Department of Natural Resources, Fernow Hall, Cornell University, Ithaca, NY, 14853, USA; Department of Microbiology, Wing Hall, Cornell University, Ithaca, NY, 14853, USA; Department of Pharmaceutical Sciences, PO Box 6000, Binghamton University, Binghamton, NY, 13902, USA.
Department of Natural Resources, Fernow Hall, Cornell University, Ithaca, NY, 14853, USA.
Talanta. 2019 Dec 1;205:120168. doi: 10.1016/j.talanta.2019.120168. Epub 2019 Jul 22.
Deficiencies in thiamine (vitamin B1) cause a host of neurological and reproductive impairments yielding morbidity and mortality across environmental and clinical realms. In a technique analogous to immunomagnetic separation, we introduce the use of thiamine periplasmic binding protein (TBP)-conjugated magnetic beads to isolate thiamine from complex matrices. TBP expressed in Escherichia coli is highly specific to thiamine and provides an alternative to antibodies for this non-immunogenic target. After incubation with the sample and removal of unbound matrix constituents, thiamine is simultaneously released and converted to its fluorescent oxidation product thiochrome by alkaline potassium ferricyanide. Subsequent measurement of fluorescence at thiochrome-specific wavelengths provides a second layer of specificity for the detection of thiamine. Thiamine could be quantified at concentrations as low as 5 nM ranging up to 240 nM. Within, we apply this technique to selectively capture and quantify thiamine in complex salmonid fish egg and tissue matrices. Our results showed no measurable non-specific binding to the beads by endogenous fluorophores in the fish egg matrix. Thiamine levels as low as 0.2 nmol/g of fish egg can be detected using this approach, which is sufficient to assess deficiencies causing morbidity and mortality in fish that occur at 1.0 nmol/g of egg. This practical method may find application in other resource limited settings for clinical, food, or dietary supplement analyses.
硫胺素（维生素 B1）缺乏会导致一系列神经和生殖系统损伤，在环境和临床领域引发发病率和死亡率。在类似于免疫磁分离的技术中，我们引入了使用硫胺素周质结合蛋白（TBP）缀合的磁珠从复杂基质中分离硫胺素。在大肠杆菌中表达的 TBP 对硫胺素具有高度特异性，为这个非免疫原性靶标提供了抗体以外的选择。与样品孵育并去除未结合的基质成分后，硫胺素同时被释放，并通过碱性钾铁氰化物转化为其荧光氧化产物噻嗪。随后测量噻嗪特有的波长的荧光提供了检测硫胺素的第二层特异性。硫胺素的浓度可以低至 5 nM 定量，最高可达 240 nM。在本文中，我们将该技术应用于选择性捕获和定量复杂的鲑鱼卵和组织基质中的硫胺素。我们的结果表明，鱼卵基质中的内源性荧光团对珠子没有可测量的非特异性结合。使用这种方法可以检测到低至 0.2 nmol/g 鱼卵的硫胺素水平，足以评估在 1.0 nmol/g 卵中导致鱼类发病率和死亡率的硫胺素缺乏症。这种实用的方法可能会在其他资源有限的临床、食品或膳食补充剂分析环境中找到应用。
