Dual-channel platform for simultaneous separation of heavy metals and creatinine, preparation and application

Abstract

The present application relates to a double-channel platform for simultaneous separation of heavy metals and creatinine, preparation and application, and belongs to the technical field of sample pretreatment and sample detection. The present application simultaneously loads an electromembrane extraction technology and a liquid membrane extraction technology on a paper chip. In the separation process, the whole paper chip is divided into two layers, the upper layer is loaded with two-channel receiving phases, the lower layer is loaded with a sample phase, and a supporting membrane is clamped between the two layers of paper bases. Different membrane solvents are coated on the corresponding positions of the supporting membrane for selective separation. The sample phase and the receiving phase of the electromembrane extraction channel are respectively connected with electrodes, and the power is connected after the addition is completed. After the separation and enrichment are completed, the corresponding color developing agent is added in the receiving liquid, and the RGB color model is used to complete the quantification of the target object. The double-channel analysis device realizes the simultaneous rapid separation and detection of heavy metals and creatinine in urine.

Description

Technical Field

[0001] This invention belongs to the field of sample pretreatment and sample detection technology, and more specifically, relates to a dual-channel platform for the simultaneous separation of heavy metals and creatinine, its preparation and application, and particularly relates to a dual-channel separation technology that utilizes electroporation and liquid membrane extraction to rapidly separate heavy metals and creatinine in urine, and uses an RGB color model to quantify the target analytes. Background Technology

[0002] With rapid industrial development, the current situation and trends of heavy metal pollution have become extremely serious, and the country is investing more and more in heavy metal pollution control. Heavy metal pollutants in the environment mainly come from industrial emissions, agricultural production, water pollution, and landfills, while humans exposed to the environment primarily ingest heavy metal pollutants through food, water, and air. Heavy metals can accumulate in the human body and cause significant harm to the nervous, respiratory, cardiovascular, and urinary systems, leading to the development of various diseases. To prevent the harmful effects of heavy metals, timely monitoring of the body's heavy metal levels is crucial, especially for high-risk groups such as workers in smelters or children living near industrial sites.

[0003] Currently, heavy metal exposure levels are mostly detected through hospital checkups, which typically use the concentration of metals in serum samples as a standard to determine the body's metal levels. Heavy metal exposure is a long-term process, making regular screening essential for high-risk individuals. Considering the conflict between psychological resistance and the need for testing, there is an urgent need to develop a device suitable for individual heavy metal screening.

[0004] Compared to serum and other samples, urine samples, as indicators of human health, have a significant advantage as being non-invasive and suitable for testing. While the concentration of heavy metals in urine can serve as a standard for assessing heavy metal exposure levels, the concentration needs to be corrected for due to factors such as diet and urine concentration. Urinary creatinine is stably excreted in the body and can be used to correct for heavy metal concentrations in urine. However, the concentration of heavy metals in urine is relatively low, and traditional detection methods require large instruments, making them unsuitable for individual self-screening. Urinary creatinine can be detected directly using colorimetric methods, but matrix interference is a problem. Furthermore, in routine testing, urinary creatinine and heavy metals in urine cannot be detected simultaneously in the same sample; separate samples are required, which increases testing costs and sample uncertainty.

[0005] Membrane extraction, as a sample pretreatment technique, offers advantages such as short extraction time and high specificity, and has been widely applied in the pretreatment of various samples, including water, plants, food, biological fluids, and tissues. Liquid membrane extraction utilizes the difference in partition coefficients between ionic and non-ionic analytes in aqueous and organic phases to achieve the separation of target substances. Electrolytic membrane extraction, building upon liquid membrane extraction, uses an electric field to drive the extraction, replacing passive diffusion. Membrane extraction technology can achieve simultaneous separation of multiple substances and solves the problem of matrix interference during detection, showing great potential for the development of rapid detection devices.

[0006] In the rapid detection of biological samples, paper-based microarrays are a simple, convenient, and low-cost detection technology. Utilizing paper as a matrix, they combine chemical, biological, and optical principles to detect and analyze target substances. Paper-based detection devices offer advantages such as simple operation, speed, high sensitivity, and low cost, and are widely used in rapid diagnostics and environmental pollution monitoring when combined with the RGB color model. However, current literature lacks reports on how to combine paper-based microarray detection with membrane extraction technology for efficient and accurate simultaneous detection of multiple substances in a sample. Summary of the Invention

[0007] This invention provides a dual-channel platform for the simultaneous separation and detection of heavy metals and creatinine in urine. It combines membrane extraction technology with a paper-based microarray, employing electroporation and liquid membrane extraction techniques to simultaneously separate heavy metals and creatinine in urine. This invention achieves simultaneous analysis of heavy metals and creatinine in urine, solving the challenge of simultaneous separation of these two substances. Furthermore, it eliminates matrix interference during detection, eliminates the need for large instruments, and enables the assessment of in vivo heavy metal exposure through non-invasive urine samples.

