Self-driven paper chip for quantitative detection of pollutants based on surface energy gradient and its application

Abstract

The application relates to a self-driven pollutant quantitative detection paper chip based on a surface energy gradient and application, which comprises a hydrophilic-hydrophobic patterned sampling separation area of a test paper, a specific recognition fluorescent marker area, a hydrophilic-hydrophobic patterned enrichment area, a detection point area, a quality control area and a quantitative scale. The sampling separation area improves the chromatographic speed and separation coefficient of the target pollutant by controlling the pattern type and surface energy. The specific recognition fluorescent marker comprises a DNA recognition sequence with specific recognition capability for the pollutant and a fluorescent marker and marker release sequence designed based on the DNA recognition sequence; the hydrophilic-hydrophobic patterned enrichment area is coupled with capillary force self-driven detection through pattern and size control, and is designed based on the detection requirements of different target pollutants to improve the detection limit and keep consistent with the quality control. The application can greatly improve the separation and enrichment coefficient of the paper chip, improve the qualitative and quantitative detection capability of the paper chip, and improve the application of the paper chip in the environmental monitoring field.

Description

Technical Field

[0001] The present invention relates to the field of fluorescence detection, and in particular to a self-driven pollutant quantitative detection paper chip based on surface energy gradient and its application. Background Art

[0002] Traditional test strips, such as pH strips, are limited to qualitative and semi-quantitative measurements of specific analytes due to the limitations of naked-eye observation. These strips rely on paper chromatography to separate and enrich samples, resulting in poor separation capabilities, low enrichment coefficients, and high minimum detection limits. This significantly limits their application in rapid on-site pollutant detection, emergency response to sudden pollution incidents, and environmental monitoring in remote areas. They are particularly difficult to apply to the detection of trace pollutants in water.

[0003] Hydrophilic-hydrophobic patterning is an emerging technology for precisely manipulating droplets. By regulating the regional hydrophilicity and hydrophobicity of a paper substrate, it can guide the directional transport and enrichment of samples. This can significantly improve the sensitivity and detection limit of test strips. Microfluidics, a technology that precisely manipulates microfluidics, enables mixing, separation, and enrichment, and enables automated sample analysis driven by capillary forces and chromatography. Summary of the Invention

[0004] The present invention provides a self-driven pollutant quantitative detection paper chip based on surface energy gradient and its application. By coupling hydrophilic and hydrophobic patterns with multiple surface energy gradients and a paper-based microfluidic system, the paper chip can significantly improve the separation and enrichment coefficients, significantly enhance its qualitative and quantitative detection capabilities, and thus enhance its application in the field of environmental monitoring.

[0005] The technical solution of the present invention is a self-driven pollutant quantitative detection paper chip based on the integration of multiple surface energy gradient patterns, characterized in that it includes a hydrophilic and hydrophobic patterned sampling and separation area, a specific recognition fluorescent marker area, a hydrophilic and hydrophobic patterned enrichment area, a detection point area, a quality control area and a quantitative scale; wherein the sampling and separation area improves the chromatography speed and separation coefficient of the target pollutants by regulating the hydrophilic and hydrophobic pattern type and the surface energy of the hydrophilic and hydrophobic materials, the specific recognition fluorescent marker includes a DNA recognition sequence with specific recognition ability for pollutants and fluorescent-labeled nanoparticles and fluorescent-labeled DNA release sequences designed based on this, and the hydrophilic and hydrophobic patterned enrichment area uses capillary force to self-drive chromatography to the detection area through the stage-by-stage merging of flow channels and the gradual reduction of flow channel size.

[0006] Furthermore, the pattern type in the hydrophilic-hydrophobic patterned sampling and separation area is one or more of a dot-shaped, line-shaped or triangular array; when the surface energy is controlled, low-surface-energy hydrophobic molecules and high-surface-energy hydrophilic molecules are selected as the basis for constructing the patterned flow channel, the hydrophilic molecules are silane coupling agent KH792 or silane coupling agent KH560, and the hydrophobic molecules are 1H, 1H, 2H, 2H-perfluorodecyltrichlorosilane or 1H, 1H, 2H, 2H-perfluorooctyltriethoxysilane; the coating of the hydrophilic-hydrophobic molecules is coupled to the paper base by inkjet printing, and the hydrophilic-hydrophobic molecules are heat-treated in the flow channel area by hot stamping technology to chemically bond them to the paper chip; or the hydrophilic-hydrophobic molecules are first completely bonded to the paper chip by ultraviolet stamping, and then the excess hydrophilic-hydrophobic molecules in the flow channel part are removed by ultraviolet stamping to achieve the purpose of patterned integration.

