CN107014790B - Method for detecting bioavailability of copper complex by phytoplankton - Google Patents

Method for detecting bioavailability of copper complex by phytoplankton Download PDF

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CN107014790B
CN107014790B CN201710131006.6A CN201710131006A CN107014790B CN 107014790 B CN107014790 B CN 107014790B CN 201710131006 A CN201710131006 A CN 201710131006A CN 107014790 B CN107014790 B CN 107014790B
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mother liquor
copper
phytoplankton
cds
vitamin
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CN107014790A (en
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李顺兴
郑凤英
刘凤娇
王震华
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Minnan Normal University
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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"

Abstract

The invention discloses a method for detecting bioavailability of copper complexes by phytoplankton, which has the advantages of stable CDs fluorescence signal, small size, good water solubility, low biotoxicity and the like compared with other fluorescence quantum dots, and can effectively overcome the technical defects of spectrophotometry, atomic absorption, anodic stripping voltammetry, oscillography, ICP-MS and the like.

Description

Method for detecting bioavailability of copper complex by phytoplankton
Technical Field
The invention belongs to the technical field of trace metal organism utilization, and particularly relates to a method for detecting bioavailability of copper complexes by phytoplankton.
Background
Trace metals are vital to marine life, and their biogeochemical cycles and their transport in the ocean affect the material circulation and energy transport in the food chain and the marine ecosystem architecture. Large marine chemistry programs such as geoteraces have developed studies on the distribution and transformation of trace metals and their isotopes in the sea and how they affect the function of marine ecosystems, global carbon cycle and climate change. The metals in the seawater exist mainly in a strong complex form, only a small part of the metals exist in a free ion form, and the metals can be absorbed and utilized by phytoplankton only in the free ion form. Therefore, it is important to investigate the bioavailability of metal complexes by phytoplankton.
Copper ions are typical representatives of the trace metals, and currently, commonly used copper ion detection methods include a 2, 9-dimethyl-1, 10-phenanthroline spectrophotometry method, a flame atomic absorption method, an APDC-MIBK extraction flame atomic absorption method, a graphite furnace atomic absorption method, an anodic stripping voltammetry, an oscillography polarography, an ICP-MS and the like. The detection technologies have high precision, but have the defects of expensive experimental equipment, complex experimental process, large detection workload, many limited factors and the like.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for detecting the bioavailability of copper complexes by phytoplankton.
The technical scheme of the invention is as follows:
a method for detecting the bioavailability of copper complexes by phytoplankton, comprising the following steps:
(1) culturing typical phytoplankton offshore in an f/2 culture medium, wherein the f/2 culture medium contains CDs for marking typical phytoplankton offshore, and obtaining a CDs marked culture solution, wherein the specific culture conditions are as follows: the f/2 culture medium contains 14-16 mg/L CDs, and the CDs are placed in a medium with the illumination intensity of 130-150 mu mol phosns (m)2s)-1Culturing in a biochemical incubator at the temperature of 18-20 ℃ and the light-dark ratio of 12-15 h: 8-10 h, stirring the seaweed suspension at 80-120 rpm to simulate the flow of seawater, and simultaneously reducing the adsorption of CDs on the wall of the container;
(2) taking a copper chloride dihydrate solution as a copper ion standard use solution, and obtaining a linear working curve according to the change of the fluorescence intensity of the CDs in the copper ion standard use solutions with different concentrations: y 2294.9x +876.32, linear range: 20-1500ng/L, wherein x represents the concentration of copper ions, and y represents the change of fluorescence intensity;
