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CN-122012084-A - Dual red fluorescence ratio detection Cu2+Tetrahedral DNA-silver cluster probe and its preparation method

CN122012084ACN 122012084 ACN122012084 ACN 122012084ACN-122012084-A

Abstract

The invention discloses a tetrahedral DNA-silver cluster probe for detecting Cu 2+ by double red fluorescence ratio and a preparation method thereof, wherein the tetrahedral DNA-silver cluster probe can emit strong red fluorescence and weaker near infrared red fluorescence at the same time, and can detect Cu 2+ by rapid ratio fluorescence. The tetrahedral DNA template comprises four sequences of S1, S2, S3 and S4, wherein ,S1:5'-AAAAAAACCCCCCCCCCCCCGCTTCAGACTTCACAGCTGCATCGGATGGCAGCCCATATTACTCCT-3';S2:5'-TTCCAGGACGCACAGAGCCATCCGATGCAGCATCTCAATGCAATGGC-3';S3:5'-CTGTGCGTCCTGGAAACAATTCGCTCTCACTAAGGAGTAATATGGGC-3';S4:5'-GTGAAGTCTGAAGCGAAGTGAGAGCGAATTGAGCCATTGCATTGAGA-3'. the tetrahedral DNA-silver cluster probe for detecting Cu 2+ by using the double red fluorescence ratio has excellent selectivity to Cu 2+ , and has been used for rapid detection of trace Cu 2+ in an actual water body sample, and the detection result is sensitive and accurate.

Inventors

  • REN WANG
  • JIANG MENGWEI
  • WANG HUIZHU
  • ZHANG YING
  • LONG LI
  • JIN XIN
  • CHEN MINGYI
  • BI SIJIE
  • CHEN YAO

Assignees

  • 四川轻化工大学

Dates

Publication Date
20260512
Application Date
20260206

Claims (10)

