A new sensitive spectrophotometric method for determination of saliva and blood glucose

Journal Pre-proof A new sensitive spectrophotometric method for determination of saliva and blood glucose

Parvin Mohammadnejad, Saeed Aminzadeh, Kamahldin Haghbeen

Soleimani

Asl,

Saeed

PII:

S1386-1425(19)31287-9

DOI:

https://doi.org/10.1016/j.saa.2019.117897

Reference:

SAA 117897

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received date:

25 July 2019

Revised date:

3 November 2019

Accepted date:

2 December 2019

Please cite this article as: P. Mohammadnejad, S.S. Asl, S. Aminzadeh, et al., A new sensitive spectrophotometric method for determination of saliva and blood glucose, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy(2019), https://doi.org/10.1016/j.saa.2019.117897

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2019 Published by Elsevier.

Journal Pre-proof A new sensitive spectrophotometric method for determination of saliva and blood glucose

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Parvin Mohammadnejad, Saeed Soleimani Asl, Saeed Aminzadeh, Kamahldin Haghbeen*

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National Institute for Genetic Engineering and Biotechnology, P.O. Box: 14965/161, Tehran,

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Iran

Corresponding Author: Kamahldin Haghbeen (Phone: +98 21 44787372, Fax: +98 21 44787399, E-mail: [email protected] and [email protected] P. Mohammadnejad and S. Soleimani Asl contributed equally to this work.

Journal Pre-proof

ABSTRACT There is an increasing need for accurate and inexpensive glucometers as the world moves toward personalized medicine. Among the existing technologies, photometric based devices are more desired due to the cost-effectiveness, ease-of-use and the potential to be adopted in the smart-

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phone technology for remote sensing and self-monitoring purposes. However, the accuracy,

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precision, and reproducibility of the results of these devices are heavily dependent on the details of the chosen glucose measuring method. Considering the delicate problems with the current

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spectrophotometric methods, a new method was developed for more precise, accurate, and fast

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measurement of blood glucose via the coupled reactions of glucose oxidase and peroxidase using

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4-[(4-Hydroxy-3-methoxyphenyl) azo]-benzenesulfonic acid (GASA) as the substrate. Stability of GASA and its oxidized products along with its direct and fast consumption by peroxidase,

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made it possible to determine blood glucose concentration in less than 20 s with high

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reproducibility. The low detection limit of GASA method (0.36 mg dL-1) with a linear range

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from 0.36 to 399.6 mg.dL-1 also allowed determination of salivary glucose concentration (SGC). As compared with the blood samples, the SGC results were more dispersed, especially for the diabetic participants, assumingly due to the diverse nature of salivary samples. However, a good correlation coefficient of 0.81 for non-diabetic individuals showed that it is accurate enough to recognize non-diabetic from diabetic condition. Results of this study disclose the potential application of GASA method as a reliable alternative for the current spectrophotometric methods with the ability to be adopted in miniaturized glucometers.

Journal Pre-proof Keywords: photometric method; peroxidase; glucose oxidase; diabetic; non-diabetic 1. Introduction Diabetes Mellitus as a major cause of morbidity, disability, and mortality is characterized by elevated glucose level in blood. The accepted range of normal blood glucose level is 87.95 to 123.85 mg dL-1. To avoid serious complications of this chronic disease, monitoring of blood

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glucose level is crucial [1]. Among the various detection methods, enzyme-based approaches

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have received more attention, in particular coupled-enzymatic reactions of glucose oxidase (GOX, EC 1.1.3.4) and peroxidase (POX, EC 1.11.1.x) [2] due to the merits associated with the

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parameters such as substrate specificity, rate of the reaction, range of pH tolerance, product

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inhibition, availability and prices of the enzymes [3-5]. Coupled-enzymatic determination of

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glucose starts with the oxidation of glucose by GOX, which produces H2O2 as a by-product. Hydrogen peroxide, then, is used by POX to oxidize an electron-donor substrate (edS-H) such as

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an aromatic organic compound [6].

