Point-of-Care Diagnostic Biosensors to Monitor Anti-SARS-CoV-2 Neutralizing IgG/sIgA Antibodies and Antioxidant Activity in Saliva

Monitoring biomarkers is a great way to assess daily physical condition, and using saliva instead of blood samples is more advantageous as the process is simple and allows individuals to test themselves. In the present study, we analyzed the titers of neutralizing antibodies, IgG and secretory IgA (sIgA), in response to the SARS-CoV-2 vaccine, in saliva. A total of 19 saliva and serum samples were collected over a 10-month period 3 weeks after the first vaccine, 8 months after the second vaccine, and 1 month after the third vaccine. The ranges of antibody concentrations post-vaccination were: serum IgG: 81–15,000 U/mL, salivary IgG: 3.4–330 U/mL, and salivary IgA: 58–870 ng/mL. A sharp increase in salivary IgG levels was observed after the second vaccination. sIgA levels also showed an increasing trend. A correlation with trends in serum IgG levels was observed, indicating the possibility of using saliva to routinely assess vaccine efficacy. The electrochemical immunosensor assay developed in this study based on the gold-linked electrochemical immunoassay, and the antioxidant activity measurement based on luminol electrochemiluminescence (ECL), can be performed using portable devices, which would prove useful for individual-based diagnosis using saliva samples.


Introduction
Early diagnosis in the field and at home can contribute to disease detection, the prevention of severe diseases, and the control of infection. While physical and blood tests are used to diagnose diseases, the use of saliva instead of blood would facilitate the screening of a large number of individuals as well as providing diagnoses in dispersed areas, such as point-of-care (POC) and home-based testing. In particular, saliva collection does not require a medical professional, as is the case in blood sampling, and saliva selfcollection can enable routine medical services in remote areas without reliance on urban clinics and hospitals. There are two main types of antibodies detected in saliva: secretory IgA (sIgA) and IgG. sIgA is produced locally in the salivary glands, while most IgG in saliva is transferred into saliva through the gingival crevices after being produced in the plasma cells. SARS-CoV-2 is transmitted to humans via the oral and nasal cavities, causing severe acute respiratory syndrome. The squamous epithelium of the tongue and periodontal tissue of the gums contain epithelial cells that express angiotensin-converting enzyme-2 (ACE-2), the receptor for SARS-CoV-2. Saliva also contains substances such as lactoferrin, lysozyme, and most abundantly, sIgA, which inhibit infection. We have identified SARS-CoV-2 crossreactive IgA spike proteins in the saliva of people without COVID-19, suggesting that sIgA lation with the amount of antibody. This method was invented by our group and is called the gold-linked electrochemical immunoassay (GLEIA), which enables the measurement of gonadotropin [29], C-reactive protein [30], and sIgA [31,32]. This method utilizes the electrochemical activity and antibody-modifying function of gold nanoparticles and has been shown to function effectively as an immunosensor on a scale of 10-100 nm. Based on this principle, high sensitivity has been achieved using printed electrodes modified with carbon nanotubes (CNTs) [33] and graphene [34]. Scheme 1. This scheme indicates the electrochemical detection of anti−SARS−CoV−2 IgG and IgA (left part) and the electrochemiluminescent detection of antioxidant activity (right part). Both electrochemical and luminescent measuring devices were capable of POC monitoring with disposable, mass-producible printed electrodes. The principle underlying the measurements is explained in the text.
On the other hand, to measure antioxidant activity, as shown in the scheme below (right), an electrode reaction was used to generate reactive oxygen species from dissolved oxygen that would then react with oxidized luminol to form high-energy intermediates by eventually forming ions emitting at 420 nm. Any molecule with antioxidant activity consumes the ROS produced on the electrode, thus quenching the luminescence from luminol. Nagatani et al. reported that the amount of quenched luminescence correlated with the antioxidant activity, and that the antioxidant activity of food can be measured rapidly (in approximately 2 min) [35]. Whereas other methods require the addition of an external reagent to generate oxygen-active species, this method allows for the generation of oxygen-active species from dissolved oxygen simply by applying an electrical potential. In addition, luminol luminescence can be induced by oxygen-active species simply by changing the potential on the same electrode. In a previous study, we created a calibration curve for trolox, a standard for antioxidant capacity, measured the luminescence intensity in 22 different types of drinking water, and calculated the antioxidant capacity from the calibration curve. The obtained results were compared with the antioxidant capacity This method was invented by our group and is called the gold-linked electrochemical immunoassay (GLEIA), which enables the measurement of gonadotropin [29], Creactive protein [30], and sIgA [31,32]. This method utilizes the electrochemical activity and antibody-modifying function of gold nanoparticles and has been shown to function effectively as an immunosensor on a scale of 10-100 nm. Based on this principle, high sensitivity has been achieved using printed electrodes modified with carbon nanotubes (CNTs) [33] and graphene [34].
On the other hand, to measure antioxidant activity, as shown in the scheme below (right), an electrode reaction was used to generate reactive oxygen species from dissolved oxygen that would then react with oxidized luminol to form high-energy intermediates by eventually forming ions emitting at 420 nm. Any molecule with antioxidant activity consumes the ROS produced on the electrode, thus quenching the luminescence from luminol. Nagatani et al. reported that the amount of quenched luminescence correlated with the antioxidant activity, and that the antioxidant activity of food can be measured rapidly (in approximately 2 min) [35]. Whereas other methods require the addition of an external reagent to generate oxygen-active species, this method allows for the generation of oxygen-active species from dissolved oxygen simply by applying an electrical potential. In addition, luminol luminescence can be induced by oxygen-active species simply by changing the potential on the same electrode. In a previous study, we created a calibration curve for trolox, a standard for antioxidant capacity, measured the luminescence intensity in 22 different types of drinking water, and calculated the antioxidant capacity from the calibration curve. The obtained results were compared with the antioxidant capacity obtained via ORAC (Oxygen Radical Absorbance Capacity), and a high correlation was observed. A correlation was also observed for the scavenging capacity for superoxide radicals and hydroxyl radicals by ESR [35]. A pocket-sized electrochemical measuring device and palm-sized electrochemiluminescence device were used and operated under PC control. In addition, printed electrodes, which are mass-producible, inexpensive, and disposable, were used to ensure safety from biological human samples, including infectious samples.
In this study, an electrochemical biosensor system was developed for the rapid detection of neutralizing antibodies (sIgA and IgG) against SARS-CoV-2 and antioxidant activity in saliva samples, which can easily be collected daily. The results obtained were evaluated to demonstrate the usefulness of the biosensor for POC diagnosis.

