**2. Application of research**

In the absence of detailed information in the research literature, this study seeks to better understand the specific properties of saliva viscosity, and how saliva viscosity reacted to factors that are integral in the creation of lab protocols; specifically, how the samples are stored. It is not uncommon for biological samples to be frozen or cooled, and this makes sense with respect to slowing down bacterial contamination and maintaining biological molecules of interest. It is well understood that many human proteins, enzymes, vitamins, degrade over time [9] and that the degradation over time can be diminished by freezing or cooling samples beyond given temperature degradation thresholds, to allow for long-term storage.

Changes within the aggregates commonly found in human saliva, such as mucins, may also be affected by temperature and shear forces. Enzymes, such as salivary alpha-amylase, and hormones, such as cortisol, will degrade over time unless this process is inhibited, typically by freezing samples to 20 degrees Celsius, or below [10]. However, it is important to determine the effects of sub zero temperatures on viscosity itself, as the viscosity of the saliva may be impactful in how the aggregates are measured via point-of-care salivary biosensors. This was reflected in the 2013 Robles et al. study which found that the most consistent and reliable salivary alphaamylase biosensor data was obtained from frozen and centrifuged passive saliva samples, rather than samples that were collected as fresh, passive, drool [11]. The authors hypothesize this discrepancy to be due to various factors of the saliva itself (such as mucin molecules), which may interfere with the device in question by preventing close binding to the sensor surface, as it attempts to detect quantities of salivary alpha-amylase. Also, this hypothesis reflects the prediction that salivary viscosity is an important factor in molecular measurements. While the aggregation effect was disadvantageous when in reference to the assessing quantities of the enzyme salivary alpha-amylase, it may actually be preferable when measuring quantities of different gases within saliva samples. This further delineates the importance of taking a closer look at physical salivary properties, in order to approach proposed handling methodologies appropriately, depending on the given purpose of the saliva sampling.

### **3. Protocols**

As previously mentioned, we hoped to better understand the properties of saliva viscosity in regards to different methodologies in sample preservation or usage. For this reason, two cycles of laboratory trials were conducted, with the aim of determining how saliva viscosity was affected. The first study aim is to determine how time alone affects saliva samples. The second trial aims at determining whether freezing and subsequent thawing affect the viscosity of the sample, as well. Both phases of data collection were done at a Biomedical Engineering laboratory, at Arizona State University, Tempe, and human saliva samples were collected within this department.

Participants were instructed to not eat within an hour of sample collection, and were then asked to drink approximately 100 mL of water immediately prior to saliva collection. This was to prevent short-term dehydration effects from confounding our variables. In addition, this aided in the ease of saliva collection. Participants were then asked to collect approximately 15 mL of passive saliva over a span of 25 minutes, into a plastic vial. The goal for saliva collection included diminishing the amount of air bubbles trapped within the saliva, by collecting the saliva very carefully, slowly, and with as little movement as possible. Foam-like saliva that is saturated with small air bubbles was not included in the overall 15 mL amount of passive saliva collected.

The viscosity of the collected saliva sample was measured using a Size 350 Cannon-Fenske apparatus, with a capillary radius of 0.045 cm, a shear rate of 2.08 1/s at 10cSt, and a viscometer constant of 0.5 cSt/s, which was cleaned and dried prior to commencing the viscosity protocol. The saliva sample was then poured into the apparatus, and allowed to flow through, while efflux time was measured concomitantly, indicating the time required for the meniscus of the viscous fluid to flow between the designated markings. This viscometer procedure was replicated 10 times consecutively for each collected sample, after which each sample was frozen and subsequently thawed the following day, at which point the viscometer procedure was performed again. A series of viscosity measurements of a 50% glycerol/water control solution was tested in the same manner to act as a control variable.

The kinematic viscosity of each trial was calculated using the efflux time and viscosity constant in the following relationship:

$$\text{Kinematic viscosity} = (\text{viscector constant}) \times (\text{efflux time} \ (\text{s})) \tag{1}$$

This procedure reflected the first half of the research questions, as to how saliva viscosity changes with time [12]. By comparing the time elapsed for the viscosity of human saliva with the glycerol/water solution, we are able to visualize how viscosity properties may be affected by the presence of the aggregates in unprocessed human saliva, which are not present in the glycerol/water mixture.

### **4. Results**

Data was collected for each measurement of salivary viscosity. The initial graph (**Figure 1**) represents the methodology behind the first research question; how does

#### **Figure 1.**

*This figure evaluates how salivary viscosity changes with time over subsequent trials following collection. This arbitrary time is demonstrated in sequential trials, as trials were completed one after the other following collection. This saliva kinematic viscosity is contrasted with that of a 50% glycerol water solution, acting as a control. In the passive saliva trials, we see a stark decrease in kinematic viscosity with each subsequent trial, however, the glycerol/ water solution shows a slight variation within an expected range given the experimental apparatus and simple laboratory control of room temperature (22 C).*

#### **Figure 2.**

*This figure demonstrates the statistically significant discrepancy between fresh and thawed passive saliva samples at 22 C. each bar represents an average of 10 trials. One outlier was removed from the thawed trial group as it was handled outside the guidelines of the lab protocol.*

salivary viscosity behave over time? **Figure 2** represents the viscosity of saliva (as average kinematic viscosity), with freshly collected samples of saliva, compared to with samples that were frozen, and subsequently, thawed for analysis.

