阅读说明：本技术 一种二价铅离子免仪器定量检测方法 (Instrument-free quantitative detection method for divalent lead ions ) 是由 张云 姚茂茂 聂瑾芳 庄苗苗 于 2019-09-29 设计创作，主要内容包括：本发明公开了一种二价铅离子免仪器定量检测方法。利用Pb<Sup>2+</Sup>特异性结合修饰在微珠表面的DNA酶链后切割底物DNA链以释放催化探针用于原位产生气体产物,进而介导多通道纸芯片纸体润湿性变化以选择性调控彩色试剂在不同纸通道中流动长度的机制。Pb<Sup>2+</Sup>浓度与不同通道中彩色试剂的流动长度差值成正比关系。通过目视直接读取纸芯片中与彩色试剂流动长度相关的刻度数目和差值,就能实现nM水平Pb<Sup>2+</Sup>的免仪器定量检测。本发明方法操作简单、成本低廉、分析快速、不需使用专业分析仪器设备、适于Pb<Sup>2+</Sup>的现场分析和即时检测。本方法能直接推广应用于环境监测、食品安全、医学诊断等诸多领域里各类溶液样品中Pb<Sup>2+</Sup>的简单、经济、快速、灵敏、特异的便携式定量检测。(The invention discloses an instrument-free quantitative detection method for divalent lead ions. Utilizing Pb 2+ Specifically binds and modifies the DNA strand on the surface of the bead, then cuts the substrate DNA strand to release a catalytic probe for generating gas products in situ, and further mediates the change of the wettability of the paper body of the multi-channel paper chip to selectively regulate and control the flow length of the color reagent in different paper channels. Pb 2+ The concentration is in direct proportion to the difference of the flow lengths of the color reagents in different channels. The nM level of Pb can be achieved by visual direct reading of the number and difference of the graduations in the paper chip relating to the length of the flow of the color reagent 2+ The quantitative detection without instrument. The method has the advantages of simple operation, low cost, quick analysis, no need of professional analytical instruments and equipment, and suitability for Pb 2+ On-site analysis and point-of-care testing. The method can be directly popularized and applied to Pb in various solution samples in various fields of environmental monitoring, food safety, medical diagnosis and the like 2+ The method is simple, economical, rapid, sensitive and specific portable quantitative detection.)

1. An instrument-free quantitative detection method for divalent lead ions is characterized by comprising the following specific steps:

(1) preparing a multi-channel paper chip, and dripping and fixing a substrate DNA chain with a modified end-labeled catalytic probe and Pb in a round micro-area in a rectangular sample-adding channel²⁺Functionalized microbeads dependent on the DNA polymerase chain;

(2) sequentially fixing a reaction reagent 1, a reaction reagent 2 and a color reagent in three circular micro-areas with the same size of the rectangular analysis channel on the upper part of the paper chip, and sequentially fixing the reaction reagent 1, an inert reagent and a color reagent in three circular micro-areas with the same size of the rectangular analysis channel on the lower part of the paper chip;

(3) placing the tip of the sample loading channel of the paper chip at Pb²⁺The equal volume of the mixed solution of the sample solution and the buffer solution with the pH value of 7 is used for several seconds, then the flowing condition of the color reagent in the two analysis channels is observed, the scale number related to the flowing length of the color reagent is read after the flowing condition is stopped, and the obtained flowing length difference value and Pb are obtained²⁺Is in positive correlation, thereby realizing Pb²⁺Quantitative detection without instrument;

the paper chip is one of a hollow type and a hydrophobic substance patterning type, has a Y-shaped structure, and mainly comprises a communicated rectangular sample adding channel and an upper rectangular analysis channel and a lower rectangular analysis channel which are identical in shape;

