfluidic chip device and method for detecting urea nitrogen. It adopts the concept of microfluidic analysis chip and integrates functional components such as LED light source, lens, beam splitter, reflector, reaction chamber, sample chamber, control chamber, microfluidic channel, photodetector, and main control chip on the chip like an integrated circuit through microfabrication technology. This embodies the idea of ​​transferring the entire laboratory's functions to the chip.

[0010] The technical solution proposed by the present invention to achieve the above objectives is as follows:

[0011] An integrated microfluidic chip device for detecting urea nitrogen includes a substrate 1, a main board 2, a cover plate 3, a photodetector 12, and optical components. The cover plate 3 is bonded to the main board 2, and the main board 2 is bonded to the substrate 1. The main board 2 has an optical path channel 7, a microfluidic channel 8, a reaction chamber 9, a sample chamber 10, a control chamber 11, and a groove 15. The photodetector 12 is fixed within the groove 15. The optical components include an LED light source 4, a lens 5, a beam splitter 6, and a refractor 18, wherein the LED light source 4, lens 5, beam splitter 6, and refractor 18 are sequentially fixed within the optical path channel 7. The light emitted from lamp source 4 enters lens 5 and is transmitted through lens 5, becoming parallel light before exiting. The exiting light enters beam splitter 6. Part of the light split by beam splitter 6 enters control chamber 11, and the other part enters refracting mirror 18. The light after being refracted by refracting mirror 18 enters sample chamber 10. After passing through sample chamber 10, the light enters the corresponding photodetector 12. The light entering control chamber 11 passes through control chamber 11 and enters the corresponding photodetector 12. Sample chamber 10 is connected to reaction chamber 9 and to the outside world through microfluidic channels 8.

[0012] The cover plate 3 is etched with a first injection port 16 and a second injection port 17, which are respectively connected to the reaction chamber 9 and the control chamber 11.

[0013] The optical path channel 7 includes an upper channel 71, a lower channel 72, and a connecting vertical channel 73. The upper channel 71 is located above the lower channel 72, and the left ends of the two are aligned. The right side of the upper channel 71 is tilted. The right side of the lower channel 72 is open to the outside. The bottom right end of the upper channel 71 is connected to the lower channel 72 through the connecting vertical channel 73. The LED light source 4 and the lens 5 are fixed from right to left in the lower channel 72 on the right side of the connecting vertical channel 73. The beam splitter 6 is tilted in the lower channel 7 below the connecting vertical channel 73. A refractor 18 is provided at the tilted part of the upper channel 71 above the beam splitter 6, and the refractor 18 is parallel to the beam splitter 6.

[0014] Both the beam splitter 6 and the reflector 18 are set at an angle of 45° to the vertical plane.

[0015] The bottom of the substrate 1 is etched with a bottom clip 13 for attaching the photodetector 12 by photolithography.

[0016] The photodetectors 12 are placed side by side in the groove 15, so that they are aligned with the center points of the sample chamber 10 and the control chamber 11 respectively, so as to ensure that the beam is transmitted and finally enters the two photodetectors 12, and their tops are locked in the corresponding bottom buckles 13.

[0017] The motherboard 2 is made of glass.

[0018] The photodetector 12 is a light intensity-sensitive photoelectric device, such as an active pixel sensor, a charge-coupled sensor, a photosensitive CMOS device, a photodiode, a polycrystalline silicon photodiode, or a photodiode embedded in a substrate.

[0019] Cross-shaped structures 14 for alignment are provided at the diagonal positions of the substrate 1, the main board 2, and the cover plate 3, respectively.

[0020] The manufacturing method of the motherboard 2 is as follows:

[0021] Step 1: Using photolithography, the reaction chamber 9, sample chamber 10, control chamber 11, microfluidic channel 8, optical path channel 7, and groove 15 are etched on the motherboard 2, and a groove for placing optical devices is etched in the optical path channel 7.

[0022] Step 2: Insert the LED light source 4, lens 5, and beam splitter 6 into the corresponding grooves in the optical path channel 7, and glue them together for reinforcement. Then, attach the refracting mirror 18 to the position reserved on the right side of the upper channel 71 of the optical path channel 7 at an angle of 45° to the vertical plane.