𝐺𝑂𝑋

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D-glucose + O2 →

D-gluconic acid + H2O2 𝑃𝑂𝑋

H2O2 + 2 edS-H →

2 H2O + 2 edS•

As a result of one-electron oxidation by the complex of POX and H2O2 [7], the organic substrate turns into a radical intermediate (edS•) which immediately interacts with its surrounding compounds. These reactions can be engineered to form chromophoric products. Consequently, the color formation can be monitored by a simple spectrophotometer. Then, the resulting data is interpreted in terms of the rate of the enzymatic reaction and glucose concentration if the extinction coefficient () of the chromophoric substance is known [8]. A renowned example is

Journal Pre-proof ortho-dianisidine which produces a chromophoric substance with a max at 550 nm (= 11300 M1

cm-1) [9].

Another well-known example can be seen in Trinder method [10, 11] which is still being widely used in clinical labs, especially in the developing countries, for determination of blood sugar. In Trinder method, phenol is used as the organic substrate. After enzymatic oxidation, phenoxyl radical is coupled with 4-aminoantipyrine (4-AA) forming a quinone-imine (quinoid), a red color

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substance (Fig. 1A) with max at 516 nm (= 11300 M-1 cm-1) [12]. Trinder method has been well

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studied and adapted to different autoanalyzers [2]. Despite its advantages, there are some

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drawbacks associated with the basic chemical reaction of the method. Similar to ortho-

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dianisidine, both phenol and 4-AA are toxic compounds [13, 14] and 4-AA is able to react with

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other existing phenolic compounds in blood serum (such as dopamine and dobutamine) [15]. In addition to the incubation time required in Trinder method (20 min at 25 °C), the low stability of

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the quinoid product (Fig. 1A) at the required pH for the blood glucose determination makes the

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method prone to production of results with low reproducibility [16].

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A delicate problem with the existing spectrophotometric glucose determination relates to the fact that the chromophoric substance is formed as a result of the chemical reactions of edS• with its surrounding molecules and not directly from the enzymatic oxidation of edS-H. Therefore, in addition to the instability of the chromophoric product, there is a chance for edS• to produce more than one product [13, 17]. It has been recently shown that the enzymatic activity of POX can be directly and precisely followed using aniline diazo compounds as the substrates. Considering the advantages of that study and the fact that the diazo substances are pH sensitive and the accuracy of the kinetic results is profoundly dependent on the extent of the overlap between the spectra of the azo substrate and the sample solution, a new spectrophotometric

Journal Pre-proof method has been developed for precise measurement of blood sugar using a guaiacol diazo derivative, 4-[(4-Hydroxy-3-methoxyphenyl) azo]-benzenesulfonic acid (GASA, Fig. 1B) in the coupled reactions of GOX and POX. It is also demonstrated here that GASA method is sensitive enough to be used for reliable measuring glucose in salivary samples. The advantages of this method make it a prime candidate for optical based glucose measurement devices.

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2. Materials and methods

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Horseradish POX (EC 1.11.1.7; 100 U mg-1), GOX (EC 1.1.3.4; 5 kU mg-1), 4aminoantipyrine (4-AA), phenol, glucose and all the required chemicals for the experiments were

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obtained from Merck (Darmstadt, Germany). GASA was synthesized as described before [18].

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Spectrophotometric measurements were conducted on a Specord 50 spectrophotometer (Analytik

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Jena, Germany). Extinction coefficient of GASA was determined at 460 nm in phosphate buffer

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solutions (PBS) at pH 7.6 and 20 °C.

2.1. Preparation of the enzymes and GASA solutions

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Freshly prepared enzyme solutions were used in this work. The stock solutions of POX (1

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mg) and GOX (0.5 mg) in PBS (1 mL, 0.01 M, pH 7.6) were prepared separately and kept on ice during the experiments. The dye solution was prepared by dissolving GASA (4 mg) in either PBS or Tris-base buffer (5 mL, 0.01 M, pH 7.6). The solutions were degassed and the stability of the resulting solution was examined by monitoring their UV/VIS spectra at 20 °C during 3 days. No change in the absorption spectra of GASA solution, due to precipitation or auto-oxidation, was observed. 2.2. Preparation of the blood serum and saliva samples

Journal Pre-proof To setup the methods, both saliva and blood samples were obtained from the laboratory of Taban Interdisciplinary Diabetes Clinic. Samples were obtained from the volunteers under fasting conditions. Whole blood (4 mL) was drawn into vacutainer tube containing no anticoagulant. To allow clotting, it was maintained at room temperature for 30 minutes. After centrifugation (15 min at 20000 ×g, 4°C), the supernatant (serum) was aspirated and kept at 4 °C. Saliva samples were collected from two groups of volunteers of age 25-50 years, including