[0008] According to a first aspect of the present invention, a dual-channel platform for the simultaneous separation of heavy metals and creatinine is provided. The dual-channel platform comprises, from top to bottom, a receiving phase layer, a supporting membrane, and a sample phase layer. Both the receiving phase layer and the sample phase layer are paper-based, and the supporting membrane is a hydrophobic membrane. The upper and lower surfaces of the receiving phase layer each include two non-communicating hydrophilic regions with hydrophobic surrounding areas. The two non-communicating hydrophilic regions on the upper surface of the receiving phase layer serve as receiving phases for heavy metals and creatinine, respectively. The upper and lower surfaces of the sample phase layer each include a hydrophilic region with hydrophobic surrounding areas. The hydrophilic region on the upper surface of the sample phase layer serves as the sample phase. The sample phase and the receiving phase (for heavy metals) are respectively used to connect to a wire, thereby forming an electroporation extraction pathway.

[0009] Preferably, the receiving phase for heavy metals and the receiving phase for creatinine have the same area, both being 25 mm². 2 -50mm 2 .

[0010] Preferably, the area of ​​the sample phase is 60 mm². 2 -150 mm 2 .

[0011] Preferably, the support membrane is a polypropylene fiber membrane, a polyethylene fiber membrane, or a polyester fiber membrane.

[0012] According to another aspect of the present invention, a method for preparing the dual-channel platform for simultaneous separation of heavy metals and creatinine as described in any one of the claims is provided, comprising the following steps: (1) Two filter papers are used as the receiving phase layer and the sample phase layer, respectively. Two non-connected hydrophilic regions with hydrophobic areas around the top and bottom surfaces of the receiving phase layer are constructed. The hydrophilic area on the top surface is used as the receiving phase for heavy metals and creatinine, respectively. A hydrophilic area with hydrophobic areas around the top and bottom surfaces of the sample phase layer is constructed. The hydrophilic area on the top surface is used as the sample phase. (2) Place the support membrane between the receiving phase layer and the sample phase layer described in step (1), and fix the two wires in the receiving phase and the sample phase, which are heavy metals, respectively, to construct the electro-membrane extraction pathway, thus obtaining the dual-channel platform.

[0013] Preferably, in step (2), after coating the support membrane with membrane solvent at the positions corresponding to the two receivers, the support membrane is placed between the receiver phase layer and the sample phase layer described in step (1); wherein the membrane solvent coated at the position corresponding to the heavy metal receiver is n-octanol, 2-nitrophenyl octyl ether, n-heptol or n-nonanol, and the membrane solvent coated at the position corresponding to the creatinine receiver is n-hexanol, n-octanol or n-heptol.

[0014] According to another aspect of the present invention, an application of the dual-channel platform for simultaneous separation of heavy metals and creatinine as described in any one of the claims is provided, comprising the following steps: (1) Add the test solution to the sample phase, add water to the receiving phase which is the heavy metal, and add acid to the receiving phase which is the creatinine; (2) When the power is turned on, the heavy metals enter the receiving phase from the sample phase through the electric field force, and the creatinine enters the receiving phase from the sample phase by utilizing the difference in the partition coefficient between the aqueous phase and the organic phase, thereby realizing the separation of heavy metals and creatinine in the test solution.

[0015] Preferably, the heavy metal is lead.

[0016] Preferably, after step (2), the method further includes adding a colorimetric agent to each of the two receiving phases to develop color, and then calculating the normalized RGB values ​​of the two receiving phases using the RGB color model; then calculating the concentration values ​​of lead and creatinine using the standard curve, and the ratio of the lead concentration value to the creatinine concentration value is the corrected lead concentration.

[0017] In summary, compared with the prior art, the above-described technical solutions conceived by this invention mainly possess the following technical advantages: (1) This invention is the first to combine electroporation and liquid membrane extraction technologies with paper-based chips, expanding the role of membrane extraction technology in sample pretreatment. This dual-channel analytical device enables the simultaneous and rapid separation and detection of multiple targets in urine.

