微流控与重组酶聚合酶扩增用于原肌球蛋白悬浮颗粒物的微量样品采样与分析方法研究

Aerosolization of allergen proteins during the aquatic product processing has been identified as an essential cause for occupational allergy and respiratory disease among workers involved in fishery processing activities. Thus, the pollution of airborne allergen aerosol affects workers’ health in a fishery operation setting and potentially hinders the development of aquatic food industry. As a way to deal with it, the advancement in monitoring technology can greatly improve workplace health and safety service. However, it is still quite difficult to rapidly collect and quantify seafood allergen aerosol owing to its low concentration in air. So far, conventional methods for air sampling and analysis, which have been widely used in the analysis of microorganism densities in aerosols, are not best suited to the analysis of seafood allergen aerosol.. We herein express our serious concern about the personal health risk caused by aerosolized crustaceans allergen such as tropomyosin. The specific aim of this proposal is to establish a novel approach that can achieve the fast collection and accurate quantification of aerosolized tropomyosin particulate which is generated during the processing of shrimps such as Penaeus vannamei Boone and Procambarus clarkii. To this end, the state-of-the-art microfluidics and Recombinase Polymerase Amplification (termed as RPA), which is a nucleic acid isothermal amplification technology, will be employed. In this proposal, the ultimate goal is to develop microfluidics chip assays with 1 μg/m3 limit of detection and 2-hour operation time. The success of our proposed research will make substantial contribution to evaluating air quality and monitoring threat level in a fishery processing setting, and thereby prevent occupational disease and maintain sustainable development of aquatic food industry.

鱼虾蟹贝类水产品加工过程中会产生大量经由空气传播的过敏原悬浮颗粒物。这些过敏原（如原肌球蛋白）颗粒物经呼吸道进入人体后能够引起过敏性哮喘、荨麻疹等症状，严重危害水产加工及水产科研人员的健康。此类有害产物在空气中含量较低且无法像微生物样品那样被培养扩增，因此不适宜直接套用微生物悬浮颗粒物的分析方法，即空气采样器捕获微量样品—微生物培养扩增—微生物分析。.本课题以如何结合运用生物化学分析原理，建立针对性的新方法，实现虾类水产品加工中产生的原肌球蛋白悬浮颗粒物的现场快速分析这一科学问题为主线，提出微流控与重组酶等温扩增相结合的采样与分析方法。该方法具有试剂消耗量少、成本低、设备便携等优势，预计分析时间2小时，检测限达1微克每立方米空气，适用于现场快速分析和工作场所空气质量的评价，进而可监控和预防水产食品加工过程产生的有害产物对人员造成的健康威胁，保证充足的劳动力以维持水产食品行业的可持续发展。

生物气溶胶是指含有微生物或过敏原蛋白生物大分子等生命活性物质的气溶胶。包括细菌、真菌、病毒、尘螨以及致敏花粉、霉菌孢子、蕨类孢子、寄生虫卵和动植物碎裂分解体等。生物气溶胶可以借助空气介质进行扩散和传输，并在空气中存在较长时间，引起多种人类疾病，主要有：肺炎、肺结核、鹦鹉热、流感、肝炎、狂犬病、哮喘、荨麻疹、过敏性鼻炎、皮炎、结膜炎等。生物气溶胶的采样与分析鉴定技术是生物气溶胶暴露评价的基础。本课题研究思路为运用微流控技术、质谱分析和等温核酸扩增方法原理，建立针对性的气溶胶采集和分析方法，实现对多种生物气溶胶等进行采集和分析，提出了微流控与重组酶等温扩增、LC-MS/MALDI-TOF-MS质谱分析多种手段相结合的先进采样与分析方法。.本课题设计并制作了具有鱼骨结构的螺旋通道微流控芯片，集成气泵、流量调节器等元件制作了一种新型气溶胶采样器；应用该设备可高效快速采集副溶血性弧菌、单增李斯特菌、沙门氏菌微生物气溶胶以及南美白对虾原肌球蛋白气溶胶；使用多重荧光定量、重组酶聚合酶扩增、质谱分析等对采集到的样品进行表征和分析；开发了一种基于重组酶聚合酶扩增的霍乱弧菌检测方法；以微石英天平器件为传感结构，制备了基于聚多巴胺功能化的介孔二氧化硅纳米颗粒的气体传感器。本项目通过上述一系列研究，实现了对多种生物气溶胶样品高效采样与精确分析。

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