(3) dividing CDs marked culture solution into two groups, wherein each group is divided into a plurality of same parts, wherein: one part was used as control, and one part was added with Cu2+The rest is added with different copper complexes respectively; placing the first group in a dark place, placing the second group under a xenon lamp for continuous illumination for 2h, taking out the second group at fixed time intervals, and observing the change of the fluorescence intensity of the second group relative to different copper complexes of the first group;
(4) and (3) substituting the fluorescence intensity changes of different copper complexes obtained in the step (3) into the linear working curve obtained in the step (2) to obtain corresponding copper ion concentrations, so that the utilization conditions of phytoplankton on different copper complexes can be obtained.
In a preferred embodiment of the invention, the off-shore phytoplankton representatives include the Haematococcus williamsii CCMA-102 and the Chlorella vulgaris CCMA-410.
In a preferred embodiment of the invention, the f/2 culture medium is composed of sodium nitrate mother liquor with the concentration of 75.0g/L, sodium phosphate mother liquor with the concentration of 5.0g/L, sodium silicate mother liquor with the concentration of 30.0g/L, trace element mother liquor, vitamin mother liquor and seawater, and the volume ratio of the sodium nitrate mother liquor, the sodium phosphate mother liquor, the sodium silicate mother liquor, the trace metal mother liquor, the vitamin mother liquor and the seawater is 2: 1: 2000, wherein the trace element mother liquor contains 3.15g/L of ferric chloride, 9.8g/L of copper sulfate, 6.3g/L of sodium molybdate, 22.0g/L of zinc sulfate, 10.0g/L of cobalt chloride and 180.0g/L of manganese chloride, and the vitamin mother liquor contains 121.0g/L of vitamin B, 0.1g/L of biotin and 0.2g/L of thiamine hydrochloride.
In a preferred embodiment of the present invention, the fixed time interval in the step (3) is 30 min.
In a preferred embodiment of the present invention, the copper complex comprises Cu-Por, Cu-H2Lp and EDTA-Cu.
The invention has the beneficial effects that: the method not only can overcome the defects of technologies such as spectrophotometry, atomic absorption method, anodic stripping voltammetry, oscillography polarography, ICP-MS and the like, but also has the advantages of wide detection linear range, high precision, low detection limit and the like.
Drawings
FIG. 1 is a graph showing the selectivity of fluorescence quenching of metal ions by carbon quantum dots in example 1 of the present invention.
Fig. 2 is a reaction mechanism diagram of carbon quantum dots and copper ions in example 1 of the present invention.
FIG. 3 is a graph showing the stability of the fluorescence signal of the carbon quantum dot to the copper complex in example 1 of the present invention.
FIG. 4 is a graph showing the intracellular copper ion concentration in the dark treatment group in example 1 of the present invention.
FIG. 5 is a graph showing the concentration of copper ions in the cells of the phototransistors in example 1.
Detailed Description
The technical solution of the present invention will be further illustrated and described below with reference to the accompanying drawings by means of specific embodiments.
Example 1:
1. selectivity of carbon quantum dots (CDs) for metal ion fluorescence quenching
Preparing a series of 10 mu mol/L-1Ionic solution: cu2+、Fe3+、Hg2+、Ag+、Ni2+、Zn2+、Co2+、Cd2+、pb2+、K+、Na+、Ca2+、Mg2+. And then adding CDs into each ionic solution system respectively to examine the fluorescence signal intensity in the system, as shown in figure 1, wherein the reaction mechanism of carbon quantum dots and copper ions is shown in figure 2.
2. And (3) analyzing the stability of the carbon quantum dot to the fluorescence signal of the copper complex:
respectively prepare 1 mmol. L-1Porphyrin, phthalic acid, ethylenediamine tetraacetic acid, then Cu2+Complexing 2d, Cu-Por and Cu-H at a ratio of 1: 1 under 40% ambient light2Lp, EDTA-Cu and Cu2+5ml of each sample was put into a 10ml colorimetric tube, and the fluorescence signal was measured every 5min after the addition of CDs in this order for 2 hours, and the results are shown in FIG. 3.