[0018] (2) This invention utilizes electroporation and liquid membrane extraction technologies to achieve simultaneous separation and detection of heavy metals and creatinine in urine, solving the problems of large instruments required for conventional heavy metal detection and matrix interference in creatinine detection.

[0019] (3) Preferably, this invention is divided into two parts: first, using membrane extraction technology to simultaneously separate heavy metals and creatinine in urine; and second, using chemiluminescence to rapidly detect heavy metals and creatinine separately. During the separation process, the entire paper chip is divided into two layers: the upper layer contains the receiving phase of two channels, and the lower layer contains the sample phase. A polypropylene fiber membrane with a microporous structure is sandwiched between the two paper layers as a support membrane. Different membrane solvents are coated on the support membrane at positions corresponding to the receiving channels for selective separation. Electrodes are connected to the receiving phase of the sample phase and the electro-membrane extraction channel, respectively. After sample addition, a power supply is connected to enable the circuit. Liquid membrane extraction technology utilizes the difference in partition coefficients between ionic and non-ionic analytes in the aqueous and organic phases to achieve the separation of target analytes. Electro-membrane extraction is an electro-extraction mode based on liquid membrane extraction, where electric field driving force replaces passive diffusion as the main driving force for extraction. After separation and enrichment are completed, a corresponding colorimetric reagent is added to the receiving liquid, and the RGB color model is used to quantify the target analytes. This dual-channel analytical device achieves simultaneous and rapid separation and detection of heavy metals and creatinine in urine. This invention enables the simultaneous analysis of heavy metals and creatinine in urine, solving the problem of separating heavy metals and creatinine in urine at the same time. It also eliminates matrix interference during detection, eliminates the need for large instruments, and realizes the assessment of heavy metal exposure in the body through non-invasive urine samples.

[0020] (4) The miniaturized device designed in this invention is suitable for personal self-screening of heavy metals and provides ideas for rapid detection of multiple indicators in personal medical care. Attached Figure Description

[0021] Figure 1 This is a schematic diagram illustrating the analysis of the present invention.

[0022] Figure 2 This is for the construction of the platform of the present invention (including a 3D shell).

[0023] Figure 3 This is a schematic diagram illustrating the principle of membrane extraction technology used in this invention.

[0024] Figure 4 The graph shows the colorimetric reaction of creatinine and lead ions in urine and its changes over time, as an example of the present invention.

[0025] Figure 5 This invention provides an example of analyzing standard curves for creatinine and lead ions in urine, and comparing them with conventional methods. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0027] The present invention discloses the preparation of a dual-channel platform for the simultaneous separation and detection of heavy metals and creatinine in urine, comprising the following steps: (1) Cut the Whatman qualitative filter paper and mark the corresponding hydrophilic and hydrophobic regions. In this study, paraffin is used as a hydrophobic polymer. The upper paper has two hydrophilic regions as receiving phases, and the lower paper has one hydrophilic region as sample phase. That is, the upper layer of the paper chip is a dual receiving phase, which is independent of each other; the lower layer is a single sample phase, which is used to load the test liquid.

[0028] (2) After coating the support membrane with membrane solvent, place it between two layers of paper base; (3) Fix two wires in the sample phase and an upper receiving phase respectively for electroporation extraction pathway; (4) Place the entire paper base into the 3D printed resin shell for fixing the dual-channel paper base.

[0029] In some embodiments, the two receiving phases prepared are of the same area but are not connected, and the area is maintained at 25 mm². 2 -50mm 2 .

[0030] In some embodiments, the area of ​​the prepared sample phase is larger than the area of ​​the receiving phase, but does not exceed the area of ​​the supporting membrane, and is maintained at 60 mm. 2 -150mm 2 .

[0031] In some embodiments, two different membrane solvents, 5 μL each, are applied to the corresponding receiving phase positions.

[0032] In some embodiments, the 3D-printed shell is constructed to match the paper base for fixation.

[0033] In some embodiments, the wires used for circuit connection are selected from materials such as platinum wire and silver wire, which have good ductility and conductivity and are inert.

[0034] The application of the dual-channel platform device for simultaneous separation and detection of heavy metals and creatinine in urine in this invention includes the following steps: (1) Take the dual-channel platform device for sample addition. Add the test solution to the sample phase and the receiving solution to the receiving phase respectively. Turn on the power to start separation. Heavy metals and creatinine are transferred from the sample phase to the receiving phase respectively. (2) After separation, add color developer to develop color, take pictures of the color development area and then use RGB color model for detection.