[0023] The cover plate 3 is made of silicon or glass and has a first injection port 16 and a second injection port 17 that are respectively connected to the reaction chamber 9 and the control chamber 11.

[0024] A method for detecting urea nitrogen using an integrated microfluidic chip device includes the following steps:

[0025] Step 1: Inject the serum biochemical sample of urea to be tested and the required reaction reagents into the reaction chamber 9 through the first injection port 16, and then inject the same amount of reaction reagents into the control chamber 11 through the second injection port 17.

[0026] Step 2: Before the absorbance measurement begins, after the liquid in the reaction chamber 9 has reacted completely, an injection pressure is applied to the first injection port 16 so that the mixture in the reaction chamber 9 is sent out of the reaction chamber 9 and into the sample chamber 10 under the action of the injection pressure.

[0027] Step 3: LED light source 4 emits light of a specific frequency, which is made parallel by lens 5. The parallel light is split into two beams by beam splitter 6. One beam enters control chamber 11, and the other beam is refracted by refractor 18 and enters sample chamber 10. The two beams pass through different solutions in control chamber 11 and sample chamber 10, and finally enter different photodetectors 12 respectively.

[0028] Step four: After the light output from the sample chamber 10 and the control chamber 11 enters different photodetectors 12, the light signals are detected by the two photodetectors 12 and the light intensity values ​​are output.

[0029] Plot the absorbance curves of the sample solution in sample chamber 10 and the control solution in control chamber 11 with the wavelength of the incident light emitted by LED light source 4 as the abscissa and absorbance as the ordinate; plot the two curves in the same coordinate system; then plot the absorbance difference graph with wavelength as the abscissa and the difference between the absorbance of the sample solution and the absorbance of the control solution as the ordinate.

[0030] The absorbance is calculated according to the following formula:

[0031] A = lg(I0 / I1) = k * b * c

[0032] Where I0 represents the incident light intensity, which is determined by the LED light source itself; I1 represents the emitted light intensity, which is measured by the photodetector; k represents the molar absorption coefficient of the light-absorbing substance solution; b is the thickness of the light-absorbing substance solution in the sample chamber 10 or the control chamber 11; c is the concentration of the light-absorbing substance solution; and lg is the logarithm.

[0033] Step 5: Different biochemical substances react with their corresponding reagents, resulting in different absorption peaks on the absorbance curves. The highest peak is found using an absorbance difference graph, with its horizontal axis corresponding to the wavelength of the absorbed light. This axis is then used to locate the corresponding absorbance on the sample solution's absorbance curve. After obtaining the absorbance of the diazine derivative, the reaction product in reaction chamber 9, using the absorbance curve, the concentration of the diazine derivative is calculated using the following formula: C = A / (k*b), in mol / L. The concentration of urea, a specific component in the serum biochemical sample, is C. 尿素 =C 二嗪衍生物 .

[0034] In step one, the required reaction reagents for the urea biochemical sample to be tested are one or more of diacetyl oxime, acid reagent, and urea nitrogen reagent.

[0035] The beneficial effects of this invention are:

[0036] 1. Extremely high efficiency: This chip completes the determination, separation and other complex operations of urea nitrogen in a few seconds to tens of seconds.

[0037] 2. Significantly reduced sample and reagent consumption: In urea nitrogen detection, this reduces analytical costs and the consumption of expensive biological reagents, and also reduces environmental pollution.

[0038] 3. Significantly reduced manufacturing and single-test costs: Due to the small chip size and minimal material consumption, the use of mass production processes significantly reduces the cost of the analysis system.

[0039] 4. Through the examples, it was found that the chip can meet the requirements of biochemical analysis, thus demonstrating the feasibility of the integrated optical detection system.

[0040] 5. The miniaturization of microfluidic analysis systems offers advantages in terms of scale. Attached Figure Description

[0041] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the content of the embodiments of the present invention and these drawings without creative effort.

[0042] Figure 1 This is a schematic diagram of the structure of the present invention.

[0043] Figure 2 This is a schematic diagram of the substrate structure of the present invention.