3. The specific detection method of this embodiment is as follows:
(1) culturing typical inshore phytoplankton (Alaska sea algae CCMA-102 and Chlorella CCMA-410) in f/2 culture medium (comprising 75.0g/L sodium nitrate mother liquor, 5.0g/L sodium phosphate mother liquor, 30.0g/L sodium silicate mother liquor, microelement mother liquor, vitamin mother liquor and seawater, wherein the volume ratio of the sodium nitrate mother liquor, the sodium phosphate mother liquor, the sodium silicate mother liquor, the microelement mother liquor, the vitamin mother liquor and the seawater is 2: 1: 2000, wherein the microelement mother liquor contains 3.15g/L ferric chloride, 9.8g/L copper sulfate, 6.3g/L sodium molybdate, 22.0g/L zinc sulfate, 10.0g/L cobalt chloride, 180.0g/L manganese chloride, the vitamin mother liquor contains vitamin B121.0g/L, 0.1g/L biotin and 0.2g/L thiamine hydrochloride), obtaining CDs marked culture solution; the specific culture conditions were as follows: the f/2 medium contained 15mg/L CDs and was placed in a medium at a light intensity of 140. mu. mol phos-ins (m)2s)-1The light-dark ratio is 14 h: culturing in a biochemical incubator at 19 ℃ for 10h, stirring the seaweed suspension at 100rpm to simulate the flow of seawater, and simultaneously reducing the adsorption of CDs on the wall of the container;
(2) taking a copper chloride dihydrate solution as a copper ion standard use solution, and obtaining a linear working curve according to the change of the fluorescence intensity of the CDs in the copper ion standard use solutions with different concentrations: y 2294.9x +876.32, linear range: 20-1500ng/L, wherein x represents the concentration of copper ions, and y represents the change of fluorescence intensity;
(3) dividing CDs marked culture solution into two groups, wherein each group is divided into 5 same parts, wherein: one part was used as control, and one part was added with Cu2+The rest is added with copper complexes Cu-Por and Cu-H respectively2Lp and EDTA-Cu, the above copper complex and Cu2+The preparation method comprises the following steps: 0.0703g protoporphyrin (Pro) and 0.1661 phthalic acid (H) were weighed respectively2LP) 0.3722g disodium ethylene diamine tetraacetate (EDTA-Na)2) 0.0341g of copper chloride dihydrate was dissolved in 50mL of water, mixed well and stored in a reagent bottle. Then with Cu2+Complexing for 2d under 40% ambient light according to the proportion of 1: 1; placing the first group in the dark, placing the second group under xenon lamp for continuous illumination for 2h, taking out every 30min, and observing the fluorescence intensity change of the second group relative to different copper complexes of the first group, as shown in FIGS. 4 and 5;
(4) and (3) substituting the fluorescence intensity changes of different copper complexes obtained in the step (3) into the linear working curve obtained in the step (2) to obtain corresponding copper ion concentrations, so that the utilization conditions of phytoplankton on different copper complexes can be obtained.
In order to verify the applicability of the carbon quantum dots in the method of the present invention to the detection of copper ion concentration, the present example additionally performs the analysis and determination of the actual water sample according to the conventional inductively coupled plasma mass spectrometry (ICP-MS) and the method, and the results are shown in table 1:
TABLE 1 analysis of copper ion concentration in actual water samples
Figure BDA0001239864340000041
In conclusion, the method of the embodiment has the advantages of stable fluorescence signal, small size, good water solubility, low biological toxicity and the like compared with other fluorescence quantum dots, and can effectively overcome the technical defects of spectrophotometry, atomic absorption method, anodic stripping voltammetry, oscillography, ICP-MS and the like. The copper chloride dihydrate solution is used as the standard copper ion use solution, and a linear working curve can be obtained in different concentration ranges: y 2294.9x +876.32, linear range: 20-1500 ng/L. The detection result of the method and inductively coupled plasma mass spectrometry (ICP-MS) has no significant difference, and the detection limit is as low as 19.5 ng/L.
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims.