### **5. Discussion**

There are several points of interest with regards to the conclusions drawn from the experimental design that reflect the difference in how fresh saliva behaves, compared to fresh saliva, and compared to saliva that had been frozen and re-thawed. Such comparisons are reflected in the literature by the 2019 study by Johannsen et al. which found that salivary viscosity measurements varied depending on whether the saliva was untreated or subject to magnet-beating prior to viscosity measurements at low shear rates [13].

One aspect that is of interest is whether there is a methodology that can guide how fresh saliva can be processed in order to have a consistent flow property or ensure that the major aggregates, presumably due to entangled mucin chains, can be minimized quickly so that a rapid test can be performed (**Figure 3**).

Bansil et al. [14] provide a molecular interpretation on how the structure of mucin leads to entanglements among the biopolymers in solution and create a range of viscosity effects depending upon concentration and mucin type. They suggest that a dilute solution of mucin generally has viscoelastic behavior that depends upon shear rate. A more general approach to the viscosity behavior of biopolymer solutions is given by Picout and Ross-Murphy [15] who provide experimental verification of the Cross equation:

$$\eta = \eta\_{\infty} + (\ \eta\_o - \eta\_{\infty})/[\mathbf{1} + (\lambda^m \mathbf{y}^m)] \tag{2}$$

with lambda being a time constant and gamma the shear rate.

Comparing the Cross equation to the data shown in this study in **Figure 2** suggests that repeated shearing with a Cannon-Fenske viscometer is a cumulative effect, so one may conjecture the following modified forms of the Cross equation could explain the apparent viscosity change in saliva after repeated shearing due to the capillary flow within the viscometer.

$$\eta = \eta\_{\text{os}} + (\eta\_{\text{o}} - \eta\_{\text{os}}) / \sum\_{n=0}^{k} [1 + k \ (\lambda^m \boldsymbol{\gamma}^m)] \tag{3}$$

**Figure 3.**

*This figure demonstrates the cross equation fit, using the limits of η*<sup>∞</sup> *and η*0*. The independent variable is demonstrated to be 1/(1 + aT).*

or

$$\eta = \eta\_{\text{os}} + (\eta\_o - \eta\_{\text{os}}) / \{ [\mathbf{1} + (\boldsymbol{\lambda}^m \boldsymbol{\gamma}^m) T] \} \tag{4}$$

where n is an integer and k is the number of repeat measurements in the first equation and T is the total elapsed time of shear for the combined repeat measurements. It is not as important to determine which equation may be better at predicting data than to understand how to use the concept to prepare saliva samples in a variety of ways rather than relying solely on freezing or centrifugation which can be cumbersome and time consuming.

The potential utility of these modified Cross equations is in determining a way to rapidly shear saliva samples using a simple microfluidic device or mixer, rather than subjecting the saliva to freezing or flowing the saliva through a longer tube to simulate the cumulative shear thinning shown in **Figure 2**. The shear rate experienced in the Cannon Fenske viscometer used for these experiments is on the order of 2 s�<sup>1</sup> , so based on the **Figure 2** data of approximately a total time of 80 seconds of shearing is needed in order to reach a stable and minimum kinematic viscosity a device capable of deliver a shear rate of 200 s�<sup>1</sup> which is well within the reach of portable and low cost homogenizers [16–19].

### **6. Conclusions**

Salivary viscosity can be an important parameter to consider when designing diagnostic devices for rapid testing. Consideration should be given to the fact that not only is saliva viscoelastic, but its apparent viscosity can change by mild shearing over a period of time. Shearing at rates as low as 2 s�<sup>1</sup> can decrease its kinematic viscosity by more than half, which could change some kinetics of enzyme action, sensor signal development, or diffusive transport. After approximately a total time of 80 seconds of shearing at 2 s�<sup>1</sup> can lead to a stable and minimum kinematic viscosity. The concept of biopolymer viscosity behavior being modeled by the Cross equation suggests that a device capable of delivering shear rates of 200 s�<sup>1</sup> and above may be able to modify the mucin superstructure sufficiently to provide saliva samples with consistent apparent viscosities. Microfluidic devices or low cost handheld homogenizers could very quickly deliver the needed shearing action in order to provide a more consistent saliva sample, in terms of its viscous properties.

## **Acknowledgements**

The institutions involved in the findings for this paper are Arizona State University, Tempe, and New Mexico State University. The data was collected at a Arizona State University Biomedical Engineering laboratory in Tempe, Arizona. This project was done under the supervision of Professor Antonio A. Garcia, who is now Associate Dean of Engineering at New Mexico State University in Las Cruces, New Mexico.

### **Conflict of interest**

The authors declare no conflict of interest.