[0044] Figure 3 This is a schematic diagram of the motherboard structure of the present invention.

[0045] Figure 4 This is a schematic diagram of the cover plate structure of the present invention.

[0046] Figure 5 This is a diagram showing the structured optical path on the motherboard of this invention.

[0047] The components include: substrate 1, main board 2, cover plate 3, LED light source 4, lens 5, beam splitter 6, optical path channel 7, upper channel 71, lower channel 72, connecting vertical channel 73, microfluidic channel 8, reaction chamber 9, sample chamber 10, control chamber 11, photodetector 12, bottom buckle 13, cross-shaped structure 14, groove 15, first injection port 16, second injection port 17, and refractor 18. Detailed Implementation

[0048] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. Furthermore, it should be noted that, for ease of description, the accompanying drawings show only the parts relevant to the present invention, and not all of the structures.

[0049] Example 1

[0050] Materials used to fabricate microfluidic analysis chips include silicon and glass. Because glass has good light transmittance, and microchannel networks and grooves can be etched onto glass and quartz using photolithography and etching techniques, silicon and glass are the main materials used for the fabrication.

[0051] like Figure 1-4 As shown, an integrated microfluidic chip device for detecting urea nitrogen includes a substrate 1, a main board 2, a cover plate 3, a photodetector 12, and optical components. The cover plate 3 is bonded to the main board 2, and the main board 2 is bonded to the substrate 1. The main board 2 has an optical path channel 7, a microfluidic channel 8, a reaction chamber 9, a sample chamber 10, a control chamber 11, and a groove 15. The photodetector 12 is fixed within the groove 15. The optical components include an LED light source 4, a lens 5, a beam splitter 6, and a refractor 18. The LED light source 4, lens 5, beam splitter 6, and refractor 18 are sequentially fixed within the optical path channel 7. Figure 5 As shown, the light emitted by the LED light source 4 enters the lens 5. After being transmitted through the lens 5, the light becomes parallel light and exits. The exiting light enters the beam splitter 6. After being split by the beam splitter 6, part of the light enters the control chamber 11, and the other part enters the refracting mirror 18. The light after being deflected by the refracting mirror 18 enters the sample chamber 10. After passing through the sample chamber 10, the light enters the corresponding photodetector 12. The light entering the control chamber 11 passes through the control chamber 11 and enters the corresponding photodetector 12. The sample chamber 10 is connected to the reaction chamber 9 and to the outside world through the microfluidic channel 8.

[0052] The cover plate 3 is etched with a first injection port 16 and a second injection port 17, which are respectively connected to the reaction chamber 9 and the control chamber 11.

[0053] The optical path channel 7 includes an upper channel 71, a lower channel 72, and a connecting vertical channel 73. The upper channel 71 is located above the lower channel 72, and the left ends of the two are aligned. The right side of the upper channel 71 is inclined. The right side of the lower channel 72 is open to the outside. The bottom right end of the upper channel 71 is connected to the lower channel 72 through the connecting vertical channel 73. The LED light source 4 and the lens 5 are fixed from right to left in the corresponding grooves in the lower channel 72 on the right side of the connecting vertical channel 73 (the grooves are etched in the optical path channel 7). The beam splitter 6 is inclined in the corresponding groove in the lower channel 7 below the connecting vertical channel 73. A refractor 18 is provided at the inclined part of the upper channel 71 above the beam splitter 6, and the refractor 18 is parallel to the beam splitter 6.

[0054] Both the beam splitter 6 and the reflector 18 are set at an angle of 45° to the vertical plane to ensure that light is transmitted in a straight line along the predetermined optical path 7.

[0055] The substrate 1, as Figure 1 As shown, it is made of silicon. A silicon wafer of appropriate size is selected, and a bottom clip 13 for attaching the photodetector 12 is etched on its bottom using a photolithography method.

[0056] The photodetectors 12 are placed side by side in the groove 15, so that they are aligned with the center points of the sample chamber 10 and the control chamber 11 respectively, to ensure that the beam is transmitted and finally enters the two photodetectors 12, and their tops are engaged in the corresponding bottom buckles 13 (the bottom buckles 13 are the grooves).