[0007] Furthermore, the hydrophilic-hydrophobic patterned sampling and separation area includes an unpatterned paper-based sampling area and a hydrophilic-hydrophobic patterned separation area, and the hydrophilic pattern and the hydrophobic pattern are alternately arranged in the separation area to increase the interfacial energy difference to improve the separation efficiency; behind the hydrophilic-hydrophobic patterned sampling and separation area is an unpatterned paper-based separation flow channel, which is adjacent to the specific identification fluorescent marker area.

[0008] Furthermore, the specific recognition fluorescent marker region contains a DNA recognition sequence with specific recognition ability for pollutants. The DNA recognition sequence is combined with nanoparticles loaded in the specific recognition region, or combined with test paper with surface-modified amino groups; the DNA recognition sequence is chemically modified with thiol groups; the types of nanoparticles include but are not limited to nanospheres, nanorods, nanobipyramids, and nanowires.

[0009] Furthermore, the fluorescent marker is attached to the nanoparticle via a DNA release sequence, which is incompletely complementary to the DNA recognition sequence modified on the surface of the nanoparticle or test paper. The fluorescently labeled incompletely complementary DNA release sequence is loaded onto the nanoparticle through base pairing hybridization; when the sample flows through the specific recognition area, the DNA recognition sequence binds to the contaminant to form a more stable complex. The incompletely complementary DNA release sequence is released into the solution during this process and then chromatographed to the concentration and detection area to amplify the sample signal.

[0010] Furthermore, the hydrophilic-hydrophobic patterned enrichment region uses the hydrophilic region as the chromatographic flow channel for the analytical substrate. Through the merging of flow channels and the gradual reduction of flow channel size, coupled with capillary force-driven chromatography, the chromatography is finally chromatographed to the detection point area, thereby enhancing the color depth of the detection area and reducing the detection limit. Hydrophobic enrichment patterns with different enrichment coefficients are designed according to the distribution of different pollutants and detection requirements. Specifically, hydrophobic enrichment patterns with different enrichment coefficients are designed based on the national standard limit values ​​of different pollutants in different water bodies and the lowest concentration observed by the naked eye in the detection point area, that is, the lowest concentration observed by the naked eye is divided by the national standard limit value.

[0011] Furthermore, the detection point area is pre-loaded with a DNA capture sequence in the stationary phase through chemical modification and inkjet printing technology to capture the fluorescent label sequence released from the specific recognition site. The DNA capture sequence is bound to the test paper with amino groups modified on the surface or the nanoparticles without surface modification through the thiol group, and the DNA capture sequence is not completely paired with the DNA release sequence.

[0012] Furthermore, the quality control area is loaded with a fluorescent marker sequence with the highest detection concentration by inkjet printing.

[0013] Furthermore, the quantitative ruler uses the color of fluorescent molecules as a ruler, and marks the colors after detecting pollutants at different concentrations.

[0014] The present invention also relates to the application of the pollutant quantitative detection paper chip in the detection of trace pollutants in water at the nanomolar to micromolar level.

[0015] The beneficial effects of the present invention are:

[0016] The present invention utilizes hydrophilic and hydrophobic molecules with different surface energies to construct patterned hydrophilic and hydrophobic flow channels on the surface of a paper chip through inkjet printing, hot embossing, and UV embossing technologies. Furthermore, by regulating the type, size, and surface energy of the flow channels, rapid chromatography, separation, capillary self-drive, and capture of target pollutants are achieved, ultimately enabling visual qualitative and fluorescent quantitative detection of pollutants. This patterning technology expands the quantitative detection capabilities of the test paper, as well as its ability to rapidly conduct on-site detection and provide early warning of pollution, thus providing powerful technical support for detection in remote areas and early warning of sudden pollution incidents. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a schematic structural diagram of the self-driven pollutant quantitative detection paper chip integrated with multiple surface energy gradient patterns in Example 1 of the present invention.

[0018] Figure 2 for Figure 1 Schematic diagram of the hydrophilic pattern morphology of the separation sampling area.

[0019] Figure 3 Schematic diagram of fluorescent markers for specific recognition of fluorescent marker regions.

[0020] Figure 4 Schematic diagram of the hydrophobic pattern morphology of the enriched area.

[0021] Figure 5 Schematic diagram of the detection area.

[0022] Figure 6 Schematic diagram of the quality control area.