the length of the rectangular sample loading channel of the paper chip is 1 ~ 2 cm, the width is 1.5 ~ 2.5.5 mm, and the middle part of the rectangular sample loading channel is designed into a circular micro-area with the diameter of 4 ~ 7mm for dropwise adding and fixing a substrate DNA chain with a modified terminal labeling catalytic probe on the surface and Pb²⁺Dependent on the function of the DNA enzyme chainThe functionalized microbeads are one of inorganic matters and organic matters, the particle size of the functionalized microbeads is 1 ~ 5 mu m, and the functionalized microbeads do not flow in a paper channel along with liquid;

the catalytic probe, the reaction reagent 1 and the reaction reagent 2 can generate a gas product which is insoluble in a reaction solution after being mixed in a second circular micro-area of the upper analysis channel, wherein the length of the upper analysis channel is 5 ~ 7 cm, the width of the upper analysis channel is 1.5 ~ 2.5.5 mm, and the diameter of the second circular micro-area is 4 ~ 7 mm;

the inert reagent has similar wettability with the reaction reagent 2, and does not generate chemical and biological reactions after being mixed with the catalytic probe and the reaction reagent 1 in the second circular micro-area of the lower analysis channel;

the color reagent is dropwise added and fixed in the whole hydrophilic paper body of the last round paper micro-area in the two analysis channels of the paper chip, a color print formed by the color reagent in the paper body has good water solubility, and the color solution formed after being dissolved by the reaction solution can spontaneously flow to the rear rectangular hydrophilic paper channel from the round hydrophilic paper body under the action of capillary;

the Pb²⁺The reaction that occurs after the sample solution is absorbed by the paper chip is Pb²⁺The catalytic probe can be released to be mixed with the reaction reagent 1 and the reaction reagent 2 in the upper analysis channel in the second circular paper micro-area, the three are mixed to generate a gas product insoluble in a reaction solution, the hydrophobicity of the paper in the circular paper micro-area is enhanced, the volume of the reaction solution flowing to the third circular paper micro-area to dissolve the color reagent and in the rear rectangular channel is reduced or completely prevented, and more reaction solutions with constant total volume flow to the lower analysis channel.

Technical Field

The invention belongs to the technical field of nano chemical sensing, and particularly relates to an instrument-free quantitative detection method for divalent lead ions.

Background

The global environmental pollution problem is becoming more severe. From divalent lead ions (Pb)²⁺) Water pollution caused by heavy metal ionDyeing is one of the most serious environmental pollution problems, and seriously threatens human health. For example, excess Pb in the human body²⁺It will affect the development of children brain and nervous system, increase the risk of hypertension and kidney damage in adults, and cause abortion, stillbirth, premature labor and low birth weight in pregnant women. The lead content in drinking water must not exceed 0.01 mg/L (about 48 nM) as defined in Standard sanitary Standard for Drinking Water (GB 5749 and 2006). For securing Pb in drinking water, food and environment²⁺No influence on human health and life safety, Pb²⁺The quantitative detection of the method is significant. Existing conventional gold standard Pb²⁺The quantitative detection technology mainly comprises inductively coupled plasma mass spectrometry, atomic absorption spectrometry, atomic emission spectrometry, atomic fluorescence spectrometry and the like. However, these quantitative methods have problems of expensive analysis cost, tedious and time-consuming operation steps, and unsuitability for home use and field analysis.

Disclosure of Invention

The invention aims to provide an instrument-free quantitative detection method for divalent lead ions, aiming at the defects of the existing quantitative technology.

The idea of the invention is as follows: by Pb²⁺After specifically binding to its DNA strand, the corresponding substrate DNA strand is cleaved to release the catalytic probe pre-labeled at one end of the substrate DNA strand. The probe then participates in subsequent catalytic reaction in the paper chip to generate gas products in situ, and further selectively regulates and controls the flow length of the color reagent in different channels of the paper chip by mediating the change of the wettability of the paper body of the paper chip. Pb²⁺The concentration is in direct proportion to the difference of the flow lengths of the color reagents in different channels. The number of scales related to the flow length of the color reagent in different channels of the paper chip is read by visual observation to replace expensive and portable professional analytical instruments such as an inductively coupled plasma mass spectrometer, an atomic absorption spectrometer and an atomic fluorescence spectrometer to read signals (the flow length of the color reagent), so that Pb can be read²⁺The method is simple, rapid, low in cost and portable, and can be used for accurate quantitative analysis.