[0057] The motherboard 2 is made of glass.

[0058] The photodetector 12 is a light intensity-sensitive photoelectric device, such as an active pixel sensor, a charge-coupled sensor, a photosensitive CMOS device, a photodiode, a polycrystalline silicon photodiode, or a photodiode embedded in a substrate.

[0059] The photolithography method for the bottom buckle 13 is as follows:

[0060] Photoresist is applied to the area of ​​substrate 1 where the etched groove (the groove of bottom buckle 13) needs to be etched. The mask is aligned with the area of ​​bottom buckle 13 to be processed and then exposed. Finally, the photoresist is rinsed with developer to remove the exposed part. Then, substrate 1 is immersed in stripping solution. Before stripping the photoresist, wet etching is performed with HF solution. Then, the photoresist is stripped with a constant temperature oscillator.

[0061] To facilitate the cooperation of the substrate 1, the main board 2 and the cover plate 3, cross-shaped structures 14 for alignment are provided at the diagonal positions of the substrate 1, the main board 2 and the cover plate 3 respectively.

[0062] The entrance to the optical path channel 7 is located at the edge of the motherboard 2.

[0063] The optical path channel 7 and the microfluidic channel 8 serve as chambers and channels, enabling liquid to enter and exit each chamber under pressure.

[0064] The manufacturing steps for the motherboard 2 are as follows:

[0065] Step 1: Using photolithography, the reaction chamber 9, sample chamber 10, control chamber 11, microfluidic channel 8, optical path channel 7, and groove 15 for placing photodetector 12 are etched on the motherboard 2, and a groove for placing optical device is etched in the optical path channel 7.

[0066] Step 2: Insert the LED light source 4, lens 5, and beam splitter 6 into the corresponding grooves in the optical path channel 7, and glue them together for reinforcement. Then, attach the refracting mirror 18 to the position reserved on the right side of the upper channel 71 of the optical path channel 7 at an angle of 45° to the vertical plane.

[0067] The cover plate 3, made of silicon or glass, is etched with a first injection port 16 and a second injection port 17, which are respectively connected to the reaction chamber 9 and the control chamber 11. Samples can be injected into the reaction chamber 9 and the control chamber 11 through the first injection port 16 and the second injection port 17, respectively.

[0068] The manufacturing steps of the cover plate 3 are as follows:

[0069] Step 1: The first injection port 16 and the second injection port 17, which are respectively connected to the reaction chamber 9 and the control chamber 11, are etched on the cover plate 3 by photolithography.

[0070] Step 2: Fix the main control board 15, which is responsible for communication and data processing, onto the cover plate 3.

[0071] In step two, the main control board 15 is fixed either by a detachable snap-fit ​​structure or by a non-detachable structure. The main control board 15 is responsible for analyzing the current signal transmitted from the photodetector 12 and converting the current signal into a light intensity value.

[0072] A method for detecting urea nitrogen using an integrated microfluidic chip device includes the following steps:

[0073] Step 1: Inject the serum biochemical sample of urea to be tested and the required reaction reagents into the reaction chamber 9 through the first injection port 16, and then inject the same amount of reaction reagents into the control chamber 11 through the second injection port 17.

[0074] Step 2: Before the absorbance measurement begins, after the liquid in the reaction chamber 9 has reacted completely, apply injection pressure to the first injection port 16 (the injection pressure can be increased by injecting gas into the first injection port 16) so that the mixture in the reaction chamber 9 is sent out of the reaction chamber 9 and into the sample chamber 10 under the action of injection pressure.

[0075] Step 3: LED light source 4 emits light of a specific frequency, which is made parallel by lens 5. The parallel light is split into two beams by beam splitter 6. One beam enters control chamber 11, and the other beam is refracted by refractor 18 and enters sample chamber 10. The two beams pass through different solutions in control chamber 11 and sample chamber 10, and finally enter different photodetectors 12 respectively.

[0076] Step four: Because the solution absorbs the input light, the transmitted light is attenuated; the light output from the sample chamber 10 and the control chamber 11 enters different photodetectors 12 respectively, and the light signal is detected by the two photodetectors 12, and the light intensity value is output to the external computer for processing.