Electrodes and Instruments
A pocket-sized potentiostat (miniSTAT100) and a palm-sized electrochemiluminometer (BDTeCL-XP) were obtained from BioDevice Technology (Ishikawa, Japan). Both pictures are shown in Scheme 1. Screen-printed electrodes (EP-P, EP-PP) with an integrated working electrode (2.64 mm 2 diameter), counter electrode, and Ag/AgCl reference electrode, with a total length of 11 mm, were also obtained from BioDevice Technology (Ishikawa, Japan). Two UV-visible spectrometers, U-2900 (Hitachi, Japan) and DS-11 (DeNovix, Wilmington, DE, USA), were used for the quantification of gold nanoparticles and proteins, respectively. A micro-high-speed cooling centrifuge (KUBOTA3700, Kubota, Tokyo, Japan) was also used in this study for the preparation of antibody-coated gold nanoparticles.

Preparation of Secondary Antibody-Coated Gold Nanoparticles
Anti-human IgA and anti-human IgG antibodies were used as secondary antibodies. Secondary antibody-modified gold nanoparticles were prepared using our previously reported method [31]. The antibodies (50 µg/mL) were dissolved in 5 mM phosphate buffer (pH 7.5), added to the Au nanoparticle solution, and then, incubated for 10 min at room temperature. This is known as the Au conjugate, which was mixed with 0.1 mL of 10% BSA in phosphate buffer and 0.05 mL of 1% PEG (0.1 mL) in phosphate buffer. Au and anti-IgA/anti-IgG conjugates were collected via centrifugation (8000× g for 15 min at 4 • C). Then, the Au anti-IgA/anti-IgG conjugate was suspended in 1 mL of preservation solution (1% BSA, 0.05% PEG, 0.1% NaN 3 , and 150 mM NaCl in 20 mM Tris-HCl buffer, pH 8.2) and collected again in the same manner. For the stock solution, the Au anti-IgA/anti-IgG conjugate was suspended in the preservation solution and the optical density was adjusted to OD 520 = 6. The Au anti-IgA/anti-IgG conjugate was diluted three times with trehalose (OD 520 = 2), and 3 µL of this solution was added to each well of the 96-well plate. The plate was then dried under vacuum for 5 min. In this case, 60 nm diameter gold nanoparticles were selected, because they showed a better response than the 20 nm and 100 nm AuNPs from our previous study [32].

