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Lead ion detection chip based on deoxyribonuclease as well as making and application methods

A deoxynuclease and detection chip technology, applied in the field of biological analysis, can solve the problems of cumbersome operation and unsuitability for high-throughput detection, and achieve the advantages of simple production method, low reagent consumption, high detection sensitivity and specificity Effect

Active Publication Date: 2011-04-27
SHANGHAI INST OF MICROSYSTEM & INFORMATION TECH CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Due to the characteristics of DNAzyme, such as easy synthesis and modification, good stability and little environmental pollution, its application in metal ion detection has attracted much attention, among which the more typical one is the detection of Pb 2+ Specific 8-17E DNAzyme (Brown, A.K.; Li, J.; Pavot, C.M.B.; Lu, Y. Biochemtstry 2003, 42, 7152-7161.), and thus developed a variety of detection Pb based on 8-17E DNAzyme 2+ Methods: such as homogeneous fluorescence method (Li, J.; Lu, Y.J.Am.Chem.Soc.2000, 122, 10466-10467), nano-gold aggregation colorimetry (Liu, J.W.; Lu, Y.J.Am.Chem. Soc.2003, 125, 6642-6643), a method for assembling 8-17E DNAzym on a gold surface for liquid phase fluorescence detection (Swearingen, C.B.; Wernette, D.P.; et al.Anal.Chem.2005, 77(2), 442-448.) A method for assembling 8-17E DNAzym on the surface of PMMA in a microfluidic chip for fluorescence detection (Dalavoy, T.S.; Wernette, D.P.; et al.LabChip, 2008, 8, 786-793.) These methods are common It has the characteristics of good selectivity and strong specificity, but the operation is cumbersome and not suitable for high-throughput detection

Method used

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  • Lead ion detection chip based on deoxyribonuclease as well as making and application methods
  • Lead ion detection chip based on deoxyribonuclease as well as making and application methods
  • Lead ion detection chip based on deoxyribonuclease as well as making and application methods

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Effect test

Embodiment 1

[0026] Embodiment 1: utilize probe Pb-1 and Pb-2 to prepare Pb 2+ Detection chip.

[0027] Dissolve 40 μM Pb-2 in water, then mix it with the same volume of Spotting Solution, use Cartesian’s microarray chip production system to array on the surface of the aldehyde-modified glass slide, and place it at room temperature at 70% relative humidity Store for 48-72 hours at room temperature for fixation, then immerse the slide in 0.2% SDS at room temperature for a few minutes, then immerse in pure water for a few minutes, then immerse in 0.2% SDS twice, each time for 2 minutes, and then immerse in pure water Twice, 2min each time, let dry. Pb-1 was diluted with hybridization solution (10mM Tris-HCl, pH 7.2, 1M NaCl) to a final concentration of 5 μM, dropped on the chip, covered with a cover slip, and hybridized at room temperature for 12-16 hours. Then put the chip at 4°C for 30min, then at 25°C for 15min. Then wash with 0.2% SDS, 2×SSC, 0.2×SSC for 3 minutes, and dry it for late...

Embodiment 2

[0028] Embodiment 2: utilize probe Pb-3 and Pb-4 to prepare Pb 2+ Detection chip.

[0029] Dissolve 40 μM Pb-4 in water, then mix it with the same volume of Spotting Solution, use Cartesian’s microarray chip production system to array on the surface of aldehyde-modified glass slides, store at room temperature and 70% relative humidity Fix for 48-72 hours, then immerse the slide in 0.2% SDS at room temperature for a few minutes, then immerse in pure water for a few minutes, then immerse in 0.2% SDS twice, each time for 2 minutes, then immerse in pure water twice , each 2min, dry. Dilute Pb-3 with hybridization solution (10mM Tris-HCl, pH 7.2, 100mM NaCl) to a final concentration of 5μM, drop on the chip, cover with a cover glass, and hybridize at room temperature for 12-16h, then place the chip at 4°C, 30min, then 15min at 25°C. Then wash with 0.2% SDS, 2×SSC, 0.2×SSC for 3 minutes, and dry it for later use.

Embodiment 3

[0030] Embodiment 3: Using the chip prepared by probe Pb-1 and Pb-2 to detect different concentrations of Pb 2+ .

[0031] Prepare different concentrations of Pb by diluting with 10mM Tris-HCl, pH 7.2, 50mM NaCl 2+ , with different concentrations of Pb 2+ The solution was placed on the prepared chip spots at 4°C for 1 hour, and then left at room temperature for 15 minutes. And washed 3 times with buffer, and dried. Scan the photos with the chip signal analysis system Scanarray 3000 of General Scanning Company ( figure 2 A) and analyze the results ( figure 2 B).

[0032] The results showed that in Pb 2+ In the presence of ions, the fluorescence intensity at the chip spot weakens, when Pb 2+ When the concentration was 10nM, compared with the fluorescence intensity of the buffer group, the fluorescence intensity at the spot was weakened by 40%. 2+ As the concentration increases, the fluorescence signal gradually decreases. The chip detects Pb 2+ The limit of detection...

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Abstract

The invention relates to a Pb<2+> detection chip based on deoxyribonuclease as well as making and application methods. The invention is characterized in that a 17DS substrate chain of a 17E enzyme chain of deoxyribonuclease 8-17 with strong specificity response is cut off, so that part or all of the cut-off 17DS substrate chain falls off. Since the 17DS substrate chain is previously labeled by fluorescence, the cut-off operation weakens the fluorescence signal. The application method is characterized by comprising the following steps: when the lead ion detection chip is used for lead ion detection, putting the sample to be detected on the chip, holding for a period of time, scanning the chip by using a chip signal analysis system, and analyzing the fluorescence signal. The Pb<2+> is detected through the variation of the fluorescence signal; and the fluorescence signal gets weakened as the concentration of Pb<2+> in the sample gets higher. The detection range of the concentration of Pb<2+> is 1nM-10mu M.

Description

technical field [0001] The invention relates to the manufacture and application method of a DNAZyme-based lead ion detection chip, which belongs to the technical field of biological analysis. Background technique [0002] Because lead is widely used in fuels, building materials, coatings, paints, and industrial processes, lead is ubiquitous in the environment, including soil, water, and even the food chain. Lead pollution has always been a very serious environmental problem. Once the inorganic pollutants represented by lead ions enter the environment, they will not be decomposed quickly in the environment like organic pollutants. They may remain in the environment for a long time, resulting in persistent pollution. And the lead ion that enters human body also exists huge harm to the health of human body, and when the lead content in human body exceeds a certain level, will seriously affect people's health, especially the health of children. Lead poisoning damages at least ...

Claims

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Application Information

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IPC IPC(8): C12Q1/44G01N21/64
Inventor 娄新徽刘美英杜娟赵建龙
Owner SHANGHAI INST OF MICROSYSTEM & INFORMATION TECH CHINESE ACAD OF SCI
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