Determination of human serum total protein via electrophoresis titration and capacitively coupled contactless conductivity detection

doi:10.3724/SP.J.1123.2023.04015

Abstract

Abstract:

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.

Key words: electrophoresis titration, capacitively coupled contactless conductivity detection, serum total protein

CLC Number:

O658

ZHANG Ruihua, GUO Zehua, ZHANG Qiang, ZHA Genhan, CAO Chengxi, FAN Liuyin, LIU Weiwen. Determination of human serum total protein via electrophoresis titration and capacitively coupled contactless conductivity detection[J]. Chinese Journal of Chromatography, 2023, 41(8): 707-713.

Figures/Tables 6

Fig. 1 Schematic diagram of electrophoresis titration- capacitively coupled contactless conductivity detection (ET-C4D) system

Fig. 2 Schematic diagrams of (a) ET-C4D principle and (b) output signal

Fig. 3 Results of performance testing of the detection system using human serum albumin (HSA) a. ET-C4D result curve of HSA in the content range of 0-5.00 g/L; b. quantitative curve of HSA content-conductivity signal; c. The descending edge in Fig. 3a is derived to locate the trough position; d. quantitative curve of HSA content-time signal. Experimental conditions: cathode liquid, 200 mmol/L NaOH; anode liquid, 20 mmol/L KCl; filled gel, polyacrylamide gel (PAG, 15% total acrylamide and 4% cross-linker); background buffer liquid, 2 mmol/L KCl; voltage, 150 V.

Table 1 Linear ranges, stabilities and LODs of ET-C4D conductivity quantification and time quantification for HSA

Method	Linear range/ (g/L)	RSD/% (n=3)	LOD/ (g/L)	Relative error^*/%
Conductivity	0.25-3.00	1.90	0.01	5.10
quantification
Time quantification	0.25-3.00	1.30	0.01	7.20

Fig. 4 ET-C4D results and standard curves of serum total protein a. ET-C4D result curve of serum total protein; b. quantitative curve of serum total protein content-conductivity signal; c. The descending edge in Fig. 4a is derived to locate the trough position; d. quantitative curve of serum total protein content-time signal. CTP: mass concentration of serum total protein. Experimental conditions were the same as that in Fig. 3.

Table 2 Detection results of clinical serum samples

Serum sample	Biuret reagent standard method/ (g/L)	ET-C⁴D method/ (g/L)	RSD/% (n=3)	Relative error/%
1	71	67.86	2.27	4.43
2	73	71.89	2.15	1.52
3	75	73.23	3.23	2.36
4	77	73.90	2.09	4.02

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