CN114544577A - Micro-fluidic paper-based aptamer sensor based on fluorescence/colorimetric dual-mode detection and used for Pb2+Detection of (2) - Google Patents
Micro-fluidic paper-based aptamer sensor based on fluorescence/colorimetric dual-mode detection and used for Pb2+Detection of (2) Download PDFInfo
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- CN114544577A CN114544577A CN202210203900.0A CN202210203900A CN114544577A CN 114544577 A CN114544577 A CN 114544577A CN 202210203900 A CN202210203900 A CN 202210203900A CN 114544577 A CN114544577 A CN 114544577A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
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- G—PHYSICS
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Abstract
The invention relates to a micro-fluidic paper-based aptamer sensor based on fluorescence/colorimetric dual-mode detection for Pb2+Detection of (3). Pb2+When present, it specifically binds to a fluorescent-labeled aptamer (fluorescent probe) immobilized in advance on a fluorescent region with high affinity. Then, complementary DNA (cDNA) labeled with a quencher is added to form a double strand with the aptamer which does not form a G-quadruplex. The ligation bridge is switched on and unbound cDNA is washed into the colorimetric area by the aggregation buffer. At this time, the fluorescence signal of the sensor is protected by the G-quadruplex, and the gold nano-material in the colorimetric region keeps red under the absorption of the cDNA single strand. The microfluidic paper-based aptamer sensor based on fluorescence/colorimetric dual-mode detection is easy to miniaturize, low in cost and strong in selectivity, and can detect Pb in2+The method has strong practical application potential in field real-time detection.
Description
Technical Field
The invention belongs to the technical field of harmful substance detection, and particularly relates to a preparation method of a microfluidic paper-based aptamer sensor based on fluorescence/colorimetric dual-mode detection.
Background
Environmental and food pollution caused by heavy metals has attracted much attention as a serious global threat. As one of the most toxic and most polluting metal contaminants, lead can be enriched in the vital organs of the human body by the biological chain, and even at low concentrations can cause cardiovascular, renal and neurological diseases, which constitute a great risk to human health.
Measurement of Pb2+The conventional analysis methods include graphite furnace atomic absorption spectrometry (GF-AAS), inductively coupled plasma-mass spectrometry (ICP-MS), Flame Atomic Absorption Spectrometry (FAAS), High Performance Liquid Chromatography (HPLC), and the like. However, the technologies have the defects of complex equipment and procedure, long time consumption, high detection cost and the like, and the application of the technologies is limited. Therefore, development of simple, economical, time-saving Pb2+Analytical techniques are a great need.
For improving the selectivity and cost-effectiveness of the analytical method, the introduction of para-Pb2+Aptamers with high affinity and specificity are a good choice. Paper-based analytical equipment has shown great promise in many fields due to its low cost, ease of miniaturized design, and real-time detection. The microfluidic paper-based aptamer sensor based on fluorescence/colorimetric dual-mode detection is combined with the simplicity and easy miniaturization of the paper-based sensor, the low cost and high selectivity of the aptamer, the quick response of a fluorescence method and the field detection of a colorimetric method, and the detection is carried out on Pb in the presence of Pb2+The method has strong practical application potential in detection.
Disclosure of Invention
The invention relates to a preparation method of a microfluidic paper-based aptamer sensor based on fluorescence/colorimetric dual-mode detection.
Based on fluorescence/colorimetric dual-mode detectionMicrofluidic paper-based aptamer sensor for Pb2+The sensor is characterized by comprising the following steps:
preparing a microfluidic paper-based fluorescence/colorimetric dual-mode aptamer sensor: paper is used as a substrate, and two circular hydrophobic areas are manufactured to be used as fluorescence and colorimetric reaction areas. The aptamer marked by the fluorescent group is used as a fluorescent probe, and the nanogold material is used as a colorimetric probe. In the fluorescent reaction zone, the aptamer is first fixed on the paper-based detection hole. Washing with a washing buffer was performed after each reaction to remove unbound material. And simultaneously depositing red nano-gold materials in a colorimetric reaction zone. The fluorescence reaction zone and the colorimetric reaction zone are fixed through a breakable connecting bridge, and the connecting bridge is broken in the process of preparing the sensor.