  1. 1. A tetrahedral DNA-silver cluster probe for detecting Cu 2+ by double red fluorescence ratio, which is characterized by comprising a tetrahedral DNA template capable of simultaneously emitting strong red fluorescence and weak near infrared red fluorescence, wherein the tetrahedral DNA template comprises four sequences of S1, S2, S3 and S4; Wherein the S1 sequence is 5'-AAAAAAACCCCCCCCCCCCCGCTTCAGACTTCACAGCTGCATCGGATGGCAGCCCATATTACTCCT-3'; The S2 sequence is 5'-TTCCAGGACGCACAGAGCCATCCGATGCAGCATCTCAATGCAATGGC-3'; the S3 sequence is 5'-CTGTGCGTCCTGGAAACAATTCGCTCTCACTAAGGAGTAATATGGGC-3'; the S4 sequence is 5'-GTGAAGTCTGAAGCGAAGTGAGAGCGAATTGAGCCATTGCATTGAGA-3'.
  2. 2. A method for preparing the tetrahedral DNA-silver cluster probe for detecting Cu 2+ by using the dual red fluorescence ratio according to claim 1, comprising the steps of: Adding a TDF-DNA cluster template solution and an AgNO 3 solution which are subjected to hybridization reaction into a Tris-HAc buffer solution, uniformly mixing, reacting for a period of time at room temperature in a dark place, adding a NaBH 4 solution which is prepared at present, incubating overnight at room temperature in a dark place, obtaining a TDF-DNA template AgNCs probe, namely a tetrahedral DNA-silver cluster probe of double red fluorescence ratio detection Cu 2+ , and refrigerating and preserving.
  3. 3. The method for preparing the tetrahedral DNA-silver cluster probe for detecting Cu 2+ by using the double red fluorescence ratio according to claim 2, wherein the final molar ratio of TDF-DNA cluster template, agNO 3 and NaBH 4 is 1:48:12.
  4. 4. The method for preparing the tetrahedral DNA-silver cluster probe for detecting Cu 2+ by using the double red fluorescence ratio according to claim 2, wherein the reaction time at room temperature and in the dark is 30-90 minutes after the Tris-HAc buffer solution, the TDF-DNA cluster template solution and the AgNO 3 solution are uniformly mixed.
  5. 5. The method for preparing the tetrahedral DNA-silver cluster probe for detecting Cu 2+ by using the double red fluorescence ratio according to claim 2 is characterized in that the method for preparing the TDF-DNA cluster template solution comprises the steps of uniformly mixing DNA sequences S1, S2, S3 and S4 with equal concentration in equal proportion, heating in a constant-temperature water bath with the temperature of 90-100 ℃ for 10-15 minutes, and cooling to obtain the TDF-DNA silver cluster template solution.
  6. 6. The method for preparing the tetrahedral DNA-silver cluster probe for detecting Cu 2+ by using the double red fluorescence ratio according to claim 3, wherein the concentration of Tris-HAc buffer solution is 5-30.0 mmol/L, the pH is 6.5-8.0, the concentration of TDF-DNA cluster template solution is 0.5-2.5 mu mol/L, the concentration of AgNO 3 solution is 0.5-5.0 mmol/L, the concentration of NaBH 4 solution is 0.5-5.0 mmol/L, and the volume ratio of Tris-HAc buffer solution to TDF-DNA cluster template solution is 1-2:1-4.
  7. 7. Use of the tetrahedral DNA-silver cluster probe of the dual red fluorescence ratio assay Cu 2+ of claim 1, wherein the tetrahedral DNA-silver cluster probe is used for rapid ratio, visualized fluorescence assay Cu 2+ .
  8. 8. The application of the tetrahedral DNA-silver cluster probe for detecting Cu 2+ by using the dual red fluorescence ratio according to claim 7 is characterized by that the tetrahedral DNA-silver cluster probe solution and Tris-HAc buffer solution are mixed according to the volume ratio of 1-2:1-4, then the standard solution of Cu 2+ with different concentrations or the sample solution containing Cu 2+ are added, after uniform mixing, the mixture is left to stand at room temperature for reaction for 1-30 minutes, the fluorescence photographs of the system under different Cu 2+ contents are recorded by a mobile phone, and then the concentration of Cu 2+ in the sample solution is rapidly semi-quantitative or quantitative by naked eyes or mobile phone software Image J according to the fluorescence color card of the Cu 2+ standard solution.
  9. 9. The method for detecting the tetrahedral DNA-silver cluster probe of the Cu 2+ by using the dual red fluorescence ratio according to the application method of the invention is characterized in that the specific application steps comprise the steps of mixing the tetrahedral DNA-silver cluster probe solution with Tris-HAc buffer solution according to the volume ratio of 1-2:1-4, then adding standard solutions of Cu 2+ with different concentrations or sample solutions containing Cu 2+ , standing at room temperature for reaction for 1-15 minutes after mixing uniformly, measuring the dual emission fluorescence spectrum of the system, establishing a standard curve according to the relationship between the fluorescence intensity ratio of red dual emission peaks and the concentration of Cu 2+ , and accurately quantifying the concentration of Cu 2+ in the sample solution according to the standard curve.
  10. 10. The use of a tetrahedral DNA-silver cluster probe for the detection of Cu 2+ by a dual red fluorescence ratio according to claim 8 or 9, wherein the tetrahedral DNA-silver cluster probe has an optimal fluorescence excitation wavelength of 540nm and 620nm, respectively, and the tetrahedral DNA-silver cluster probe has a maximum fluorescence emission wavelength of 617nm and 704nm, respectively; If the Cu 2+ concentration of the sample solution is too high, the sample is re-detected after being properly diluted with secondary distilled water.