[0077] Plot the absorbance curves (absorption spectra) of the sample solution in sample chamber 10 and the control solution in control chamber 11 on a computer, with the wavelength of the incident light emitted by LED light source 4 as the x-axis and absorbance as the y-axis; and plot the two curves on the same coordinate system; (where the control solution is used to eliminate the attenuation caused by the refraction and reflection of light in the container and the influence of other irrelevant substances in the solution on light absorption). Then, plot the absorbance difference graph with wavelength as the x-axis and the difference between the absorbance of the sample solution and the absorbance of the control solution as the y-axis; the plotting of the above data charts can be achieved by the corresponding software on the computer.

[0078] The absorbance is calculated according to the following formula:

[0079] A = lg(I0 / I1) = k * b * c

[0080] Where I0 represents the incident light intensity, which is determined by the LED light source itself; I1 represents the emitted light intensity, which is measured by the photodetector; k represents the molar absorption coefficient of the light-absorbing substance solution (obtained by referring to a table); b is the thickness of the light-absorbing substance solution in the sample chamber 10 or control chamber 11; c is the concentration of the light-absorbing substance solution; and lg is the logarithm.

[0081] The horizontal axis usually represents wavelength (nm) to indicate the specific frequency or wavelength of the input light.

[0082] The vertical axis represents absorbance (usually denoted by A), a dimensionless relative value used to indicate the degree to which a sample absorbs light of a specific wavelength or frequency. Utilizing the selective absorption properties of substances interacting with light, the appearance of an absorption peak indicates that the substance absorbs light of that wavelength to its maximum extent.

[0083] Step 5: Different biochemical substances react with their corresponding reagents, resulting in different absorption peaks on the absorbance curves. The highest peak is found using an absorbance difference graph, with its horizontal axis corresponding to the wavelength of the absorbed light. This axis is then used to locate the corresponding absorbance on the sample solution's absorbance curve. After obtaining the absorbance of the diazine derivative, the reaction product in reaction chamber 9, using the absorbance curve, the concentration of the diazine derivative is calculated using the following formula: C = A / (k*b), in mol / L. The concentration of urea, a specific component in the serum biochemical sample, is C. 尿素 =C 二嗪衍生物 .

[0084] The content of urea, a specific component, in the serum biochemical sample is determined to be C using the following reaction formula. 尿素 =C 二嗪衍生物 :

[0085]

[0086]

[0087] In step one, the required reaction reagents for the urea biochemical sample to be tested are one or more of diacetyl oxime, acid reagent, and urea nitrogen reagent.

[0088] Example 2

[0089] The following description, in conjunction with the accompanying drawings and preferred embodiments, further illustrates Example 2:

[0090] An integrated microfluidic chip device for detecting urea nitrogen comprises an LED light source 4, a lens 5, a beam splitter 6, a reflector 7, a reaction chamber 9, a sample chamber 10, a control chamber 11, a microfluidic channel 8, a photodetector 12, and a main control chip 15.

[0091] On the microfluidic chip, the serum biochemical sample and reaction reagents used in the experiment enter the reaction chamber 9 through the first injection port 16, while the serum biochemical sample and reaction reagents of the control sample enter the control chamber 11 through the second injection port 17. Before the absorbance measurement begins, the completely reacted mixture is injected under pressure and sent out of the reaction chamber 9 into the sample chamber 10. The LED light source 4 emits light within a specific wavelength range, which is parallelized by the lens 5. The light is then split into two beams by the beam splitter 6. One beam enters the control chamber 11, and the other enters the sample chamber 10. The two beams pass through different solutions and finally enter different photodetectors 12.

[0092] The photodetector 12 has a resolution of 2 nm and a detection spectral range of approximately 400 to 800 nm, with an external interface for output to a computer. Because the solution absorbs the input light, the transmitted light attenuates. After the output light enters the photodetector 12, it is visualized on the computer through photoelectric signal conversion, and an absorbance curve is plotted. Different biochemical substances react with reagents, corresponding to different absorption peaks. By comparing the position and size of the absorption peaks, the content of specific components can be determined. The entire chip adopts a packaged structure and a miniaturized design, with an overall size of 10 cm * 10 cm. This represents a significant reduction in volume compared to traditional analytical instruments, and also allows for a substantial reduction in sample volume.