Microfluidic paper-based fluorescence/colorimetric dual-mode aptamer sensor pair Pb2+The detection of (2): adding Pb in the fluorescence reaction area of the sensor2+Of time, Pb2+Specifically binds to the aptamer with high affinity. Adding complementary DNA (cDNA) marked with a quenching group, and forming double-stranded DNA (dsDNA) by base complementary pairing with the aptamer which does not form G-quadruplex. The fluorescent group is close to the quenching group to generate Fluorescence Resonance Energy Transfer (FRET), and the fluorescence is quenched. At this time, the connecting bridge is connected, the unbound cDNA is flushed into the colorimetric reaction zone through the aggregation buffer solution, and a macroscopic color reaction can be generated according to the Local Surface Plasmon Resonance (LSPR) characteristic of the nanogold material deposited in the colorimetric reaction zone. Therefore, in the absence of Pb2+When the cDNA and the aptamer are completely complementary, the fluorescence signal is weak, and the nanogold material turns blue under the action of the aggregation buffer solution; and Pb2+After the reaction with the aptamer, the G-quadruplex protects the fluorescence from being quenched, the fluorescence signal is strong, and the unbound cDNA can keep the red color of the nanogold material.
The paper base material is one of printing paper, qualitative filter paper and chromatographic paper; the hydrophobic region is formed by one of an ink-jet printing method, a marker method and a wax printing method.
The fixing method of the aptamer in the fluorescence reaction area is one of physical adsorption, Schiff base reaction and streptavidin-biotin reaction.
The diameter of the fluorescence and colorimetric reaction zone is 2-7 mm.
Said Pb2+The reaction temperature of the aptamer chain is 20-50 ℃, the reaction time is 10-60 min, the volume range is 2-20 mu L, and the concentration range is 5-15 mu mol/L.
The washing buffer solution is one or two of Tris-HCl, PBS and HEPES, the volume range is 5-20 mu L, and the pH range is 6.0-8.0.
The nano-gold material is one of gold nano-particles (Au NPs) and gold nano-rods (Au NRs).
The reaction temperature of the complementary strand is 20-50 ℃, the reaction time is 10-60 min, the volume range is 2-20 mu L, and the concentration range is 5-15 mu mol/L.
Said Pb2+The volume range is 2-20 mu L, and the reaction time is 10-60 min.
The aggregation buffer solution is water containing sodium chloride, Tris-HCl, PBS, HEPES solution, and sucrose/MgCl2One of the buffers has a volume range of 5-20 μ L and a pH range of 6.0-8.0.
In the sensor of the present invention, Pb labeled with a fluorescent group2+The aptamer chain is used as a fluorescent probe, the Au NPs is used as a colorimetric probe, the microfluidic paper-based material is used as a substrate of a sensor, and the substrate is used for Pb together with other materials2+Compared with the aptamer sensor, the aptamer sensor has the advantages of low cost, easy miniaturization design and real-time detection.
Drawings
FIG. 1 is a process diagram of the preparation and detection of a microfluidic paper-based aptamer sensor based on fluorescence/colorimetric dual-mode detection.
FIG. 2 shows the presence or absence of Pb2+A change in signal from the fluorescent detection zone in the presence.
FIG. 3 the different cDNA concentrations give the color change in the presence of the same amount of Au NPs. (a) The cDNA concentrations of (d) were 0, 2, 5 and 10. mu.M, respectively.
FIG. 4 fluorescent Signal vs. Pb2+Variation in concentration (Pb of a-f)2+Concentrations were 0, 0.01, 0.1, 0.5, 5, 50 μ M, respectively).
FIG. 5 ratio of variations in fluorescence signal versus different concentrations of Pb2+A linear relationship constructed between the logarithms of (c).
Detailed Description
The invention is described below with reference to specific examples:
example 1
The method comprises the following specific steps:
(1) preparing a microfluidic paper-based fluorescence/colorimetric dual-mode aptamer sensor: and drawing the hydrophobic area of the paper base by a mark pen. And dripping a streptavidin solution dissolved in a Tris buffer solution into the fluorescence reaction area, and incubating for a period of time to ensure that the streptavidin is uniformly coated on the surface of the paper base. After being washed by Tris buffer solution, the aptamer marked by biotin is dripped in a fluorescence reaction area to fix the recognition element. And dripping 20 nm Au NPs into the colorimetric reaction zone.
