Rapid detection of methylglyoxal in blood samples

描述(申请人提供)：糖尿病是一种严重的危及生命的疾病，由葡萄糖的不适当吸收和代谢引起。血糖水平升高会导致有毒的糖降解产物在血液中积累，如甲基乙二醛(MG)。镁是一种活性分子，可以修饰和灭活血液蛋白，进而导致高血压、神经疾病和心脏病。因此，MG已被证明是许多糖尿病相关并发症的极好生物标志物。遗憾的是，目前所有直接测量血液中MG水平的方法都非常昂贵、耗时和费力。为了充分发挥MG的诊断潜力，需要建立血液等生物医学样品中MG的常规检测方法。我们提议的第一阶段研究将创造一种独特的测试来测量血液样本中的MG水平，如血浆和血清。这一独特的测试将使研究人员能够在蛋白质和血管系统受到损害之前，方便地研究反应性MG在血液中的积累情况。我们的新检测方法将使用重组细菌氧化还原酶(MG-R)，该酶能够以96孔板的形式将MG特异和快速地转化为可检测的比色信号。为了提高灵敏度，我们将把我们的酶检测反应与NADP+/NADPH可见的比色放大环路连接起来，将分析灵敏度提高100倍以上。将使用大鼠和小鼠的血浆和血清对该检测进行优化和验证，这些血清中添加了已知数量的MG。经过验证后，我们将推出一种商业化的快速检测试剂盒，直接检测临床前和其他生物医学样本中的MG。

DESCRIPTION (provided by applicant): Diabetes is a serious, life-threatening disease caused by improper absorption and metabolism of glucose. Increased glucose levels can lead to an accumulation of toxic sugar degradation products, such as methylglyoxal (MG), in the bloodstream. MG is a reactive molecule which can modify and inactivate blood proteins which in turn leads to hypertension, neuropathy, and heart disease. Consequently, MG has been shown to be an excellent biomarker for a number of diabetes-related complications. Unfortunately, all current methods to directly measure MG levels in blood are very expensive, time-consuming and laborious. To fully realize the diagnostic potential of MG, rapid analytical methods are needed for the routine determination of MG in biomedical samples such as blood. Our proposed Phase I research will create a unique test to measure MG levels in blood samples such as plasma and serum. This unique test will permit researchers to conveniently study the accumulation of reactive MG in blood before damage to proteins and vasculature has occurred. Our novel assay will use a recombinant bacterial oxidoreductase enzyme (MG-R) capable of specifically and rapidly converting MG into a detectable colorimetric signal in a 96 well plate assay format. To boost sensitivity, we will link our enzymatic detection reaction to an NADP+/NADPH visible colorimetric amplification loop to increase assay sensitivity more than 100-fold. The assay will be optimized and validated using rat and mouse plasma and serum spiked with known amounts of MG. After validation, we will commercially launch a rapid test kit to directly detect MG in preclinical and other biomedical samples.