[0023] Figure 7 Schematic diagram of the quantitative scale. DETAILED DESCRIPTION

[0024] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.

[0025] Example 1

[0026] This embodiment provides a self-driven pollutant quantitative detection paper chip integrating multiple surface energy gradient patterns for single target pollutant detection, such as Figure 1 As shown in the figure, the paper-based detection chip mainly includes sampling and separation of target detection objects, specific identification, enrichment, capture and detection, quality control, and scale area.

[0027] The sampling and separation unit consists of a non-patterned paper-based sampling area and a hydrophilic-hydrophobic patterned separation area.

[0028] Behind the sampling and separation area is a non-patterned paper-based chromatography channel, and the specific recognition area is adjacent to the chromatography channel.

[0029] The specific recognition fluorescent marker region contains a DNA recognition sequence that has specific recognition capabilities for pollutants. The DNA recognition sequence is bound to nanoparticles loaded in the specific recognition region; the DNA recognition sequence is chemically modified with thiol groups; the nanoparticles are specifically nanowires. The fluorescent marker is connected to the nanoparticles via a DNA release sequence, which is connected to the DNA recognition sequence modified on the surface of the nanoparticles or test paper by incomplete complementary pairing. The fluorescently labeled incompletely complementary DNA release sequence is loaded onto the nanoparticles through base pairing hybridization; when the sample flows through the specific recognition region, the DNA recognition sequence binds to the pollutant to form a more stable complex. The incompletely complementary DNA release sequence is released into the solution during this process and then chromatographed to the concentration and detection region to amplify the sampling signal.

[0030] The hydrophilic and hydrophobic enrichment pattern has a patterned microfluidic system. The microfluidic system concentrates the target detection object through stage-by-stage merging. The size of the microfluidic system decreases from the specific recognition area to the detection point area. The detection point area can capture the fluorescent-labeled specific DNA capture sequence through inkjet printing. The DNA capture sequence is combined with the nanowire through the thiol group.

[0031] The quality control area is loaded with a certain concentration of fluorescently labeled DNA release sequence through inkjet printing. The fluorescently labeled DNA release sequence is combined with the nanowire through the thiol group.

[0032] The quantitative ruler is loaded with fluorescently labeled DNA sequences of different concentrations by inkjet printing, and the fluorescently labeled DNA sequences are combined with the nanowires through thiol groups.

[0033] This solution targets the detection of trace pollutants in water, such as PCB77. Water samples are dripped into the sampling area to ensure the entire sampling area is moistened. Dissolved components in the water diffuse into the hydrophilic-hydrophobic patterned separation area under the action of chromatography. Finally, the target test substance is separated from other dissolved components by liquid chromatography through the separation area. Chromatography then releases a certain amount of fluorescently labeled DNA release sequences through the specific recognition area. The released sequences pass through the enrichment flow channel and achieve signal enrichment through step-by-step merging. The fluorescently labeled DNA release sequences are chromatographed to the detection area and bind to the capture sequence in the stationary phase. The color depth of the detection point is compared with the color depth of the quality control point to determine whether the test has been detected. The content is preliminarily determined by comparing the color depth of the quantitative scale. When quantitative detection is required, a portable fluorescence detector can be used to determine the precise content.

[0034] The design of the patterned separation area was based on the analysis of the surface energy of the main dissolved components and target pollutants in the water. The low-surface-energy hydrophobic molecule 1H, 1H, 2H, 2H-perfluorodecyltrichlorosilane with a high separation distribution coefficient and the high-surface-energy hydrophilic molecule KH560 were selected as the basis for the construction of the patterned flow channel. Due to the hydrophobic properties of PCB77, a separation area with a hydrophilic dot-line pattern was designed to promote the separation of PCB77 from other hydrophilic substances. The chip detection limit was designed to be 1 μM. This scheme uses rhodamine as a fluorescent marker, whose minimum visible concentration is 1 mM, and the designed enrichment factor is 6.4×10 3 Paper-based quantitative detection chip.

[0035] Due to the different diffusion rates of different dissolved components in the flow channel and the different adsorption and resolution capabilities in the hydrophobic solid phase, hydrophilic solid phase and liquid phase, their distribution coefficients are different, such as Figure 2As shown, the dotted and line hydrophilic pattern preferentially adsorbs solutes with strong hydrophilicity and has a lower affinity for the target detection substance PCB77. After multiple separation cycles, PCB77 in the sample is finally quickly separated by chromatography.

