Rapid and Simple Analysis of the Human Pepsin Secondary Structure Using a Portable Raman Spectrometer

Abstract

Human pepsin is a digestive protease that plays an important role in the human digestive system. The secondary structure of human pepsin determines its bioactivity. Therefore, an in-depth understanding of human pepsin secondary structure changes is particularly important for the further improvement of the efficiency of human pepsin biological function. However, the complexity and diversity of the human pepsin secondary structure make its analysis difficult. Herein, a convenient method has been developed to quickly detect the secondary structure of human pepsin using a portable Raman spectrometer. According to the change of surface-enhanced Raman spectroscopy (SERS) signal intensity and activity of human pepsin at different pH values, we analyze the change of the human pepsin secondary structure. The results show that the content of the β-sheet gradually increased with the increase in the pH in the active range, which is in good agreement with circular dichroism (CD) measurements. The change of the secondary structure improves the sensitivity of human pepsin SERS detection. Meanwhile, human pepsin is a commonly used disease marker for the noninvasive diagnosis of gastroesophageal reflux disease (GERD); the detection limit of human pepsin we obtained is 2 μg/mL by the abovementioned method. The real clinical detection scenario is also simulated by spiking pepsin solution in saliva, and the standard recovery rate is 80.7–92.3%. These results show the great prospect of our method in studying the protein secondary structure and furthermore promote the application of SERS in clinical diagnosis.

Portable and Rapid Fluorescence Turn-On Detection of Total Pepsin in Saliva Based on Strong Electrostatic Interactions

“…To further investigate the accuracy and feasibility of the SG–pepsin system for the detection of pepsin in real saliva samples from different volunteers, the saliva samples were determined and compared with the assay kit . As shown in Table , pepsin was not detected in the saliva samples because there is almost no pepsin in the saliva of normal people .…”

“…However, when the temperature reaches 90 °C, the intensity of the band gradually decreases, and the band is red-shifted. For the secondary structure of the protein, the polypeptide bonds are arranged in one direction, and the different arrangement determines the cleavage of the peptide bond energy level transition . The secondary structure of pepsin treated at a high temperature is destroyed, suggesting that the fluorescence enhancement of SG is correlated with the primary structure of pepsin rather than bioactivity.…”

“…Because the saliva environment is very complex, it contains many substances, such as some proteins and inorganic ions. , Second, we investigated the effects of several common interfering ions and proteins on the determination of pepsin (Figure b). The results showed that the method had no significant effect on the analyzed fluorescence signal intensity, indicating that the method has good anti-interference ability.…”

“…First, 0.1 M sodium citrate was used to collect saliva to reduce matrix interference as much as possible . Second, the collected saliva samples were diluted 100-fold with water before use.…”

“…Therefore, to detect pepsin, it is usually necessary to reduce the pH of the acid solution and reactivate it, but this increases the running time and leads to inaccurate measurements . Li’s group recently developed a method to detect the secondary structure of human pepsin at different pH values using a portable Raman spectrometer, but the materials synthesis required and the sensitivity of the method could be improved. It remains a challenge to accurately detect pepsin by using rapid and sensitive assays at a neutral pH.…”