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10 diabetics and 10 non-diabetics. To determine salivary glucose concentration, 1 mL of whole

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unstimulated saliva was used. The sample was centrifuged (5 min at 10000 ×g, 4 °C) and the

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resulting supernatant was subjected to the glucose measurements. In order to find correlation

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between salivary and blood glucose concentration, concurrent blood monitoring was also carried

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out for each volunteer by the lab. The statistical analysis was performed using Microsoft Excel software and results presented as mean ± standard deviation. All the sample preparations were

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carried out under the guidelines of Bioethics Committee of National institute of Genetic

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Engineering and Biotechnology of Iran (Approval number: IR.NIGEB.EC.1398.6.24 B).

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2.3. Glucose determination

To set up GASA method, test samples were examined by Trinder method [19] and by a commercial Glucose Assay Kit [Glucose (GOD-POX), Pars Azmun Company, Tehran, Iran]. The standard curve for Trinder method was obtained (r2 = 0.99, Fig. S1A, supplementary document) according to the reference [20]. The accuracy of Trinder method was double checked by the commercial kit. According to the vendor’s procedure, Glucose Assay Kit solution (1000 μL) was added to 10 μL of the serum sample. The resulting mixture was incubated at 25 °C for 20 minutes. Then, subjected to spectrophotometric reading at 516 nm. Glucose concentration was calculated from the standard curve introduced by the vendor.

Journal Pre-proof 2.4. GASA method The serum of the blood sample (30 L) was added to the assay solution made from PBS (0.01mM, pH 7.6) containing POX (0.005 pM), GOX (0.021 pM) and GASA (0.04 mM) in a total volume of 3 mL at 20 °C. After 20 seconds, the change in the optical density at 460 nm was recorded. The glucose concentration was calculated from the corresponding standard curve

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obtained in this work (r2 = 0.99, Fig. S1B, supplementary document).

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To evaluate the GASA method for determination of salivary glucose concentration (SGC), after drawing 1 mL of the fresh saliva and centrifugation, 50 µL of the resulting supernatant was

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used in the assay solution. The glucose concentration was calculated from the corresponding

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standard curves of Trinder and GASA methods (Fig. S2A and B, supplementary document).

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3. Results and discussion

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3.1. Rationale of the method

The current technologies for the determination of blood glucose are based either on

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electrochemical or photometric methods. Direct oxidation of glucose by GOX has been exploited

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for fabrication of electrochemical sensors to be used in personal digital measurement devices [21]. But, in addition to the susceptibility of the electrochemical methods to some serious interference, the reproducibility of the results declines as the device ages [22]. In contrast to the construction of an electrochemical electrode, making photometric/colorimetric devices are easier and more cost effective [23]. However, the accuracy of these devices relies on the quality of the measurement method as well as the engineering details. Trinder method is one of the routine enzymatic-based techniques, used in diagnostic laboratories [2]. Nonetheless, due to the associated problems mentioned in the Introduction, it has not been

Journal Pre-proof used in miniaturized personal glucometers. Some researchers developed colorimetric methods which are based on the reaction of H2O2 (the by-product of glucose oxidation by GOX) with an indicator dye such as methyl red [24]. The resulting color change can be monitored by a mobile phone and translated to glucose concentration using the red-green-blue (RGB) profiling method. This young technology is promising but the reaction of H2O2 with the dye is not selective. Additionally, the decomposition extent can be immensely varied in the presence of copper, iron

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and calcium ions, especially upon exposure to light [25, 26].

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Considering these challenges, GASA neither falls into auto-oxidation at the required pH (7.6) for

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blood glucose measurements, nor does it show any reaction with H2O2 at pH range of 6 to 8 (Fig.

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2A). Interestingly, the spectra of the enzymatic oxidation products show little overlap with the

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spectrum of GASA (Fig. 2A) as the mechanistic studies suggest benzenesulfonic acid and 2methoxy hydroquinone as the major products of the enzymatic oxidation of this diazo dye by

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POX [27]. Therefore, this method provides precise information about the amount of H2O2

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consumed during the enzymatic oxidation of GASA.