[0036] Assume that the distribution coefficient of the target pollutant to the hydrophilic line is K1, the distribution coefficient to the hydrophilic point is K2, and the distribution coefficient to the flow channel is K3. The distribution coefficient of other dissolved components in the sample to the hydrophilic line is k i1 (There is no hydrophobic barrier in this scheme, and the value is 1), and the distribution coefficient for the hydrophilic site is k i2 , the distribution coefficient of the flow channel is k i3 , where i represents the various soluble impurities with different surface energies present in the sample. All partition coefficients are referenced to water. Then the partition coefficient of the target pollutant in the separation unit composed of a single-stage hydrophilic line and hydrophilic point is:

[0037]

[0038] By coupling the separation unit array, the sample is passed through n-stage separation units one by one, and the final separation coefficient is:

[0039]

[0040] like Figure 3 The figure shows a schematic diagram of the specific recognition of the fluorescent marker area. The target pollutant is captured by designing and screening DNA nucleic acid aptamer sequences for the target pollutant. Among them, the capture sequence is a DNA sequence that fully adapts to the target pollutant. Its 5' end is modified with a thiol group and bonded to the surface of the silver nanowire. Due to its large size, the silver nanowire adsorbs and entangles with the fibers in the paper chip, does not migrate in the paper chip, and is considered to be a stationary phase. A DNA release sequence that is not completely complementary to the DNA recognition sequence is designed and synthesized to ensure its complementarity of more than 15 base pairs to ensure stability before detection. The fluorescent marker molecule rhodamine is modified at the 5' end of the DNA release sequence. When the target pollutant binds to the DNA recognition sequence, the fluorescently labeled DNA release sequence is released and chromatographed to the enrichment area.

[0041] like Figure 4 The figure shows the schematic diagram of the structure of the hydrophobic patterned enrichment area. Through the merging of the patterned flow channels, the fluorescently labeled DNA sequence released from the specific recognition area is finally capillary driven to the final flow channel. For an enrichment unit with n-level microfluidic channels, when the radius of the first-level microfluidic channel is R and the radius of the n-level microfluidic channel is r, the corresponding enrichment coefficient δ is:

[0042]

[0043] Specifically, the width of the enrichment area is set to 2 cm, the width of the hydrophobic barrier between the flow channels is 10 μm, the diameter of the first-level flow channel is 40 μm, the diameter of the n-level flow channel is 1 μm, n is 3, and the enrichment coefficient δ is 6.4×10 3 The upper limit of this enrichment factor depends on the resolution of the patterning technology.

[0044] like Figure 5 As shown, the enriched fluorescently labeled DNA release sequence diffuses to the detection area under the action of chromatography, and binds to the nanowires modified with incompletely matching DNA capture sequences in the region, resulting in color development. The purpose of qualitative and quantitative detection is achieved by determining the color.

[0045] like Figure 6 As shown, the quality control area is loaded with a certain amount of fluorescent labeling sequence through inkjet printing to mark the minimum detection limit of the detection chip and ensure that the color development ability of the detection paper chip is normal.

[0046] like Figure 7 As shown, the quantitative marking area is loaded with different concentrations of fluorescent marker sequences through inkjet printing to identify pollutants of different concentrations, facilitating preliminary determination of pollutant concentrations. When performing quantitative testing, a standard fluorescence intensity is provided for the target pollutant to improve the reliability of quantitative detection.

[0047] Example 2: The recognition sequence modified silver nanowires in Example 1 are replaced by modifying the recognition sequence on gold nanorods, which has the same effect.

[0048] Example 3: The silver nanowires modified with the fully complementary pairing sequence in Example 1 were replaced by modifying the gold nanocones with the fully complementary pairing sequence, which had the same effect.

[0049] Example 4: The recognition sequence modified silver nanowires in Example 1 were replaced by modifying the recognition sequence on the surface of the amino-modified test paper, which had the same effect.

[0050] Example 5: The silver nanowires modified with the fully complementary paired sequence in Example 1 were replaced by modifying the fully complementary paired sequence on the surface of the amino-modified test paper, which had the same effect.