[0035] In some embodiments, the sample phase is added in a volume of 80µL-150µL, and the receiving phase is added in a volume of 10µL-40µL.

[0036] In some embodiments, the separation time is 10 min to 30 min.

[0037] In some embodiments, the power supply during the separation process is a battery with a voltage of 1.5V-9V.

[0038] In some embodiments, the colorimetric reagent is added in a volume of 10µL-40µL, and the color development time is 3-10min.

[0039] This invention discloses a dual-channel platform for the simultaneous separation and detection of heavy metals and creatinine. From top to bottom, the platform comprises a receiving phase layer, a supporting membrane, and a sample phase layer. Both the receiving phase layer and the sample phase layer are paper-based, and the supporting membrane 6 is a hydrophobic membrane. The receiving phase layer includes two non-communicating hydrophilic regions on both its upper and lower surfaces, with the two non-communicating hydrophilic regions on the upper surface serving as the receiving phases for heavy metals and creatinine, respectively, i.e., the first receiving phase 3 and the second receiving phase 4. The sample phase layer includes a hydrophilic region on both its upper and lower surfaces, with the hydrophilic region on the upper surface serving as the sample phase 9. The sample phase and the receiving phase for heavy metals are connected to a lead wire 2 to form an electroporation extraction pathway. The entire dual-channel platform is housed within a resin casing 1.

[0040] A membrane solvent is applied to the support membrane at the positions corresponding to the two receivers. The first membrane solvent 7 applied to the position corresponding to the heavy metal receiver is n-octanol, 2-nitrophenyl octyl ether, n-heptol or n-nonanol, and the second membrane solvent 8 applied to the position corresponding to the creatinine receiver is n-hexanol, n-octanol or n-heptol.

[0041] The following are specific embodiments. Example

[0042] This invention relates to a dual-channel platform for the simultaneous separation and detection of heavy metals and creatinine in urine. Figure 1 Its operation steps include: (1) Preparation of paper-based device like Figure 2 Whatman qualitative filter paper, cut to shape I and III, is used to confine the sample or receiving solution to specific areas. Molten paraffin is chosen as the hydrophobic material to construct the hydrophobic region; except for the locations of the receiving and sample phases, the remaining areas are hydrophobic. Simultaneously, the solidification of the paraffin can fix the wires in place.

[0043] (2) Platform construction combined with 3D printed shell After the paper base is completed, a polypropylene fiber membrane is added between the two layers of paper as a support membrane (II), and a membrane solvent is applied to the positions corresponding to the two receivers. Then the entire paper base is placed into the resin shell for fixation.

[0044] (3) Separation of the target object After the platform is set up, 120 µL of sample is added to the sample phase, and 30 µL of receiving liquid is added to each of the two receiving phases. Connecting wires forms a pathway. Heavy metals enter the receiving phase from the sample phase via an electric field, while creatinine enters the receiving phase from the sample phase using the difference in its partition coefficient between the aqueous and organic phases. Figure 3 ).

[0045] (4) Rapid detection of target objects After the target analytes were separated, colorimetric reagents were added to the receiving phase. Once the colorimetric reaction stabilized, a photograph was taken with a mobile phone, and quantification was performed using the RGB color model. Figure 4 ) In some instances, the area of ​​the two receiving phases, i.e. the area of ​​the two hydrophilic regions, is 6mm × 6mm.

[0046] In some instances, the battery voltage is 9V.

[0047] In some instances, the amount of the two membrane solvents coated on the support liquid membrane is 5 µL.

[0048] In some instances, the sample phase was added in a volume of 120 µL.

[0049] In some instances, the sample volume of the receiving phase is 30 µL.

[0050] In some instances, the separation of the target object requires 20 minutes.

[0051] In some instances, the colorimetric reaction requires 5 minutes to stabilize. Example

[0052] The dual-channel platform for simultaneous separation and detection of heavy metals and creatinine in urine, provided by this invention, is used to detect lead and creatinine in urine. like Figure 2 Whatman qualitative filter paper, cut to shape I and III, is used to confine the sample or receiving solution to specific areas. Except for the two receiving phases (6mm × 6mm) and the sample phase (6mm × 10mm), the remaining portion is treated with molten paraffin to create a hydrophobic region. During the construction of the hydrophobic region, the solidification of the paraffin is used to fix the wires to the paper base.

[0053] After the paper base is completed, a 20mm × 10mm polypropylene fiber membrane is placed in the middle of the paper base as a support membrane. 5µL each of the selected membrane solvents n-hexanol (for creatinine extraction) and n-octanol (for lead extraction) are applied to the positions corresponding to the two receivers. The entire paper base is then fixed in place with a resin shell for later use. Figure 2 ).