(2) Microfluidic paper-based fluorescence/colorimetric dual-mode aptamer sensor pair Pb2+Detection of (2): adding Pb with different concentrations in the fluorescence reaction area of the sensor2+Solution (0-50. mu.M) and react well. Pb2+Specifically binds to the aptamer with high affinity. Equal amounts of quencher-labeled cDNA of the aptamers were dropped onto the fluorescence reaction zone, and dsDNA was formed with aptamers that did not form G-quadruplexes. The fluorescence intensity was measured by a fluorescence spectrophotometer. The bridge is now engaged and unbound cDNA is washed into the colorimetric reaction zone by the aggregation buffer. Therefore, in the absence of Pb2+When the cDNA is completely complementary with the aptamer, the fluorescence signal is weak; the Au NPs became blue by the action of the aggregation buffer, and the R/B value (red luminance/blue luminance value) was small. And Pb2+After the reaction, the G-quadruplex protects the fluorescence from being quenched, and the fluorescence signal is strong; unbound cDNA can keep Au NPs red, and R/G value is increased. The colorimetric signals are determined by naked eyes, and after the intelligent mobile phone is used for photographing, specific data can be obtained through mobile phone color analysis software. The dual change of the fluorescence signal and the colorimetric signal can qualitatively and quantitatively detect Pb2+。
Example 2
The method comprises the following specific steps:
(1) preparing a microfluidic paper-based fluorescence/colorimetric dual-mode aptamer sensor: ink-jet printing draws hydrophobic areas of the paper substrate. Firstly, soaking a paper base material in a sodium hydroxide solution, dissolving lignin, drying, and then, generating aldehyde groups on paper base cellulose by using a sodium periodate-lithium chloride mixed solution to obtain a treated fluorescence reaction area. After being washed and dried by ultrapure water, the aptamer marked by the amino group is dripped into a fluorescence reaction area, and the identification element is fixed through Schiff base reaction. And dripping 20 nm Au NPs into the colorimetric reaction zone.
(2) Microfluidic paper-based fluorescence/colorimetric dual-mode aptamer sensor pair Pb2+Detection of (2): adding Pb with different concentrations in the fluorescence reaction area of the sensor2+Solution (0-50. mu.M) and react well. Pb2+Specifically binds to the aptamer with high affinity. Equal amounts of quencher-labeled cDNA from the aptamers were dropped onto the fluorescence reaction zone to form dsDNA with aptamers that did not form G-quadruplexes. The fluorescence intensity was measured by a microplate reader. The bridge is now engaged and unbound cDNA is washed into the colorimetric reaction zone by the aggregation buffer. Therefore, in the absence of Pb2+When the cDNA is completely complementary with the aptamer, the fluorescence signal is weak; the Au NPs became blue by the action of the aggregation buffer. And Pb2+After the reaction, the G-quadruplex protects the fluorescence from being quenched, and the fluorescence signal is strong; unbound cDNA can retain Au NPs red. The colorimetric signal was visually characterized. The dual change of the fluorescence signal and the colorimetric signal can qualitatively and quantitatively detect Pb2+。
Prepared fluorescent sensor pair Pb2+The detection has high accuracy and wide linear range (1 multiplied by 10)-8~5×10-2mol/L), low detection lower limit (0.8X 10)-8mol/L) of the compound. Meanwhile, the detection result of the actual sample (such as tea) shows that the prepared sensor has very good practical application value.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (5)
1. Micro-fluidic paper-based aptamer sensor based on fluorescence/colorimetric dual-mode detection and used for Pb2+The sensor is characterized by comprising the following steps:
(1) preparing a microfluidic paper-based fluorescence/colorimetric dual-mode aptamer sensor: taking paper as a base material, and manufacturing two circular hydrophobic areas as a fluorescence and colorimetric reaction area; the aptamer marked by the fluorophore is used as a fluorescent probe, and the nanogold material is used as a colorimetric probe; in the fluorescence reaction zone, an adapter is firstly fixed on a paper-based detection hole; washing with a washing buffer solution after each reaction to remove unbound materials; simultaneously depositing red nano-gold materials in a colorimetric reaction zone; the fluorescence reaction zone and the colorimetric reaction zone are fixed through a breakable connecting bridge, and the connecting bridge is broken in the process of preparing the sensor;
(2) microfluidic paper-based fluorescence/colorimetric dual-mode aptamer sensor pair Pb2+Detection of (2): adding Pb in the fluorescence reaction area of the sensor2+Of time, Pb2+Specifically binds to the aptamer with high affinity; adding complementary DNA (cDNA) marked with a quenching group, and forming double-stranded DNA (dsDNA) by base complementary pairing with the aptamer which does not form G-quadruplex; the fluorescent group is close to the quenching group to generate Fluorescence Resonance Energy Transfer (FRET), and the fluorescence is quenched; at this time, the connecting bridge is connected, and the unbound cDNA is washed into the colorimetric reaction zone by the aggregation buffer solution, according to the deposited nano-particles in the colorimetric reaction zoneThe Local Surface Plasmon Resonance (LSPR) characteristic of the gold material can generate macroscopic color reaction; therefore, in the absence of Pb2+When the cDNA and the aptamer are completely complementary, the fluorescence signal is weak, and the nanogold material turns blue under the action of the aggregation buffer solution; and Pb2+After the reaction with the aptamer, the G-quadruplex protects the fluorescence from being quenched, the fluorescence signal is strong, and the unbound cDNA can keep the red color of the nanogold material.
