为研究添食金属或金属氧化物纳米颗粒后家蚕体内金属元素含量的变化规律,建立了微波消解结合电感耦合等离子体质谱(ICP-MS)测定家蚕血液、中肠和丝腺中Cu和Ti元素含量的方法.实验中分别给5龄3天家蚕喂食浓度为500.00 mg/L纳米Cu和纳米TiO2悬浮液;72 h后,剪破家蚕腹足收集家蚕血淋巴,并解剖收集中肠和丝腺.实验发现,Cu和Ti元素的标准曲线呈良好的线性关系,相关系数分别为0.999 95和0.999 89,两种元素的加标回收率均在96.6%~105.4%之间,相对标准偏差RSD小于3.7%.结果表明,对于纳米TiO2实验组,家蚕血液和丝腺中的Ti元素含量与空白组相比变化不大,而中肠的Ti元素含量明显增加为(181.46±9.58) μg/g,约为空白组含量(63.39±6.44) μg/g的3倍(p<0.05);在纳米Cu组中,家蚕血液中的Cu元素含量有轻微的变化,而中肠和丝腺中的含量增加较多,分别为(82.73±1.72) μg/g和(3.88±0.17) μg/g,约为空白组含量(11.68±0.46) μg/g和(1.77±0.26) μg/g 的7和2倍(p<0.05).该方法可准确地检测喂食纳米颗粒后,家蚕组织中的金属元素含量及其变化,可为进一步研究家蚕的纳米生物效应提供有力支持.%With the popularity of nanomaterials, an increasing number of nanoproducts are used in our daily life, and as a result, the opportunities of exposure to nanomaterials for humans are greatly increased.Given such wide exposure of nanoparticles (NPs), the biosecurity of NPs had attracted people's attention.During the toxicological study of NPs, it is crucial important to know their distribution in animal tissues.In order to study the variation of the metallic element contents in the Bombyx mori after feeding the metal/metal oxide NPs, a method for determination of copper and titanium in hemolymph, silk gland and midgut of the fifth-instar silkworm larvae was developed by microwave digestion-inductively coupled plasma-mass spectrometry (MD-ICP-MS).Compared with other measurement techniques, ICP-MS has many advantages, such as less sample consumption, low detection limit, high sensitivity and wide linear range.In the experiment, the 3-day-old larvae of fifth-instar Bombyx mori were fed with mulberry leaves sprayed with the suspension of Cu NPs and TiO2 NPs (at concentration of 500.00 mg/L), respectively.Seventy-two hours after treatment with the nanoparticles, the samples of the larval hemolymph, silk gland and midgut were obtained by cutting through one of the prolegs and dissecting the larvae.The samples were digested by microwave digestion with the model of Ramp to Temperature: nitric acid dosage 5 mL, maximum temperature 180 ℃, hold time 25 min, and the determination was detected by the ICP-MS under the optimum condition.In this study, the correlation coefficients for Cu and Ti are 0.999 95 and 0.999 89, respectively.The recoveries of the two elements are between 96.6% and 105.4%, and both the relative standard deviations(RSDs) are less than 3.7%.The results indicated that the contents of Ti in the hemolymph and silk gland only have a little change compared with the control group, while the content of Ti in the midgut (181.46±9.58) μg/g is about three times higher than that of the control group (63.39±6.44) μg/g for the group treated with TiO2 NPs.In the group treated with Cu NPs, the Cu content in the hemolymph has no obvious change, while the contents in the midgut (82.73±1.72) μg/g and silk gland (3.88±0.17) μg/g are about two and seven times higher than that of the control group (11.68±0.46) μg/g, (1.77±0.26) μg/g, respectively.In this paper, the contents of Cu and Ti in the hemolymph, silk gland and midgut of larvae of fifth-instar silkworm had been determined accurately by MD-ICP-MS.This may provide a useful method to study the variation of the metallic element contents in the tissues of Bombyx mori after fed with the metal/metal oxide nanoparticles, which will support the further study of biological effects of nanoparticles on silkworm.The distribution tests of NPs based on Bombyx mori can solve some problems, such as high costs and highly ethical and moral controversy.Since the metabolic mechanism of Bombyx mori was similar to mammal, the toxicity study of NPs on Bombyx mori might provide data support for the biological safety evaluation of NPs and method for reference to toxicity studies of other nanomaterials.
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