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UV-Visible spectrum of GASA shows two strong peaks at 370 and 460 nm due to the equilibrium between GASA and its anion, Fig. 2A and 2B. The extinction coefficient of the second peak at 460 nm is 9654 M-1 cm-1 (Fig. S3A, supplementary document) which is much higher than that of the quinoid ( = 6500 M-1 cm-1 at 516 nm) [12]. Changing the buffer from PBS to Tris affects only the extinction coefficients of the observed peaks (Fig. S3B, supplementary document). Phenolic compounds carrying electron-donating groups show very fast reactions with POX [27], but the sulfonic group on GASA structure (Fig. 1B) not only makes it soluble in PBS, it also subdues the rate of the enzymatic oxidation, so that the reaction can be directly monitored

Journal Pre-proof through the decrease in the optical density at 460 nm by a simple spectrophotometer (Fig. 2B). Consequently, the whole measurement finishes in less than 20 seconds without being affected by molecular oxygen or light exposure. In addition, sulfonic group increases the solubility of GASA and its decomposition products. This, in turn, reduces potential toxicity of these compounds compared to phenol and 4-AA.

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3.2. Development of the blood glucose assay

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The response of GASA to various amounts of POX in the presence of a constant concentration of H2O2 was linear (Fig. 3A). POX (5 nM = 15 pmol in 3 mL of the reaction

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mixture) was selected for the assay experiments. The GASA response to various amounts of

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H2O2 was also linear with a detection limit of 1.66 M (Fig. 3B). This is equal to 0.0057 mg dL-1

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(0.5 nmol H2O2 in 3 mL of the reaction mixture). Consequently, GASA oxidation by POX (5 nM) was coupled with the oxidation of glucose by GOX (20.83 nM). The selected amounts of

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the enzymes produced linear responses in Trinder method (Fig. S1A supplementary document).

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The responses of both Trinder and GASA methods to the oxidation of different concentrations of

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glucose were linear in a range of 0.36 – 399.6 mg.dL-1 glucose, with a detection limit of 0.36 mg dL-1(Fig. S1A and B supplementary document). This is comparable with the reported limit of detections of other methods (0.17, 0.31 and 0.51 mg. dL-1, See Table S1 in supplementary document) for SGC determination [4, 28] and well below the normal blood glucose concentration (87.95 – 123.85 mg dL-1) [1]. 3.3. Blood glucose measurements To validate the applicability of GASA method, the glucose concentration of 80 serum samples was analyzed by the lab (using Automated Trinder method) and GASA method (Table

Journal Pre-proof S2, supplementary document). The independent t-test of the mean values of the blood glucose concentration of the examined samples by GASA and Trinder methods indicated no significant difference between the data (P > 0.05, See Table S3, supplementary document). Pearson’s analysis of the data (Fig. 4A) revealed a high correlation (r = 0.95) between the lab and GASA measurements. The Bland-Altman plot indicates that more than 93% of GASA results fall between ± 7.21 mg. dL-1 differences with those of the lab (Fig. 4B). Since the analysis of the

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samples by GASA method were carried out after Trinder method, positive errors are more

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abundant in Fig. 4B. This is reflected in the mean values of Trinder (93.59 ± 14.73 mg. dL-1) and

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GASA results (90.93 ± 14.32 mg. dL-1) which means a positive bias of 2.7 mg. dL-1. This is

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3.4.Salivary glucose measurements

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assumingly originated from glycolysis during the delayed time [30].

Salivary diagnostics have been contributed in the early screening and prevention of several

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diseases [31]. Due to the promising potential of saliva applications in personal medicine [32],

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and more importantly the low limit of detection of GASA method (0.36 mg dL-1), the reliability of GASA method was assessed for noninvasively monitoring of SGC. In the present study, unstimulated whole saliva of non-diabetic and diabetic samples was evaluated by both Trinder and GASA methods (Table S4, supplementary document). The samples obtained under fasting condition to minimize the risk of possible interfering substances, especially ascorbic acid and lactic acid, which could have significant impact on pH of saliva [33]. Statistical analysis of the SGC data obtained from GASA and Trinder methods suggested insignificant differences between the results (P > 0.05, See Table S3, supplementary document).The SGC results of both non-diabetic and diabetic individuals were correlated to the corresponding fasting blood sugar