Immobilization of S Protein Antigens on the Working Electrode
The S protein (2 µL) in phosphate buffer (50 µg/mL) was dropped onto the working electrode and incubated at room temperature for 1 h to adsorb the protein. The amount of antibody adsorbed on the electrode was evaluated via electrochemical impedance spectroscopy (EIS) and 50 ug/mL was determined to be adequate for this study (see Figure S3).
Then, 10 µL of 1% BSA in phosphate buffer was dropped onto the entire electrode, and this was incubated at room temperature for 1 h to suppress non-specific adsorption.
2.5. Sandwich-Type Neutralizing Antibody and Gold Nanoparticles Modified with Anti-IgA/IgG on the Working Electrode and Electrochemical Detection of Gold Nanoparticles A sandwich-type reaction occured directly on the working electrode, as shown in Scheme 1. IgA/IgG test solutions (10 µL) were added onto the 96-well plate containing Au anti-IgA/anti-IgG conjugates, and mixed for 10 s. Then, 1.4 µL of the solution was placed on the working electrode and incubated for 15 min at room temperature for IgG detection. After rinsing with a phosphate buffer, the solution was removed. The direct redox reaction was performed in a 0.5M HCl solution (30 µL) covering the entire electrode at room temperature. The pre-oxidation of Au nanoparticles was performed at a constant potential of 1.2 V for 40 s, immediately followed by DPV, while scanning the potential range from 0.6 to 0.1 V with a step potential of 4.0 mV, a pulse amplitude of 50 mV, and a pulse period of 0.2 s. The DPV conditions were determined according to our previous report [29][30][31]. The potentials were recorded against the Ag/AgCl electrode.

ECL Measurement for Determination of Antioxidant Activity
The antioxidant activity of the saliva samples was determined by diluting them 5fold and mixing them with a luminol solution (final concentration: 100 µM, pH 8.1). The potential window of CV was kept between −800 mV and +800 mV, and the other CV operating parameters included a scan rate of 50 mV/s and a time interval of 0.1 s. The trigger signal was sent to the photon detection unit from the potentiostat at the same time as the electrochemical measurement was started, and the ECL intensity was measured every 0.1 s. Antioxidant standard curves were prepared using different concentrations of ascorbate and trolox.

Protein Concentration
Protein concentrations were determined using Bradford assay reagent with BSA as the standard control. The measuring instrument used was a DeNovix DS 11 with a wavelength of 595 nm. Saliva samples were diluted 10-20 times as required and mixed with Bradford reagent, and 3 µL was used for the measurements.

Saliva Collection from Selected Participants
We collected samples using Salivettes1 (Sarstedt AG & Co., KG, Numbrecht, Germany) in a hospital room between 9 a.m. and 12 p.m. The participants were instructed to refrain from eating, drinking, and brushing their teeth for at least 1 h before sample collection. Saliva samples were immediately centrifuged at 2000× g for 15 min, and then, stored at -80 • C. We tested the samples for SARS-CoV-2 using polymerase chain reaction (PCR). Individuals with saliva samples were confirmed to be negative for COVID-19 upon PCR testing in the study. The participants were volunteers from Kanagawa Dental University. Individuals with IgA nephropathy, selective IgA deficiency, autoimmune diseases, or coldlike symptoms within the past 2 weeks were excluded from the study. This study was approved by the Kanagawa Dental University Research Ethics Review Board (approval number: 792). This study was registered in the Japanese UMIN Clinical Trials Registry (UMIN-CTR) (approval numbers: #UMIN000047028 and UMIN000043717), and therefore, meets the ICMJE standards.