[0054] After the platform setup was completed, sample loading began. 120 µL of urine was added to the sample phase, and 30 µL each of pH 2 hydrochloric acid solution (for creatinine) and ultrapure water (for lead) were added to the two receiving phases. Then, the circuit was connected using platinum wires. A 9V battery was used as the power source, and the extraction time was 20 minutes.

[0055] like Figure 4 As shown, the colorimetric reactions used for quantification on the dual-channel platform were optimized, and the normalized RGB values ​​of the colorimetric region showed a linear relationship with the concentration of the target analyte. After separation, the optimal colorimetric reagent for creatinine was 0.4 g / L alkaline picric acid, added in 20 µL; for lead, 10 µL of 2% potassium iodide solution was added first, followed by 10 µL of 0.3 g / L ethyl violet. Color development stabilized after 5 min, and RGB values ​​were obtained by photographing and using the RGB color model.

[0056] The target substances (lead: 0~200 μg / L, creatinine: 0~5 g / L) at varying concentrations were added to artificial urine as samples. Separation was performed using the aforementioned platform device. After separation, a colorimetric reagent was added for color development, yielding normalized RGB values. The concentrations of lead and creatinine were then calculated using standard curves. The ratio of lead concentration to creatinine concentration was the corrected metal concentration.

[0057] like Figure 5 As shown, we tested the urine of eight smelter workers using the above procedure and obtained results consistent with traditional methods. The corrected lead concentrations in the urine of the eight workers were 23.1 μg / g creatinine, 17.9 μg / g creatinine, 7.1 μg / g creatinine, 5.6 μg / g creatinine, 9.1 μg / g creatinine, 37.6 μg / g creatinine, 40.1 μg / g creatinine, and 21.6 μg / g creatinine, respectively.

[0058] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for the simultaneous separation of heavy metals and creatinine in urine, characterized in that, The method utilizes a dual-channel platform to simultaneously separate heavy metals and creatinine in urine, wherein the concentration of creatinine in the urine is greater than the concentration of the heavy metal; the heavy metal is lead; the dual-channel platform consists of a receiving phase layer, a supporting membrane, and a sample phase layer from top to bottom; both the receiving phase layer and the sample phase layer are paper-based, and the supporting membrane is a hydrophobic membrane; the upper and lower surfaces of the receiving phase layer each include two non-communicating hydrophilic regions surrounded by hydrophobic areas, and the two non-communicating hydrophilic regions on the upper surface of the receiving phase layer serve as receiving phases for heavy metals and creatinine, respectively; The membrane solvent coated at the location corresponding to the heavy metal receiver is n-octanol, 2-nitrophenyl octyl ether, n-heptanol, or n-nonanol, while the membrane solvent coated at the location corresponding to the creatinine receiver is n-hexanol, n-octanol, or n-heptanol. Both the upper and lower surfaces of the sample phase layer include a hydrophilic region surrounded by hydrophobic areas, with the hydrophilic region on the upper surface of the sample phase layer serving as the sample phase. The sample phase and the heavy metal receiver phase are respectively used to connect to the wires, thereby forming an electro-membrane extraction pathway. The method includes the following steps: (1) Add the urine sample to be tested to the sample phase, add water to the receiving phase which is the heavy metal, and add acid to the receiving phase which is the creatinine; (2) When the power is turned on, the heavy metals enter the receiving phase from the sample phase through the electric field force, and the creatinine enters the receiving phase from the sample phase by utilizing the difference in the partition coefficient between the aqueous phase and the organic phase, thereby realizing the separation of heavy metals and creatinine in the test solution.

2. The method as described in claim 1, characterized in that, After step (2), the process also includes adding color-developing agents to the two receiving phases respectively, and then calculating the normalized RGB values ​​of the two receiving phases using the RGB color model; then using the standard curve to calculate the concentration values ​​of lead and creatinine, and the ratio of the lead concentration value to the creatinine concentration value is the corrected lead concentration.

3. The method as described in claim 1, characterized in that, The receiving phases for heavy metals and creatinine have the same area, both being 25 mm². 2 -50 mm 2 .

4. The method as described in claim 1, characterized in that, The area of ​​the sample phase is 60 mm. 2 -150 mm 2 .

5. The method as described in claim 1, characterized in that, The support membrane is a polypropylene fiber membrane, a polyethylene fiber membrane, or a polyester fiber membrane.

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