Claims

1. A self-driven pollutant quantitative detection paper chip based on surface energy gradient, characterized in that: It includes a hydrophilic and hydrophobic patterned sampling and separation area, a specific recognition fluorescent marker area, a hydrophilic and hydrophobic patterned enrichment area, a detection point area, a quality control area, and a quantitative scale; The sampling and separation area improves the chromatography speed and separation coefficient of the target pollutants by regulating the type of hydrophilic and hydrophobic patterns and the surface energy of hydrophilic and hydrophobic materials. The specific identification fluorescent marker includes a DNA recognition sequence with specific recognition ability for pollutants, as well as fluorescently labeled nanoparticles and fluorescently labeled DNA release sequences designed based on this. The hydrophilic and hydrophobic patterned enrichment area is self-driven by capillary force to the detection area through the stage-by-stage merging of flow channels and the gradual reduction of flow channel size.

2. The pollutant quantitative detection paper chip according to claim 1, characterized in that: The pattern type in the hydrophilic-hydrophobic patterned sampling and separation area is one or more of a dot-shaped, linear or triangular array; when regulating the surface energy, low-surface-energy hydrophobic molecules and high-surface-energy hydrophilic molecules are selected as the basis for constructing the patterned flow channel, the hydrophilic molecules are silane coupling agent KH792 or silane coupling agent KH560, and the hydrophobic molecules are 1H,1H,2H,2H-perfluorodecyltrichlorosilane or 1H,1H,2H,2H-perfluorooctyltriethoxysilane; the coating of the hydrophilic-hydrophobic molecules is coupled to the paper base by inkjet printing, and the hydrophilic-hydrophobic molecules are heat-treated in the flow channel area by hot stamping technology to chemically bond them to the paper chip; or the hydrophilic-hydrophobic molecules are first completely bonded to the paper chip by ultraviolet stamping, and then the excess hydrophilic-hydrophobic molecules in the flow channel are removed by ultraviolet stamping to achieve the purpose of patterned integration.

3. The paper chip for quantitative detection of pollutants according to claim 1, characterized in that: The hydrophilic-hydrophobic patterned sampling and separation area includes an unpatterned paper-based sampling area and a hydrophilic-hydrophobic patterned separation area. Hydrophilic patterns and hydrophobic patterns are alternately arranged in the separation area to increase the interfacial energy difference and improve the separation efficiency. Behind the hydrophilic-hydrophobic patterned sampling and separation area is an unpatterned paper-based separation flow channel, which is adjacent to the specific identification fluorescent marker area.

4. The paper chip for quantitative detection of pollutants according to claim 1, characterized in that: The specific recognition fluorescent marker region contains a DNA recognition sequence with specific recognition ability for pollutants. The DNA recognition sequence is combined with nanoparticles loaded in the specific recognition region, or combined with test paper with surface modified amino groups; the DNA recognition sequence is chemically modified with thiol groups; the types of nanoparticles include but are not limited to nanospheres, nanorods, nanobipyramids, and nanowires.

5. The paper chip for quantitative detection of pollutants according to claim 4, characterized in that: The fluorescent marker is connected to the nanoparticle through a DNA release sequence, and the DNA release sequence is connected to the DNA recognition sequence modified on the surface of the nanoparticle or the test paper by incomplete complementary pairing.

6. The paper chip for quantitative detection of pollutants according to claim 5, characterized in that: The hydrophilic-hydrophobic patterned enrichment area uses a hydrophilic area as a chromatographic flow channel for the analytical substrate, and through the step-by-step merging of the flow channels and the gradual reduction of the flow channel size, the capillary force is coupled to drive the chromatography, and finally the chromatography is carried out to the detection point area; according to the national standard limit values ​​of different pollutants in different water bodies and the lowest concentration observed by the naked eye in the detection point area, hydrophobic enrichment patterns with different enrichment coefficients are designed, that is, the lowest concentration observed by the naked eye is divided by the national standard limit value.

7. The paper chip for quantitative detection of pollutants according to claim 5, characterized in that: The detection point area is pre-loaded with a DNA capture sequence in the stationary phase through chemical modification and inkjet printing technology to capture the fluorescent label sequence released from the specific recognition site. The DNA capture sequence is combined with the test paper with amino group modification on the surface or the nanoparticles with no modification on the surface through the thiol group, and the DNA capture sequence is not completely paired with the DNA release sequence.

8. The paper chip for quantitative detection of pollutants according to claim 1, characterized in that: The quality control area is loaded with fluorescent labeling sequences by inkjet printing.

9. The paper chip for quantitative detection of pollutants according to claim 1, characterized in that: The quantitative ruler uses the color of fluorescent molecules as a ruler, and is marked by the color after the pollutants are detected at different concentrations.

10. Use of the pollutant quantitative detection paper chip according to any one of claims 1 to 9 in the detection of trace pollutants in water at the nanomolar to micromolar level.

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