Serum total protein refers to the sum of all proteins in the serum, and its content determination is relevant to human health monitoring and disease diagnosis. However, existing detection techniques present a number of limitations; for example, the Kjeldahl method suffers from the negative effects of interfering substances such as non-protein nitrogen (NPN). Although the electrophoresis titration (ET) method has solved interference problems to some extent, the current ET technique relies on optical detection methods, which increases the tediousness of the operation. This study addresses the challenge of accurate serum total protein detection by combining the traditional ET technique with capacitively coupled contactless conductivity detection (C4D). The research contributions of this work are multifold. First, it presents the first development of an ET-C4D detection system, which consists of six components: an ET power module, an ET chip, a C4D sensing module, a detection module, a data acquisition card, and software. The developed system can capture the conductivity of substances in the channel using the software developed by our laboratory during ET. The detection system can be used to quantify the total protein content in human serum without the addition of specific labeling reagents or using optical detection equipment, and its running time is approximately 300 s. Second, this research proposes the corresponding principle of the system. Under an electric field, ion migration results in different pH levels before and after the boundary, leading to a protein surface charge difference. The maintenance of the electrical neutrality of the substances in the detection channel is related to the protein surface charge; therefore, the ion concentration distribution of the substances in the detection channel changes as the protein surface charge varies. A plot of conductivity as a function of running time showed an "inverted clock shape", first falling and then rising. Owing to the addition of different types and concentrations of proteins, the microenvironment of the entire system changes, resulting in different changes in conductivity. Third, the performance of the detection system was tested using human serum albumin (HSA) standard protein, which was mixed with polyacrylamide gel (PAG) mother liquor, riboflavin, etc., and irradiated under ultraviolet light for 10 min to form a gel. The ET experiments were then carried out. The shape of the conductivity curve was consistent with the proposed principle, and the higher the HSA concentration, the lower the conductivity curve trough, followed by a lagged time of the trough. Quantitative analysis of the conductivity signals showed that the linear range was 0.25-3.00 g/L, with a linearity of up to 0.98. The limit of detection (LOD) was 0.01 g/L, the relative standard deviation (RSD) was 1.90%, and the relative error of the test values was <7.20%, indicating the good detection stability and sensitivity of the system. Clinical samples collected from healthy volunteers were used as target blood samples for serum total protein content measurement using our detection system. Blood samples from a volunteer were used to obtain a standard curve, and the serum samples of other four volunteers were selected for ET-C4D and biuret detection. The results showed that the relative errors between the two methods were within 4.43%, indicating the accuracy and reliability of the detection system. The advantages of the ET-C4D detection system proposed in this paper are as follows: (i) ET-C4D realizes the rapid detection of total serum protein content based on the ET technique; (ii) compared with the traditional protein ET technique, the ET-C4D method does not rely on specific labeling components or optical detection equipment, thereby reducing the complexity of the operation; and (iii) the output signal of ET-C4D can be used for quantitative analysis with excellent analytical performance and high accuracy. These merits highlight the potential of the developed system for clinical application and biochemical analysis.
血清总蛋白含量检测与人体健康监测和疾病诊断密切相关,本文基于电泳滴定(ET)技术结合电容耦合非接触电导检测(C4D)技术实时捕获ET过程中通道内物质的电导率变化,在不依赖指示剂和光学检测设备的情况下,定量检测人源血清总蛋白含量,ET-C4D检测总耗时约300 s。本文以人源血清白蛋白(HSA)标准品作为模式蛋白,与聚丙烯酰胺凝胶(PAG)母液、核黄素等混合后,在紫外灯光下照射10 min聚合形成凝胶,进行ET实验,耦合在ET管道外壁的非接触式电导检测电极捕捉通道内物质在电泳过程中的电导率信号,经检测模块和数据采集卡处理后送入计算机,由开发的分析测试软件根据电导率信号进行定量分析,结果表明:线性范围为0.25~3.00 g/L,线性拟合度(R2)在0.98以上,检出限(LOD)为0.01 g/L,相对标准偏差为1.90%,0.50 g/L的HSA标准品的测试值与实际值的相对误差低于7.20%,表明该检测系统具备较好的检测稳定性和灵敏度。最后,针对人源实际血样中的血清总蛋白含量进行检测,建立了相应的总蛋白标准曲线,然后选取4位志愿者的血清样本进行ET-C4D检测,并将ET-C4D检测结果与双缩脲法的检测结果进行了比对,两种方法检测结果的相对误差在4.43%以内,进一步证明了该检测系统的准确性与可用性,以及该检测系统在临床即时检测(POCT)领域的潜在应用价值和生化分析价值。
Keywords: capacitively coupled contactless conductivity detection; electrophoresis titration; serum total protein.