[0093] In this embodiment, silicon is selected to fabricate the substrate 1 and the cover plate 3, and glass is selected to fabricate the main board 2. The specific fabrication scheme is as follows:

[0094] This chip is mainly constructed by bonding three parts: substrate 1, main board 2, and cover plate 3.

[0095] The substrate 1 is made of silicon. A 10cm*10cm silicon wafer is selected, and the bottom buckle 13 for placing the photodetector is etched on it by photolithography.

[0096] The photolithography method is as follows:

[0097] Step 1: Photoresist is applied to the area on substrate 1 where the grooves need to be etched. The mask is aligned with the area to be processed and then exposed. Finally, the photoresist is rinsed with developer to remove the exposed parts. Then, substrate 1 is immersed in stripping solution. Before stripping the photoresist, wet etching is performed with HF solution. Then, the photoresist is stripped using a constant temperature oscillator.

[0098] In step one, in order to facilitate the cooperation of the substrate 1, the main board 2 and the cover plate 3, a cross-shaped structure 14 for alignment needs to be set at the diagonal position.

[0099] The main board 2, with its reaction chamber 9, sample chamber 10, control chamber 11, optical path channel 7, and microfluidic channel 8, combines with the base plate 1 and cover plate 3 to form chambers and channels, so that liquid can enter and exit each chamber under pressure.

[0100] The production steps are as follows:

[0101] Step 1: Using photolithography, the reaction chamber 9, sample chamber 10, control chamber 11, microfluidic channel 8, optical path channel 7, area 15 for placing photodetectors, and grooves for placing optical devices (LED light source 4, lens 5, beam splitter 6) are etched on the glass substrate 2.

[0102] Step 2: Insert the LED light source 4, lens 5, and beam splitter 6 into the groove and glue them together for reinforcement. Attach the refracting mirror 7 directly to the position reserved in the optical path channel 7 at an angle of 45° to the vertical plane.

[0103] In step one, in order to facilitate the cooperation of the substrate 1, the main board 2 and the cover plate 3, a cross-shaped structure 14 for alignment needs to be set at the diagonal position.

[0104] In step one, the size of the sample chamber and control chamber affects the path length. To ensure detection accuracy and chip size, their size needs to be controlled to 10*10*30. .

[0105] In step two, the beam splitter 6 and the reflector 7 are both installed at an angle of 45° to the vertical plane to ensure that the light is transmitted in a straight line along the predetermined optical path 7.

[0106] The cover plate 3 is made of silicon. A 10cm*10cm silicon wafer is selected and the injection ports 16 and 17 of the reaction chamber 9 and the control chamber 11 are etched on it.

[0107] The production steps are as follows:

[0108] Step 1: The injection ports 16 and 17 of the reaction chamber 9 and the control chamber 11 are etched on the cover plate 3 by photolithography.

[0109] Step 2: Fix the main control board 15 onto the cover plate 3.

[0110] In step one, in order to facilitate the cooperation of the substrate 1, the main board 2 and the cover plate 3, a cross-shaped structure 14 for alignment needs to be set at the diagonal position.

[0111] In step two, the main control board is fixed by a detachable snap-fit ​​structure.

[0112] Chip assembly

[0113] Step 1: Align the base plate 1 and the main plate 2 using the cross-shaped structure 14, and bond the two parts together by heating, applying voltage or pressure.

[0114] Step 2: Place the two photodetectors 12 in area 15, so that the bottom of the photodetectors 12 is locked onto the bottom buckle 13;

[0115] Step 3: Use the cross-shaped structure 14 to align the motherboard 2 and the cover plate 3, and then bond them together by heating, applying voltage or pressure to complete the chip assembly.

[0116] In step two, two photodetectors 12 are placed side by side on the bottom buckle 13 and aligned with the center points of the sample chamber 10 and the control chamber 11, respectively, to ensure that the beam of light is ultimately transmitted into the two photodetectors 12.