Choice of Antigens and Calibration for Neutralizing IgG and sIgA Antibodies
To measure antibodies that show neutralizing activity, we chose viral S proteins that reacted selectively with neutralizing antibodies on the electrode surface. Spike proteins are transmembrane proteins that contain two subunits: S1 and S2. S1 mainly contains the receptor-binding domain (RBD) and is responsible for recognizing cell surface angiotensin receptors, while S2 contains the basic elements required for membrane fusion. Three recombinant S proteins were tested in this study: the S1-mFc recombinant protein, the S1 + S2 recombinant with His-tag, and the RBD recombinant protein with Fc tag, which correspond to amino acid sequences 16-685, 16-1210, and 319-541 of the S protein, respectively. The results showed that the S1 + S2 region recombinant protein had good calibration properties for both IgG and IgA. Although the RBD region is the most useful binding site for assessing neutralizing activity, as a sensor, it showed a high background signal, even at low concentrations. This could be due to partial changes in the protein structure upon immobilization on the electrode surface. The neutralizing activity of various types of antibodies against SARS-CoV-2 in serum has been reported [36] and the results have shown that the RBD, S1 domain, full-length S protein, and S trimer have high specificity (RBD: 98%, S1: 97%, S full: 93%, and S trimer: 96%). In terms of sensitivity, the full-length S protein had greater sensitivity than the RBD to a large extent, and the S trimer had greater sensitivity than the S1 domain, but to a smaller extent.
The electrochemical immunosensor responses and calibration curves for the neutralizing antibodies sIgA and IgG are shown in Figure 1. Calibrations were obtained as Michaelis-Menten type functions via non-linear curve fitting in graphing software. The detection limit was 0.28 U/mL for IgG. This value was determined using three-sigma limits. The range of both assays was almost two orders of magnitude; therefore, the assayed sample had to be diluted. The ELISA kit had a measurement range of 2-250 U/mL of neutralizing antibody activity, indicating that our GLEIA system was more sensitive. The ELISA measurement required 3 h, including repeated washing and enzymatic reaction operations, and was not suitable as POC diagnostic equipment. In addition, saliva samples contain high concentrations of enzyme proteins and mucins (in the order of 100 µg/mL), which needed to be diluted to reduce their effect on the antigen-antibody reaction. The GLEIA system showed a high background signal without dilution ( Figure S1 in Supplementary Materials). This was attributed to the non-specific adsorption of antibody-modified gold nanoparticles on the electrode. Blocking conditions have been extensively studied during the development of GLEIA. In the present study, a 100-fold dilution was used because a 10-fold dilution showed a lower correlation coefficient with the ELISA method (less than 0.4).

Antioxidant Activity of Saliva Samples
Luminol electrochemiluminescence was used to measure the antioxidant activity of saliva. This method, reported by us, uses the luminescence produced by oxidized luminol at +0.3-0.4 V (vs. Ag/AgCl) with the reactive oxygen species produced via the reduction of dissolved oxygen at −0.8-0.5 V (vs. Ag/AgCl). Antioxidant molecules inhibit ROS production and quench luminescence. Different beverage products were tested using trolox as a standard for measuring the antioxidant activity in food [24]. The results presented here are based on ascorbic acid and trolox as a representative of antioxidant substances ( Figure 2).

Antioxidant Activity of Saliva Samples
Luminol electrochemiluminescence was used to measure the antioxidant activity of saliva. This method, reported by us, uses the luminescence produced by oxidized luminol at +0.3-0.4 V (vs. Ag/AgCl) with the reactive oxygen species produced via the reduction of dissolved oxygen at -0.8-0.5 V (vs. Ag/AgCl). Antioxidant molecules inhibit ROS production and quench luminescence. Different beverage products were tested using trolox as a standard for measuring the antioxidant activity in food [24]. The results presented here are based on ascorbic acid and trolox as a representative of antioxidant substances ( Figure 2).
Antioxidant activity was plotted as the rate of luminescence inhibition (%), using the formula: [1 − (luminescence at zero concentration − luminescence with sample)/(luminescence at zero concentration)] × 100 (%) The rate of luminescence inhibition corresponded to antioxidant activity in concentration ranges of 0.2-0.5 mM and 0.5-5 mM for ascorbic acid and trolox, respectively.

Applications for Monitoring Human Saliva Samples
Human saliva samples were collected from 10 individuals without COVID-19 infection. All 10 saliva samples were collected 3 weeks after the second dose of the vaccine. The saliva samples were tested for the neutralizing antibodies IgG and sIgA, antioxidant activity, and protein concentration (Figure 3). Each saliva sample was diluted 100-fold to suppress the effect of non-specific adsorption while measuring neutralizing antibodies. Salivary IgG concentrations were 10-100 times lower than their concentration in serum