Description

Tetrahedral DNA-silver cluster probe for detecting Cu 2+ by double red fluorescence ratio and preparation method Technical Field The invention belongs to the technical field of preparation of fluorescent nano-cluster materials, and particularly relates to a tetrahedral DNA-silver cluster probe for detecting Cu 2+ by using a double red fluorescence ratio and a preparation method thereof. Background The existence of excessive bivalent copper ions (Cu 2+) in the water body not only can destroy the biological enzyme activity and cell membrane structure of aquatic organisms and induce unbalance of the ecological system of the water body, but also can threaten human health through the food chain enrichment effect and cause gastrointestinal tract stimulation, liver injury and other series of diseases, and the content of the cupric ions is used as a core index for water quality evaluation, so that the accurate detection of the cupric ions can provide a key scientific basis for tracing the pollution of the water body, optimizing the treatment scheme and performing the landing of related water regulations, thereby having important practical significance and application value in developing the detection research of Cu 2+ in the water environment. The classical methods currently used for detecting trace/trace Cu 2+ mainly include atomic emission spectroscopy, atomic absorption spectroscopy, ultraviolet-visible spectrophotometry, inductively coupled plasma-mass spectrometry, and the like. These detection methods require complex pretreatment of the sample, are time-consuming and labor-consuming, are expensive to test, and are difficult to implement for rapid on-site detection. The preparation time of the probe for detecting Cu 2+ based on the DNA-metal nanocluster and the detection time of Cu 2+ reported at present are long (generally more than 30 min), so that the rapid field detection of trace Cu 2+ is difficult to realize. The ratio fluorescence analysis method has high accuracy, high sensitivity and good selectivity, and is widely used in the fields of environmental safety monitoring and the like in recent years. However, most of the reported ratio fluorescence methods require the preparation of two or more fluorescent probes, and the probe preparation method and steps are time-consuming and complicated, and the Cu 2+ detection is time-consuming and has high cost. Disclosure of Invention It is an object of the present invention to address at least the above problems and/or disadvantages and to provide at least the advantages described below. To achieve these objects and other advantages and in accordance with the purpose of the invention, as embodied and broadly described herein, there is provided a tetrahedral DNA-silver cluster probe for double red fluorescence ratio detection Cu 2+, which contains a tetrahedral DNA template capable of simultaneously emitting strong red fluorescence and weak near infrared red fluorescence, the tetrahedral DNA template including four sequences S1, S2, S3, S4; Wherein the S1 sequence is 5'-AAAAAAACCCCCCCCCCCCCGCTTCAGACTTCACAGCTGCATCGGATGGCAGCCCATATTACTCCT-3'; The S2 sequence is 5'-TTCCAGGACGCACAGAGCCATCCGATGCAGCATCTCAATGCAATGGC-3'; the S3 sequence is 5'-CTGTGCGTCCTGGAAACAATTCGCTCTCACTAAGGAGTAATATGGGC-3'; the S4 sequence is 5'-GTGAAGTCTGAAGCGAAGTGAGAGCGAATTGAGCCATTGCATTGAGA-3'. The preparation method of the tetrahedral DNA-silver cluster probe for detecting Cu 2+ by using the double red fluorescence ratio comprises the following steps: Adding a TDF-DNA cluster template solution and an AgNO 3 solution which are subjected to hybridization reaction into a Tris-HAc buffer solution, uniformly mixing, reacting for a period of time at room temperature in a dark place, adding a NaBH 4 solution which is prepared at present, incubating overnight at room temperature in a dark place, obtaining a TDF-DNA template AgNCs probe, namely a tetrahedral DNA-silver cluster probe of double red fluorescence ratio detection Cu 2+, and refrigerating and preserving. Preferably, the final molar ratio of TDF-DNA cluster template, agNO 3, and NaBH 4 is 1:48:12. Preferably, the Tris-HAc buffer solution, the TDF-DNA cluster template solution and the AgNO 3 solution are uniformly mixed and then subjected to light-shielding reaction at room temperature for 30-90 minutes. Preferably, the preparation method of the TDF-DNA cluster template solution comprises the steps of uniformly mixing DNA sequences S1, S2, S3 and S4 with equal concentration in equal proportion, heating in a constant-temperature water bath at 90-100 ℃ for 10-15 minutes, and cooling to obtain the TDF-DNA silver cluster template solution. Preferably, the concentration of the Tris-HAc buffer solution is 5-30.0 mmol/L, the pH is 6.5-8.0, the concentration of the TDF-DNA cluster template solution is 0.5-2.5 mu mol/L, the concentration of the AgNO 3 solution is 0.5-5.0 mmol/L, the concentration of the NaBH 4 solution is 0.5-5.0 mmol/L, and the volume ratio