Currently, non-invasive detection techniques based on saliva samples are basically targeted at DM patients. With the change of living habits, the proportion of people facing hidden dangers of DM will increase. Therefore, the importance and necessity of routine blood glucose monitoring for healthy people is self-evident. In this study we constructed a saliva-based high-sensitivity glucose detection system, and this method was used to compare the SFR and saliva glucose concentration of six different saliva collection methods for the first time.
The research of non-invasive blood glucose detection methods is one of the hot topics of great concern in the international medical and sensor fields. It will make a qualitative leap in the detection of DM and has important practical significance for the treatment of DM. Therefore, there are many people engaged in this research at home and abroad, but due to various reasons, still various defects in various detection methods, which need to be improved and developed. Besides, commercial instruments have not yet been launched. E. g. in the optical rotation method, the components such as lithium tantalum crystals used therein are greatly affected by temperature, so the environmental temperature is relatively high, which brings inconvenience to household use [23]. Ryman spectroscopy analysis method: the structure is relatively complex, and it is not easy to be accepted by patients [24]. Ion chromatography analysis method: its sensitivity, stability and accuracy are poor, and this method is susceptible to interference from other substances in saliva, which affects the detection results [25]. The ultra-micro ultraviolet spectrophotometer method in this study is a highly repeatable method with high sensitivity and high detection accuracy.
In this study, a new sensitive method was developed to realize daily non-invasive blood glucose monitoring of healthy people. An automated analysis method using TBHBA as the chromogen was developed for the detection of saliva glucose. This method not only increased the amount of sample, but also used a highly sensitive chromogen. This study found that the product has a maximum absorption at a wavelength of 520nm, and the optimal pH is 6.5, the optimal NaCl concentration is 5mg/dl. The environmental PH value will change or affect the dissociation state of the enzyme and the substrate to increase or decrease the enzyme activity. Therefore, the maximum activity of the enzyme required the corresponding optimum PH value. The main catalytic enzymes in the test solution are GOD and POD. The activity of the former was in the range of pH 4.0 to 7.0, and the activity of the latter was in the range of pH 5.0 to 9.0. The optimal PH value of the reaction test solution measured in the experiment was 6.5, which was within the range of GOD activity and POD activity. Therefore, it was theoretically speculated that the PH value of 6.5 was the comprehensive optimal PH value of the same system where the two enzymes were located. Besides, it is found that its linear range, accuracy, and precision can meet the requirements of detection, and more importantly, it makes the measurement process more standardized, reduces errors, and is simple and easy to implement.
Saliva, like plasma or serum, is a unique and complex body fluid. Sufficient saliva secretion is essential for maintaining oral health. The advantages of saliva assessment include the cost-effectiveness of non-invasive collection and screening of large populations [26]. Saliva is currently considered to be an excellent diagnostic biomarker for human characteristics. Although in this study, no research on the correlation between salivary glucose concentration and blood glucose concentration was done, a large number of scholars’ studies have demonstrated the potential of salivary glucose in blood glucose detection. For example, the saliva microbiome is considered to be as accurate as fingerprints in terms of biometrics when used to screen for normality of characteristics [27]. In some laboratory tests, saliva may be used as a substitute for blood, for example, to detect viral infections, DM[28, 29]. Some scholars have different conclusions, which are mainly caused by different saliva collection methods and time. The 6 saliva collection methods and 4 saliva collection times studied in this study can make up for the shortcomings of existing research and provide the next step for the concentration of glucose in saliva. The determination of saliva has laid a good foundation and pointed out the direction for finding the most suitable method and time to collect saliva glucose. Therefore, it can better improve the accuracy of saliva glucose detection and provide a theoretical basis for non-invasive blood glucose detection in saliva.