Applications for Monitoring Human Saliva Samples
Human saliva samples were collected from 10 individuals without COVID-19 infection. All 10 saliva samples were collected 3 weeks after the second dose of the vaccine. The saliva samples were tested for the neutralizing antibodies IgG and sIgA, antioxidant activity, and protein concentration (Figure 3). Each saliva sample was diluted 100-fold to suppress the effect of non-specific adsorption while measuring neutralizing antibodies. Salivary IgG concentrations were 10-100 times lower than their concentration in serum and were close to the detection limit for samples. However, the concentrations were below the detection limit for the ELISA kit. Individual variations in sIgA were observed at 0.334-1.97 µg/mL, which was within the range of variation reported for uninfected saliva samples [7]. As the total amount of sIgA in saliva is 65-145 µg/mL, it was also shown that only approximately 0.5-1% of sIgA has neutralizing activity. sIgA is a useful diagnostic indicator of an individual's health, due to its affinity for various pathogens as mucosal antibodies. Therefore, routine pre-onset identification of sIgA based on individual characteristics may enable early detection to prevent the onset of disease. In contrast, there was a variation of 29 ± 25-268 ± 22 U/mL (almost the same as ng/mL) for IgG-neutralizing antibodies. Because the total amount of IgG in saliva is 20-30 µg/mL [37], the neutralizing antibody corresponds to 0.1-1%. This value is similar to that of sIgA.  Antioxidant activity was determined using 5-10-fold dilutions, and the antibody tivity was measured relative to the suppression of luminescence in the test vs. the cont sample. The results showed individual variability of 17.3-82.4 (relative activity). The tioxidant activity of salivary components is made up of enzyme molecules that inhibit formation of ROS, such as SOD, catalase, and peroxidase, as well as those that bind to a Antioxidant activity was determined using 5-10-fold dilutions, and the antibody activity was measured relative to the suppression of luminescence in the test vs. the control sample. The results showed individual variability of 17.3-82.4 (relative activity). The antioxidant activity of salivary components is made up of enzyme molecules that inhibit the formation of ROS, such as SOD, catalase, and peroxidase, as well as those that bind to and remove radical species, such as vitamins A and E, uric acid (UA), and glutathione. Inflammation caused by microbial and viral infections in the oral cavity activates leukocytes, which generate reactive oxygen species and reduce the antioxidant activity of saliva [9,10]. It has been suggested that knowing the antioxidant status of saliva can be an indicator of an individual's health status. There are few reports on the changes in salivary antioxidant activity before and after the onset of COVID-19, and it would be useful to accumulate data on an individual basis. Protein levels varied between 0.76 and 3.83 mg/mL (BSA was used as standard). Sample 10 showed a high value of 3.83 mg/mL, while samples 1-9 were in the range of 0.76-1.82 mg/mL. In saliva, enzymes such as amylase (0.48 mg/mL), lysozyme (0.01 mg/mL), peroxidase (0.06 mg/mL), and the polysaccharide mucin (0.2 mg/mL) were present in high concentrations [38]. The four datasets of neutralizing activity-IgG and sIgA concentrations, antioxidant activity, and the protein concentration of the saliva samples from 10 individuals (T1-T10)-are shown in a radar chart for each individual ( Figure 4). The data are presented as the ratio of the maximum concentration of each measured item. Samples 1-3 showed relatively high sIgA and IgG concentrations and relatively low antioxidant activity and protein concentration. Samples 6-8 showed a lowvalue pattern for all items. These results were considered to be due to the individual characteristics and conditions at the time of sampling. Daily data accumulation may be useful for individual health diagnoses.   Figure 3. The data are shown as a ratio of the maximum concentration of each measured item.

Monitoring Changes in Saliva Markers over Time, after Multiple Vaccinations
We measured IgA, IgG, antioxidant activity, and protein concentration in the saliva of an individual without COVID-19 infection (without periodontal disease or subjective symptoms of oral inflammation or bleeding before and after three vaccinations over time) (Figures 5 and S2). Simultaneously, IgG measurements in the serum were performed and compared. Nineteen saliva and serum samples were collected over a 10-month period 3 weeks after the first vaccine, 8 months after the second vaccine, and 1 month after the third vaccine. The range of data measured during the study period was as follows: serum IgG: 81 U/mL (0)-15,000 U/mL(2b); salivary IgG: 3.4 U/mL (2k)-330 U/mL (2d); and sIgA: 58 ng/mL (2k)-870 ng/mL (2c). The serum IgG level was 660 U/mL 4 days after the first vaccination. IgG in the serum increased significantly to 15,000 U/mL at 1 week after the second vaccination, clearly indicating the effect of the vaccine. The salivary IgG levels increased from 33 U/mL on the day of the first vaccination to 79 U/mL 1 week later. It also increased from 140 U/mL 1 week after the second vaccination to 330 U/mL 2 weeks later. Interestingly, the peak was observed later than that for serum IgG. Following the third vaccination, an increasing trend in IgG was observed in both serum and saliva samples,  Figure 3. The data are shown as a ratio of the maximum concentration of each measured item.