Journal Pre-proof (FBS) values (Fig. 5A and 5B). These figures show a direct correlation between glucose concentrations of blood and saliva; i.e., the diabetic individuals had higher level of salivary glucose concentration. From a technical point of view, there was a reasonable correlation (r = 0.81) between the results of GASA and Trinder tests for the non-diabetic samples, Fig. 5C. Comparing the mean glucose level values of Trinder and GASA results, 1.19 ± 0.53 and 1.16 ± 0.54 mg dL-1 respectively, indicates a low positive bias of 0.03 mg dL-1 for Trinder method. But

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this difference jumps to 0.15 mg dL-1 for the diabetic people as the difference in the mean

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glucose level values were 3.47 ± 0.87 mg dL-1 and 3.32 ± 0.68 mg dL-1 for Trinder and GASA

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results, respectively. No doubt that this is caused by the wide distribution of the diabetic results.

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Nonetheless, the Bland-Altman plot of the results (Fig. 5D) illustrates high level of agreement between the data obtained from Trinder and GASA methods for all diabetic and non-diabetic

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subjects.

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It is important to mention that various mean values have been reported for SGC. Some of them are close to the results of this work and some are dissimilar. For instance, Abikshyeet et al.

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reported 4.2 mg dL-1 [34] while Gupta et al. reported 11.3 mg dL-1 for SGC of the diabetic people

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[35]. This large difference between the reported data is presumably related to the different biological nature of saliva in various individuals. Yet, the results of this work demonstrate that the fasting SGC determination by GASA method can disclose reliably the diabetes risk level.

4. Conclusion Based on the spectrophotometric properties of GASA, a new method was developed for measuring glucose in blood and salivary samples using coupled enzymatic reactions of GOX and POX. The stability of GASA and the products of its enzymatic oxidation as well as its direct

Journal Pre-proof consumption by POX give the method ability to determine accurately the glucose concentration in less than 20 seconds. Considering the fact that in GASA method only the first step of the coupled enzymatic assay of blood glucose measurement has been modified, no interferers except those known for Trinder method is anticipated. Reviewing the increasing demand for precise self-monitoring glucometers at reasonable cost, and the sufficient capability of the GASA method, it is likely that the same chemistry of the suggested method could be applied to devise

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equipment based on photometric technology, especially for non-invasively detection of the

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glucose level in real-time.

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Acknowledgement

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We would like to appreciate the sincere collaboration of Taban Interdisciplinary Diabetes Clinic

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for providing us with the serum samples and the corresponding results of the glucose concentration analysis. Financial support of this research was provided by National Institute of

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Genetic Engineering and Biotechnology (Project number 641). P. Mohammadnejad and S.

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Declaration of interest

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Soleimani Asl contributed equally to this work.

The authors declare that they have no conflicts of interest with the contents of this article.

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Journal Pre-proof Figure 1. A) The product of phenol oxidation by POX in the presence of 4-AA. B) Chemical structures of GASA Figure 2. Absorption spectra of A) GASA (a), GASA + H2O2 (b), GASA + H2O2 + POX (c) in PBS (0.01 M, pH 7) at 20 °C. B) Enzymatic oxidation of GASA in PBS (0.01 M, pH=7.6) at 20 °C.

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Fig. 3. Spectrophotometric responses of GASA during oxidation by A) various amounts of POX

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and a constant amount of hydrogen peroxide (660 µM) and B) various amounts of H2O2 and a

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constant amount of POX (5 nM) in PBS (0.01 M, pH 7.6) at 20 °C. Fig. 4. A) Pearson and B) Bland-Altman plots of the glucose analysis results of 80 blood serum

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samples.

Figure 5. Distribution of SGC results from A) Trinder and B) GASA methods against FBS for

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the diabetic and non-diabetic volunteers. C) Pearson plot of SGC results for the non-diabetic subjects. D) Bland-Altman plot of GASA and Trinder method for SGC results of the diabetic and

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non-diabetic salivary samples.

Journal Pre-proof Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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The authors declare that they have no conflicts of interest with the contents of this article.

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A guaiacol diazo derivative (GASA) was used as substrate in joint-enzymatic assay of glucose Stability and spectrophotometric properties of GASA and its oxidation products allowed accurate blood sugar determination Sensitive, fast and linear spectrophotometric responses of GASA also allowed accurate salivary glucose measurement Direct tracking of GASA oxidation, free of spectral interferences, gives precise glucose concentration GASA method advantages give it the merit to be used in photometric-based personal glucometers

Jo

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