Claims (4)

1. A method for detecting the bioavailability of a copper complex by phytoplankton, which is characterized by comprising the following steps: the method comprises the following steps:
(1) culturing typical phytoplankton offshore in an f/2 culture medium, wherein the f/2 culture medium contains CDs for marking typical phytoplankton offshore, and obtaining a CDs marked culture solution, wherein the specific culture conditions are as follows: the f/2 culture medium contains 14-16 mg/L CDs and is placed under illumination with the intensity of 130-150μmol photons （m2s）-1Culturing in a biochemical incubator at the temperature of 18-20 ℃ and the light-dark ratio of 12-15 h: 8-10 h, stirring the seaweed suspension at 80-120 rpm to simulate the flow of seawater, and simultaneously reducing the adsorption of CDs on the wall of the container;
(2) taking a copper chloride dihydrate solution as a copper ion standard use solution, and obtaining a linear working curve according to the change of the fluorescence intensity of the CDs in the copper ion standard use solutions with different concentrations: y =2294.9x +876.32, linear range: 20-1500ng/L, wherein x represents the concentration of copper ions, and y represents the change of fluorescence intensity;
(3) dividing CDs marked culture solution into two groups, wherein each group is divided into a plurality of same parts, wherein: one part was used as control, and one part was added with Cu2+The rest is added with different copper complexes respectively; placing the first group in dark place, placing the second group under xenon lamp for continuous illumination for 2h, taking out at fixed time interval, and observing the difference of the second group relative to the first groupChange in fluorescence intensity of the copper complex;
(4) substituting the fluorescence intensity changes of different copper complexes obtained in the step (3) into the linear working curve obtained in the step (2) to obtain corresponding copper ion concentrations, so as to obtain the utilization conditions of phytoplankton on different copper complexes; the copper complex comprises Cu-Por and Cu-H2Lp and EDTA-Cu.
2. The method of claim 1, wherein: the typical phytoplankton offshore includes Haptophyceae Wei-Shi CCMA-102 and Chlorella vulgaris CCMA-410.
3. The method of claim 1, wherein: the f/2 culture medium is composed of 75.0g/L sodium nitrate mother liquor, 5.0g/L sodium phosphate mother liquor, 30.0g/L sodium silicate mother liquor, trace element mother liquor, vitamin mother liquor and seawater, and the volume ratio of the sodium nitrate mother liquor to the sodium phosphate mother liquor to the sodium silicate mother liquor to the trace element mother liquor to the vitamin mother liquor to the seawater is 2: 2: 2: 2: 1: 2000, wherein the microelement mother liquor contains 3.15g/L ferric chloride, 9.8g/L copper sulfate, 6.3g/L sodium molybdate, 22.0g/L zinc sulfate, 10.0g/L cobalt chloride and 180.0g/L manganese chloride, and the vitamin mother liquor contains 121.0g/L vitamin B, 0.1g/L biotin and 0.2g/L thiamine hydrochloride.
4. The method of claim 1, wherein: the fixed time interval in the step (3) is 30 min.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4956188A (en) * 1988-10-20 1990-09-11 Zinpro Corporation Copper complexes with alpha hydroxy organic acids and their use as nutritional supplements
CN102779222A (en) * 2011-05-13 2012-11-14 苏州科技学院 Method for detecting bioavailable particulate phosphori in shallow lake

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4956188A (en) * 1988-10-20 1990-09-11 Zinpro Corporation Copper complexes with alpha hydroxy organic acids and their use as nutritional supplements
CN102779222A (en) * 2011-05-13 2012-11-14 苏州科技学院 Method for detecting bioavailable particulate phosphori in shallow lake

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
不同形态Fe 对铜绿微囊藻生长及其生物可利用性的影响;王海明 等;《农业环境科学学报》;20070531;第26卷(第3期);第1030-1031页 *
以碳点为探针荧光猝灭法测定Cu2+;毛小娇 等;《西南大学学报(自然科学版)》;20100930;第32卷(第9期);第40-42页 *

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