[0117] Measure blood urea nitrogen level:

[0118] Step 1: Add 0.01 mL of serum biochemical sample to sample chamber 10, and add 0.05 mL of 0.02% diacetyl monooxime solution, 0.5 mL of urea nitrogen reagent, and 0.01 mL of deionized water to control chamber 11.

[0119] Step 2: Add 0.05 mL of 0.02% diacetyl monooxime solution, 0.5 mL of urea nitrogen reagent, and 0.01 mL of deionized water to reaction chamber 9 to initiate the reaction.

[0120] Step 3: After waiting for 5 minutes, use a syringe to push the solution in reaction chamber 9 into sample chamber 10;

[0121] Step four: The LED light wavelength can be adjusted from 420-750nm. The absorption spectrum of the sample is plotted according to step four of the method for detecting urea nitrogen in Example 1, and the corresponding urea nitrogen content is calculated according to step five.

[0122] The preferred embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the scope of protection of the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, any person skilled in the art can make equivalent substitutions or changes based on the technical solution and inventive concept of the present invention within the scope of the technology disclosed in the present invention. These simple modifications are all within the scope of protection of the present invention.

[0123] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0124] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. An integrated microfluidic chip device for detecting urea nitrogen, characterized in that, The system includes a substrate (1), a main board (2), a cover plate (3), a photodetector (12), and optical components; wherein the cover plate (3) is bonded to the main board (2), and the main board (2) is bonded to the substrate (1); the main board (2) is provided with an optical path channel (7), a microfluidic channel (8), a reaction chamber (9), a sample chamber (10), a control chamber (11), and a groove (15); wherein the photodetector (12) is fixed in the groove (15), and the optical components include an LED light source (4), a lens (5), a beam splitter (6), and a refractor (18), wherein the LED light source (4), the lens (5), the beam splitter (6), and the refractor (18) are sequentially fixed in the optical path channel (7), and the LED light source (4), the lens (5), the beam splitter (6), and the refractor (18) are sequentially fixed in the optical path channel (7). The light emitted from the lamp source (4) shines into the lens (5). After being transmitted through the lens (5), the light becomes parallel light and exits. The exiting light enters the beam splitter (6). After being split by the beam splitter (6), part of the light enters the control chamber (11) and the other part enters the refracting mirror (18). After being deflected by the refracting mirror (18), the light enters the sample chamber (10). After passing through the sample chamber (10), the light enters the corresponding photodetector (12). The light entering the control chamber (11) passes through the control chamber (11) and enters the corresponding photodetector (12). The sample chamber (10) and the reaction chamber (9) are connected to each other and to the outside world through microfluidic channels (8). The cover plate (3) is etched with a first injection port (16) and a second injection port (17) that are respectively connected to the reaction chamber (9) and the control chamber (11). The optical path channel (7) includes an upper channel (71), a lower channel (72) and a connecting vertical channel (73). The upper channel (71) is located above the lower channel (72), and the left ends of the two are aligned. The upper channel (71) is tilted on the right side, and the lower channel (72) is connected to the outside on the right side. At the same time, the bottom right end of the upper channel (71) is connected to the lower channel (72) through the connecting vertical channel (73). The LED light source (4) and the lens (5) are fixed from right to left in the lower channel (72) on the right side of the connecting vertical channel (73). The beam splitter (6) is tilted in the lower channel (72) below the connecting vertical channel (73). A refractor (18) is provided at the tilted part of the upper channel (71) above the beam splitter (6), and the refractor (18) is parallel to the beam splitter (6). The photodetectors (12) are placed side by side in the groove (15) so that they are aligned with the center points of the sample chamber (10) and the control chamber (11) respectively, so as to ensure that the beam is transmitted and finally enters the two photodetectors (12), and their tops are locked in the corresponding bottom buckles (13).

2. The integrated microfluidic chip device for detecting urea nitrogen according to claim 1, characterized in that, Both the beam splitter (6) and the refractor (18) are set at an angle of 45° to the vertical plane.