Monitoring Changes in Saliva Markers over Time, after Multiple Vaccinations
We measured IgA, IgG, antioxidant activity, and protein concentration in the saliva of an individual without COVID-19 infection (without periodontal disease or subjective symptoms of oral inflammation or bleeding before and after three vaccinations over time) (Figures 5 and S2). Simultaneously, IgG measurements in the serum were performed and compared. Nineteen saliva and serum samples were collected over a 10-month period 3 weeks after the first vaccine, 8 months after the second vaccine, and 1 month after the third vaccine. The range of data measured during the study period was as follows: serum IgG: 81 U/mL (0)-15,000 U/mL(2b); salivary IgG: 3.4 U/mL (2k)-330 U/mL (2d); and sIgA: 58 ng/mL (2k)-870 ng/mL (2c). The serum IgG level was 660 U/mL 4 days after the first vaccination. IgG in the serum increased significantly to 15,000 U/mL at 1 week after the second vaccination, clearly indicating the effect of the vaccine. The salivary IgG levels increased from 33 U/mL on the day of the first vaccination to 79 U/mL 1 week later. It also increased from 140 U/mL 1 week after the second vaccination to 330 U/mL 2 weeks later. Interestingly, the peak was observed later than that for serum IgG. Following the third vaccination, an increasing trend in IgG was observed in both serum and saliva samples, although the increase was not as large as that after the second vaccination. These results indicate that vaccine efficacy can be monitored using saliva samples, without the need for blood sampling. As for sIgA, the concentration increased to 980 ng/mL after the second vaccination, which was approximately double the pre-vaccination level; after the third vaccination, it increased to 280 ng/mL, which was lower than that in the second vaccination, but significantly higher than the pre-vaccination level. sIgA is secreted into saliva through the salivary glands, and IgG is secreted into saliva through periodontal fluid; however, the dynamics of their presence in saliva after vaccination are not understood. The accumulation of such data over time, together with antioxidant activity and protein concentrations, could be useful as a basis for diagnosis and care based on individual characteristics.

Conclusions
In this study, we analyzed salivary titers of neutralizing antibodies against the SARS-CoV-2 vaccine, IgG and secreted IgA (sIgA), using a portable electrochemical biosensor. Thorough the continuous monitoring of saliva and serum samples from 19 subjects for 10 months, the concentration ranges of the antibodies were: serum IgG: 81-15,000 U/mL, sal-

Conclusions
In this study, we analyzed salivary titers of neutralizing antibodies against the SARS-CoV-2 vaccine, IgG and secreted IgA (sIgA), using a portable electrochemical biosensor. Thorough the continuous monitoring of saliva and serum samples from 19 subjects for 10 months, the concentration ranges of the antibodies were: serum IgG: 81-15,000 U/mL, salivary IgG: 3.4-330 U/mL, and salivary sIgA: 58-870 ng/mL. After the second vaccination, a sharp increase in serum IgG occurred, and salivary IgG and sIgA tended to follow. The tracking of saliva-neutralizing antibodies provided the possibility to determine the efficacy of the vaccine.
We also analyzed salivary antioxidant activity using a portable ECL biosensor. The results included lifestyle and individual differences among the subjects, and therefore, there is room for additional research. Daily data accumulation may be useful for individual health diagnoses.
To evaluate the utility of electrochemical biosensors, post-vaccination salivary biomarkers were monitored. Our GLEIA method and ECL biosensor show sufficient utility for on-site salivary biomarker evaluation. In the future, we will work to develop a more practical POC biosensor by expanding the number of subjects and the types of biomarkers to be evaluated.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/bios13020167/s1, Figure S1: Optimization of the dilution rate for saliva sample; Figure S2: Preliminary check of blood contamination on saliva samples and Figure S3: Optimization of primary antibody concentration using the electrochemical impedance spectroscopy (EIS).  Institutional Review Board Statement: This study was approved by the Kanagawa Dental University Research Ethics Review Board (approval number: 792). This study was also registered in the Japanese UMIN Clini-cal Trials Registry (UMIN-CTR) (approval numbers: #UMIN000047028 and UMIN000043717), and therefore, meets the ICMJE standards.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding authors upon reasonable request.