The determination of saliva glucose concentration is a prerequisite for the development of saliva as a diagnostic and prognostic tool for DM biomarker discovery. In this case, it is important to keep the technical variability caused by sample collection and processing to a minimum so that inter-subject variability in health and disease states can be assessed reproducibly [30]. Single or mixed saliva can be collected. It should be noted that many unknown factors and unstable elements will affect the properties of mixed saliva. Saliva collected directly from a single gland is stable and not affected by oral conditions. Thus, it can accurately reflect blood glucose status. Saliva from the parotid gland is easily collected under unstimulated and stimulated conditions. Dhanya et al. [31] reported that when saliva is collected under unstimulated conditions, the concentration of glucose in saliva is higher than under stimulated conditions, which is consistent with the conclusions obtained in this study. Other studies have found that there is no significant difference in the concentration of glucose in saliva collected under unstimulated and stimulated conditions [32]. Because the participants may not be easy to accept acid stimulation, and the water concentration in stimulated saliva is higher. Besides, unstimulated saliva may be more representative of a normal physiological state.
Takeda et al. [33] measured the saliva chemical concentration of healthy subjects under different conditions and found that compared with stimulated saliva, almost all metabolites in unstimulated saliva were higher. Jha et al. [34] also found that compared with stimulated saliva, the average saliva glucose level in unstimulated saliva of control and non-control DM patients was higher. Saliva collected directly from a single gland is stable and not affected by oral conditions. Therefore, it may accurately reflect blood glucose status. In this study, six saliva collection methods were used include whole saliva, parotid saliva, and sublingual/submandibular saliva in stimulated and unstimulated states. It was found that the glucose concentration of unstimulated saliva was higher than that of stimulated saliva. Secondly, the UPS glucose was higher than the USS glucose and higher than the UWS glucose.
The relative contribution of different glands to the whole saliva varies with the collection method, the degree of stimulation, age and even the time of day [35]. The variable nature of saliva secretion suggests that different methods may have to be used when studying its components or their possible role as indicators of specific physiological conditions. There is a large amount of literature on the diagnostic possibilities of saliva, but there is still no standardized technique for collecting saliva samples. In different studies, different sampling methods are often used, and many studies do not or rarely describe patient preparation or sampling procedures [36]. In addition, without proper clinical examination, the characteristics of participants are usually insufficient. Most saliva glucose research papers focus on studying the whole saliva [37] because it can be easily obtained by spitting it into a test tube or letting it drip from the mouth. Few people pay attention to ductal saliva obtained from different salivary glands [38]. Moreover, as far as we know, this is the first study focused on structurally comparing the glucose expression of whole saliva and glandular saliva in a cohort of careful characterization and clinical examination. The results indicate that different collection methods provide significant differences in the snapshots of saliva glucose.
Our results revealed a close correlation between the SPS and UPS, as well as the USS and UWS glucose. The main function of the parotid gland is to digest carbohydrates and form food masses, so the serous cells dominate, making the glands mainly secrete serous secretions with high glucose concentration. This may explain the high glucose concentration in parotid saliva. The main function of sublingual saliva is to lubricate the oral cavity and prevent chemical and mechanical influences. Therefore, there is little change before and after meals.
The limitation of our study is the relatively small sample size. Further studies with a larger sample size are necessary to confirm the correlation between blood glucose and saliva glucose, so as to design a saliva-based diagnostic test method for DM. In addition, there are still many problems in this study that need to be resolved and further explored. For example, the submandibular glands and sublingual glands are closely located, so it is difficult to separate saliva from these glands with certainty, which is why saliva is collected from both glands in the current study. How to distinguish sublingual saliva from submandibular saliva is also a direction that needs further research.
In summary, the results of this study indicate that different saliva collection methods provide significant differences in the snapshots of saliva glucose. Based on the comparison of unstimulated and stimulated saliva collection methods, it can be shown that based on the simplicity and low variability of the collection method, UPS may be a preferred collection method. The results emphasize the importance of consistency when collecting saliva samples, which should be more important than the collection method itself.