Dhyani Rajat, Shankar Krishna, Bhatt Ankita, Jain Shubham, Hussain Ajmal, Navani Naveen Kumar
Chemical Biology Laboratory, Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247667, Uttarakhand, India.
Anal Chem. 2021 Mar 16;93(10):4521-4527. doi: 10.1021/acs.analchem.0c04914. Epub 2021 Mar 3.
Clinicians require simple quantitative tools for the detection of homogentisic acid in alkaptonuria patients, a rare inherited disorder of amino acid metabolism. In this study, we report a whole-cell biosensor for homogentisic acid to detect alkaptonuria disease through the expression of green fluorescence protein. The assay system utilizes a promoter sequence (hmgA) isolated from the genome. To increase the sensitivity, the sensor module harboring was further transformed into various transposon mutants debilitated in steps involved in the metabolism of phenylalanine and tyrosine via homogentisic acid as a central intermediate. The proposed biosensor was further checked for analytical features such as sensitivity, selectivity, linearity, and precision for the quantification of homogentisic acid in spiked urine samples. The limit of detection for the developed biosensor was calculated to be 3.9 μM, which is comparable to that of the various analytical techniques currently in use. The sensor construct showed no interference from all of the amino acids and its homolog molecules. The accuracy and precision of the proposed biosensor were validated using high-performance liquid chromatography (HPLC) with satisfactory results.
临床医生需要简单的定量工具来检测尿黑酸尿症患者体内的尿黑酸，这是一种罕见的氨基酸代谢遗传性疾病。在本研究中，我们报告了一种用于尿黑酸的全细胞生物传感器，通过绿色荧光蛋白的表达来检测尿黑酸尿症。该检测系统利用从基因组中分离出的启动子序列（hmgA）。为了提高灵敏度，将携带该序列的传感器模块进一步转化为各种转座子突变体，这些突变体在以尿黑酸为中心中间体的苯丙氨酸和酪氨酸代谢过程中所涉及的步骤中功能减弱。对所提出的生物传感器的分析特性，如灵敏度、选择性、线性和精密度进行了进一步检测，以对加标尿样中的尿黑酸进行定量。所开发生物传感器的检测限经计算为3.9 μM，与目前使用的各种分析技术相当。该传感器构建体不受所有氨基酸及其同源分子的干扰。使用高效液相色谱（HPLC）对所提出生物传感器的准确性和精密度进行了验证，结果令人满意。