The method comprises the following specific steps:

(1) system for makingPreparing a multi-channel paper chip, and dripping and fixing a substrate DNA chain with a modified end-labeled catalytic probe and Pb in a round micro-area in a rectangular sample-adding channel²⁺Functionalized microbeads dependent on the DNA polymerase chain.

(2) The reaction reagent 1, the reaction reagent 2 and the color reagent are sequentially fixed in three circular micro-areas with the same size of the rectangular analysis channel on the upper part of the paper chip, and the reaction reagent 1, the inert reagent and the color reagent are sequentially fixed in three circular micro-areas with the same size of the rectangular analysis channel on the lower part of the paper chip.

(3) Placing the tip of the sample loading channel of the paper chip at Pb²⁺The equal volume of the mixed solution of the sample solution and the buffer solution with the pH value of 7 is used for several seconds, then the flowing condition of the color reagent in the two analysis channels is observed, the scale number related to the flowing length of the color reagent is read after the flowing condition is stopped, and the obtained flowing length difference value and Pb are obtained²⁺Is in positive correlation, thereby realizing Pb²⁺The quantitative detection without instrument.

The paper chip is one of a hollow type and a hydrophobic substance patterning type, has a Y-shaped structure, and mainly comprises a communicated rectangular sample adding channel and an upper rectangular analysis channel and a lower rectangular analysis channel which are identical in shape.

The length of the rectangular sample loading channel of the paper chip is 1 ~ 2 cm, the width is 1.5 ~ 2.5.5 mm, and the middle part of the rectangular sample loading channel is designed into a circular micro-area with the diameter of 4 ~ 7mm for dropwise adding and fixing a substrate DNA chain with a modified terminal labeling catalytic probe on the surface and Pb²⁺A DNA polymerase chain-dependent functionalized microbead of one of inorganic and organic substances having a particle size of 1 ~ 5 μm, while not flowing with liquid in the paper path.

The catalytic probe, the reagent 1 and the reagent 2 can generate a gas product insoluble in a reaction solution after being mixed in a second circular micro-area of the upper analysis channel, wherein the length of the upper analysis channel is 5 ~ 7 cm, the width of the upper analysis channel is 1.5 ~ 2.5.5 mm, and the diameter of the second circular micro-area is 4 ~ 7 mm.

The inert reagent has similar wettability with the reaction reagent 2, and does not generate chemical and biological reaction after being mixed with the catalytic probe and the reaction reagent 1 in the second circular micro-area of the lower analysis channel.

The color reagent is dripped and fixed in the whole hydrophilic paper body of the last round paper micro-area in the two analysis channels of the paper chip, the color blot formed by the color reagent in the paper body has good water solubility, and the color solution formed after being dissolved by the reaction solution can flow from the round hydrophilic paper body to the rear rectangular hydrophilic paper channel under the capillary action.

The Pb²⁺The reaction that occurs after the sample solution is absorbed by the paper chip is Pb²⁺The catalytic probe can be released to be mixed with the reaction reagent 1 and the reaction reagent 2 in the upper analysis channel in the second circular paper micro-area, the three are mixed to generate a gas product insoluble in a reaction solution, the hydrophobicity of the paper in the circular paper micro-area is enhanced, the volume of the reaction solution flowing to the third circular paper micro-area to dissolve the color reagent and in the rear rectangular channel is reduced or completely prevented, and more reaction solutions with constant total volume flow to the lower analysis channel.