2. The preparation method of the microfluidic paper-based aptamer sensor based on dual-mode fluorescence/colorimetry detection according to claim 1, wherein in the step (1), the paper-based material is one of printing paper, qualitative filter paper and chromatographic paper; the hydrophobic area is formed by one of an ink-jet printing method, a Mark pen method and a wax printing method; the fixing method of the aptamer in the fluorescence reaction area is one of physical adsorption, Schiff base reaction and streptavidin-biotin reaction.
3. The preparation method of the microfluidic paper-based aptamer sensor based on dual-mode fluorescence/colorimetric detection as claimed in claim 1, wherein in the step (1), the diameter of the fluorescence and colorimetric reaction zone is 2-7 mm; said Pb2+The reaction temperature of the aptamer chain is 20-50 ℃, the reaction time is 10-60 min, the volume range is 2-20 mu L, and the concentration range is 5-15 mu mol/L; the washing buffer solution is one or two of Tris-HCl, PBS and HEPES, the volume range is 5-20 mu L, and the pH range is 6.0-8.0.
4. The preparation method of the microfluidic paper-based aptamer sensor based on dual-mode fluorescence/colorimetry detection as claimed in claim 1, wherein in the step (1), the nanogold material is one of gold nanoparticles (Au NPs) and gold nanorods (Au NRs).
5. The method for preparing a microfluidic paper-based aptamer sensor based on dual-mode fluorescence/colorimetry detection according to claim 1, wherein the step (2) is carried outThe reaction temperature of the complementary strand is 20-50 ℃, the reaction time is 10-60 min, the volume range is 2-20 mu L, and the concentration range is 5-15 mu mol/L; said Pb2+The volume range is 2-20 mu L, and the reaction time is 10-60 min; the aggregation buffer solution is water containing sodium chloride, Tris-HCl, PBS, HEPES solution, and sucrose/MgCl2One of the buffers has a volume range of 5-20 μ L and a pH range of 6.0-8.0.
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Citations (3)
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|---|---|---|---|---|
| CN106018373A (en) * | 2016-07-19 | 2016-10-12 | 济南大学 | Three-dimensional metal-enhanced fluorescent/colorimetric dual-mode paper chip and ATP measurement |
| US20180217062A1 (en) * | 2017-01-31 | 2018-08-02 | Industry-University Cooperation Foundation Hanyang University | Methods for detecting endocrine disruptors using dual modes of colorimetric and fluorometric analysis |
| CN110286224A (en) * | 2019-06-06 | 2019-09-27 | 江苏大学 | It is a kind of based on lead content detection method in upper conversion-gold nano-magnetic Nano specificity system tealeaves |
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Patent Citations (3)
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|---|---|---|---|---|
| CN106018373A (en) * | 2016-07-19 | 2016-10-12 | 济南大学 | Three-dimensional metal-enhanced fluorescent/colorimetric dual-mode paper chip and ATP measurement |
| US20180217062A1 (en) * | 2017-01-31 | 2018-08-02 | Industry-University Cooperation Foundation Hanyang University | Methods for detecting endocrine disruptors using dual modes of colorimetric and fluorometric analysis |
| CN110286224A (en) * | 2019-06-06 | 2019-09-27 | 江苏大学 | It is a kind of based on lead content detection method in upper conversion-gold nano-magnetic Nano specificity system tealeaves |
Non-Patent Citations (2)
| Title |
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| CHRISTY Y. HUI ET AL.: "A Paper Sensor Printed with Multifunctional Bio/Nano Materials", 《ANGEW. CHEM. INT. ED.》 * |
| 林永青 等: "基于金纳米聚集－核酸适配体传感光度法检测铅", 《中国卫生检验杂志》 * |
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