A Calcium-Ion-Cleaving General-purpose Interval Ultrafast Amplification Visual Sensor
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A calcium ion and substrate chain technology, applied in the field of biosensors, can solve problems such as long detection cycle, large equipment, and high cost
Active Publication Date: 2020-10-02
CHINA AGRI UNIV
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Problems solved by technology
[0004] The detection methods of calcium ions in water mainly include titration analysis and atomic photometry. This method generally requires professional technicians to collect samples to the laboratory for analysis, and there are some shortcomings, such as the need for large-scale equipment and long detection cycles. , expensive, requires professionals to operate, etc.
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Embodiment 1
[0100] Example 1 Establishment of Calcium Ion-Cleaving Universal Separation Ultrafast Amplification Visual Sensor
[0101] 1. Experimental materials
[0102] SYBR Gold nucleic acid dye, nucleic acid molecular weight standard ultra-low range DNA ladder, dNTP, ExTaq DNA polymerase, 10×Taq buffer, hemin, calcium chloride, 2,2-azino-bis(3-ethyl- Benzothiazole-6-sulfonic acid) diamine salt (ABTS), H 2 o 2 , 4-Hydroxyethylpiperazineethanesulfonic acid (HEPES), MES, LiCl, Tris, potassium chloride, sodium chloride, magnesium chloride, disodium edetate, sulfuric acid, tetramethylbenzidine, urea, all Purchased from Thermo Scientific Life Technologies. All experimental water was from Milli-Q pure water system.
[0103] The sequence design is as follows (SEQ ID NO:1-7):
[0104]
[0105]
[0106] Note: The ribozyme cuts the target product and the 3' end sequence of the amplified template is complementary;
[0107] The bold base sequence added to the end of the ribozyme substra...
Embodiment 2
[0128] The specificity investigation of embodiment 2 sensor
[0129] According to the biosensor constructed in Example 1, 10nM Ca 2+ , 10 μM Mg 2+ 、Cd 2+ 、Cu 2+ , Zn 2+ 、Cr 3 + , Pb 2+ , Fe 2+ Added to the system for detection, the results show that the established Ca 2+ The biosensor has good specificity ( Figure 5 ).
Embodiment 3
[0130] Embodiment 3 standard addition experiment
[0131] Get high-purity water and detect with the biosensor constructed in embodiment 1, Ca 2+ No detection was carried out, and the standard addition experiment was carried out on it, and the results obtained by continuous measurement are shown in Table 1.
[0132] Table 1 Ca 2+ Spike recovery test results
[0133]
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Abstract
The invention provides a cutting type general partition ultrafast amplificationvisualization sensor for calcium ions. The sensor comprises a cutting system (1), an sPCR (serial port control register)amplification system, an HCR (hybridization chain reaction) system (3) and a detection system containing ABTS developing solution (4). In the sensor, a primer, a template and a probe are skillfully designed, so that the template can be ultrafast amplified under the existence of the calcium ions, and an amplified product is enabled to react by priming an HCR to form G-quadruplex in a suitable environment. Further, the sensor is used for developing by utilizing the activity of peroxidase of the G-quadruplex, the problem that the traditional PCR product is difficult in visual detection is solved,and the calcium ions are detected fast and visually. Additionally, the sensor provided by the invention has the characteristics of high specificity and high sensitivity, and a detection result is more objective and more accurate.
Description
technical field [0001] The invention relates to the technical field of biosensors, in particular to a calcium ion-cutting general-purpose barrier ultrafast amplification visualization sensor. Background technique [0002] Calcium is a metal element with symbol Ca, atomic number 20, and atomic weight 40.078. It belongs to group IIA and is silver-white crystal at room temperature. The calcium content in the earth's crust is 4.15%, occupying the fifth place. The main calcium-bearing minerals are limestone CaCO 3 , Dolomite CaCO 3 · MgCO 3 , Gypsum CaSO 4 2H 2 O, Fluorite CaF 2 , apatite Ca 5 (PO 4 ) 3 F et al. Calcium is found in eggshells, pearls, coral, some animal shells and soil. Calcium chloride in seawater accounts for 0.15%. Calcium can be used as a deoxidizer for alloys, a dehydrating agent for oils, a reducing agent for metallurgy, a desulfurization and decarburization agent for iron and iron alloys, and a getter in electronic tubes. Its compounds are wide...
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