Compared with the existing conventional gold standard Pb²⁺Compared with the quantitative detection method, the invention has the outstanding advantages that:

(1) the cost of porous paper base materials such as filter paper, chromatographic paper, nitrocellulose membrane and the like is low, the paper chips can be prepared in batches, and the size of a single chip is small and exquisite (centimeter level, convenient to store, carry and use).

(2) Quantitative detection of Pb by using paper chip²⁺The operation process of (A) is extremely simple and rapid, and only involves two steps of sample solution suction and color reagent flow length reading, thereby greatly reducing the analysis cost and realizing Pb²⁺Home testing and field analysis.

(3) The method can be directly popularized and applied to Pb in various solution samples in the fields of environmental monitoring, food safety, medical diagnosis and the like²⁺Simple, economical, rapid, sensitive and specific portable quantitative detection of analytes.

Drawings

FIG. 1 shows Pb in the present invention²⁺Schematic diagram of the principle of the instrument-free quantitative detection method.

The labels in the figure are: 1-a rectangular sample-adding channel of the paper chip; 2-a circular paper micro-area in the sample loading channel; 3-a first circular paper micro-area in the upper rectangular analysis channel; 4-a second circular paper micro-area in the upper analysis channel; 5-a third circular paper micro-area in the upper analysis channel; 6-first circular paper micro-area in lower rectangular analysis channel; 7-a second circular paper micro-area in the lower analysis channel; 8-a third circular paper micro-area in the lower analysis channel; 9-a colour reagent flow channel in the upper rectangular analysis channel; 10-a colour reagent flow channel in the lower rectangular analysis channel; 11-graduations in the paper chip to indicate the length of flow of the color reagent; 12-substrate DNA strand with terminal-labeled catalytic probe modified on surface and Pb²⁺Functionalized beads dependent on the DNA polymerase chain (12-1-beads, 12-2-substrate DNA strand, 12-3-catalytic probe labeled at the end of substrate DNA strand, 12-4-Pb²⁺Dependent DNA enzyme chain); 13-reagents 1 immobilized in the first circular paper micro-zones of the two rectangular analysis channels; 14-reagent 2 immobilized in the second circular paper micro-area of the upper analysis channel; 15-inert reagent immobilized in the second circular paper micro-area of the lower analysis channel; 16-color reagents immobilized in the third circular paper microdomains of the two rectangular analysis channels; 17-equal volume of mixed solution of sample solution and buffer solution; 18-Pb²⁺(ii) a 19-secondary products formed by the reaction of reagents 1 and 2; 20-Pb²⁺The catalytic probe released after combining the DNA polymerase chain and cutting the substrate DNA chain catalyzes the gas product which is generated by the secondary product and is not dissolved in the reaction solution; 21-the color reagent fixed in the paper chip is dissolved again by the reaction solution and flows into the two rectangular channels to form a color print; 22-flow length of color reagent in upper analysis channel; 23-flow length of color reagent in lower analysis channel; 24-difference in flow length of color reagent in upper and lower two analytical channels.

FIG. 2 is Pb using the present invention in example 1 of the present invention²⁺Respectively detecting blank water sample (blank) and 62 nM Pb without analyte ions by instrument-free quantitative detection method²⁺Water sample, other 15 kinds of metal ion water samples (ion concentration is 10 mu M), and metal ions and 62 nM Pb²⁺And (3) comparing the obtained signal values (length change difference of flow lengths of the color reagent red ink in the two analysis channels of the paper chip). Error bars in the figures are the standard deviation of the results of three replicates.

FIG. 3 is Pb using the present invention in example 2 of the present invention²⁺Instrumentally-free quantitative detection method for analyzing a series of Pb with concentration range of 1.8 ~ 500 nM²⁺Signal value (length change difference of color reagent red ink in two analysis channels of paper chip) and Pb in water sample²⁺Log value of concentration (Log [ Pb)²⁺]) The operating curve in between. Error bars in the figures are the standard deviation of the results of three replicates.

Detailed Description

The following examples further illustrate the invention but are not intended to limit the invention thereto.