3. The integrated microfluidic chip device for detecting urea nitrogen according to claim 1, characterized in that, The bottom of the substrate (1) is etched with a bottom clip (13) for attaching the photodetector (12) by photolithography.

4. The integrated microfluidic chip device for detecting urea nitrogen according to claim 1, characterized in that, The motherboard (2) is made of glass; and cross-shaped structures (14) for alignment are provided on the diagonal positions of the substrate (1), motherboard (2) and cover plate (3).

5. The integrated microfluidic chip device for detecting urea nitrogen according to claim 1, characterized in that, The photodetector (12) is a photoelectric device that is sensitive to light intensity.

6. The integrated microfluidic chip device for detecting urea nitrogen according to claim 1, characterized in that, The manufacturing method of the motherboard (2) is as follows: Step 1: Using photolithography, the reaction chamber (9), sample chamber (10), control chamber (11), microfluidic channel (8), optical path channel (7), and groove (15) are etched on the motherboard (2), and a groove for placing optical devices is etched in the optical path channel (7); Step 2: Insert the LED light source (4), lens (5), and beam splitter (6) into the corresponding grooves in the optical path channel (7) and glue them together for reinforcement. Then, attach the refracting mirror (18) to the position reserved on the right side of the upper channel (71) of the optical path channel (7) at an angle of 45° to the vertical plane.

7. A method for detecting urea nitrogen using the integrated microfluidic chip device for detecting urea nitrogen as described in any one of claims 1-6, characterized in that, Includes the following steps: Step 1: The urea serum biochemical sample to be tested and the required reaction reagents are injected into the reaction chamber (9) through the first injection port (16), and then the same amount of reaction reagents are injected into the control chamber (11) through the second injection port (17). Step 2: Before the absorbance measurement begins, after the liquid in the reaction chamber (9) has reacted completely, an injection pressure is applied to the first injection port (16) so that the mixture in the reaction chamber (9) is sent out of the reaction chamber (9) and into the sample chamber (10) under the action of the injection pressure. Step 3: The LED light source (4) emits light of a specific frequency, and the lens (5) makes it parallel light. The parallel light is split into two beams by the beam splitter (6). One beam enters the control chamber (11), and the other beam is refracted by the refracting mirror (18) and enters the sample chamber (10). The two beams pass through different solutions in the control chamber (11) and the sample chamber (10), and finally enter different photodetectors (12). Step 4: After the light output from the sample chamber (10) and the control chamber (11) enters different photodetectors (12), the light signal is detected by the two photodetectors (12) and the intensity value of the light is output. Plot the absorbance curves of the sample solution in the sample chamber (10) and the control solution in the control chamber (11) with the wavelength of the incident light emitted by the LED light source (4) as the abscissa and the absorbance as the ordinate; and plot the two curves in the same coordinate system; then plot the absorbance difference graph with the wavelength as the abscissa and the difference between the absorbance of the sample solution and the absorbance of the control solution as the ordinate. The absorbance is calculated according to the following formula: A = lg(I0 / I1) = k * b * c Where I0 represents the incident light intensity, which is determined by the LED light source itself; I1 represents the outgoing light intensity, which is measured by the photodetector; k represents the molar absorption coefficient of the light-absorbing substance solution; b is the thickness of the light-absorbing substance solution in the sample chamber (10) or control chamber (11); c is the concentration of the light-absorbing substance solution; and lg is the logarithm. Step 5: Different biochemical substances react with corresponding reagents, resulting in different absorption peaks on the absorbance curves. The highest peak is found through the absorbance difference graph, and its horizontal axis corresponds to the wavelength of the absorbed light. This axis is used to find the corresponding absorbance in the absorbance curve of the sample solution. After obtaining the absorbance of the diazine derivative reaction product in the reaction chamber (9) through the absorbance curve, the concentration of the diazine derivative is calculated using the following formula: C = A / (k*b), with the unit being mol / L. The content of the specific component urea in the serum biochemical sample is C. 尿素 =C 二嗪衍生物 .

8. The method for detecting urea nitrogen according to claim 7, characterized in that, In step one, the required reaction reagents for the urea biochemical sample to be tested are one or more of diacetyl oxime, acid reagent, and urea nitrogen reagent.

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