Sensitive Detection of Glycated Albumin in Human Serum Albumin

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Sensitive Detection of Glycated Albumin in Human Serum Albumin using Electrochemiluminescence Yuki Inoue, Mikako Inoue, Masato Saito, Hiroyuki Yoshikawa, and Eiichi Tamiya Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 08 May 2017 Downloaded from http://pubs.acs.org on May 8, 2017

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Analytical Chemistry

Sensitive Detection of Glycated Albumin in Human Serum Albumin using Electrochemiluminescence Yuki Inoue1, Mikako Inoue2, Masato Saito1, Hiroyuki Yoshikawa1, Eiichi Tamiya*1 1. Department of Applied Physics, Graduate School of Engineering, Osaka University, 2-1, Yamadaoka, Suita, Osaka 5652.

0871, Japan Department of Pharmacology and Toxicology, Faculty of Arts and Sciences, University of Toronto, 27 King’s College Circle, Toronto, ON, M5S 1A1, Canada

Fax No.: 06-6879-4087 Email: [email protected]

ABSTRACT: Monitoring of blood glucose content is vital for diabetes patients. Conventional widely used method involves invasive procedure for blood sampling. In addition, blood glucose measured by this way is affected by immediate food consumption and it does not show accurate baseline blood glucose measurement. Thus, monitoring blood glucose by non-invasive method that accurately reflects baseline blood glucose content is important. Glycated albumin (GA), a biomarker for diabetes indicating the average blood glucose over 2 weeks, can be used for semi-long-term blood glucose monitoring. Detection of GA in saliva is a non-invasive method that alleviate the use of needles for diabetic patients, however, its content in saliva is in nanomolar range. Therefore, the GA enzymatic detection method was combined with ECL method for a highly sensitive detection of GA in human serum albumin and in saliva sample. Here, the standard curve was constructed using model substrate, FZK, between 0.1 to 2 µM and GA in human serum albumin was measured in this range. Also, we successfully demonstrated the detection limit of 0.1 µM GA in human serum albumin sample using ECL which has seen improvement of about 70 times than the colorimetric methods. The detection of GA in real saliva sample suggested that sample dilution of 5 times may be necessary to suppress the ECL quenching effect by impurities.

Diabetes mellitus is a metabolic disorder affecting 415 million people world-wide in 2015, and the number is expected to increase to 642 million by 2040. Estimated number of one in two people are undiagnosed with this disease 1. The disease is a group of metabolic disorder characterized by hyperglycemia resulting from dysfunction of insulin secretion, insulin action, or both 2. Patients with diabetes are more likely to suffer from diseases affecting the eyes, nerves, heart, blood vessels, and kidneys. Thus, monitoring of glucose is necessary to avoid complications including, but not limited to, hypothermia, cardiovascular, pregnancy, and kidney diseases. Detection of glycated hemoglobin A1c (HbA1c) is most common for diabetes glycemic control 3, but the life-span of erythrocyte (120 days) hinders its use in short-span monitoring. Furthermore, direct measurement of blood glucose is available by many user-friendly devices offering cheap and fast self-glucose monitoring method, but the blood glucose content is affected immediately after food intake thus it does not accurately represent the base blood glucose content. It rather requires multiple administration during the day for accurate glycemic control, which renders the user with multiple shots of needles causing discomfort for the patient. Glycated albumin (GA) which reflects average blood glucose level over the past 2 weeks can be used for glycemic control in shorter span than HbA1c and has less fluctuation than the direct blood glucose measurement. GA monitoring is collecting attention for glycemic control as it could be a better indicator for assessing changes in serum glucose level in diabetes patients on hemodialysis 4. Non-enzymatic glycation process is a spontaneous post-translational modification by prolonged exposure to re-

ducing carbohydrates such as glucose targeting free amino group in lysine residues and N-terminal of a protein. Glycation site of serum albumin at lys-199, lys-281, lys-439, lys-525, and the N-terminus of Asp-1 are well established for in vivo glycation 5. Blood GA content range in 11-16% to HSA in healthy individuals 6, but the proportion increases to 20-30% in diabetic people 7. Therefore, the increase of GA content can be used as a tool for diagnostic of diabetes. Conventional method of glycated protein detection for glycated hemoglobin and glycated albumin include affinity chromatography 8,9, liquid chromatography 10,11, colorimetry 12–14, and immunochemistry 15. They suffer from disadvantages requiring large and expensive equipment and laborious and time consuming procedures. The method of detection specifically targeting GA in blood albumin serum was introduced by enzyme method discovered by Kouzuma et al. in 2002. The enzyme method uses albumin-specific protease digestion to cleave peptide bonds in serum albumin to individual amino acid residues, then treated with ketoamine oxidase that specifically converts glycated lysine to yield glucosone and H2O2 as product 16. Quantification of reaction product is conducted by conventional colorimetric method using TODB and 4aminoantipyrine (4AA) system measuring absorbance of the oxidized condensation product under presence of peroxidase. The extent of albumin glycation is represented as GA value defined by the percentage of glycated albumin to the total amount of albumin in serum. Commercialized glycated albumin assay kit provides practical mean to GA evaluations in clinical setting 3,17. The enzymatic method is linear in the con-

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centration range of about 10 to 35% of GA in plasma with lower limit at 0.5 g/dL or about 80 µM GA 17. The issue pertaining to measuring endogenous biomolecules mentioned above for glycemic control persists in the use of invasive sampling method to obtain blood for each measurement. Non-invasive method which uses aspirated air or other bodily fluids such as tears, saliva, and urine is sought as ideal method that alleviates the sampling stress for patients. While not requiring invasive extraction, typically, glucose and glycated proteins in saliva 18 and tears 19 is present in two orders of magnitudes lower (micro-molar range for glucose and nanomolar range in glycated proteins) than in blood, however, traditional electrochemical blood glucose monitors have detection range of up to 2.8 mM (50 mg/dL) 20 which prevents its use with these fluids. Sensing of blood glucose using tears with conventional glucose oxidase scheme 21 and fluorescence labeled wearable lens 22,23, and in saliva with plasmonic interferometry 24 have been reported in literature. Electrochemical measurement for drug efficacy characterizing the interaction between HSA and drug could be used as an indirect determination of GA 25, but non-invasive sensor targeting GA has not been reported yet. Here we report the sub-nanomolar determination of GA level in human serum albumin (HSA) using the enzymatic GA assay method combined with electrochemiluminescence (ECL) technique and attempted the detection of GA in real saliva sample. ECL is a luminescence reaction involving electron transfer of luminous species in excited state, and the light emission is recorded once the excited species return to ground state. The reaction between the electro-activated luminol and reactive oxygen species as coreactants can be used for label free detection of H2O2 26, antioxidant capacity 27, enzyme study 28, and cell studies 29. ECL method has favorable features for biosensor design as it can be conducted with simple operation and instrumentation setup, high signal-to-noise ratio, fast output time, and detection limit in sub-nanomolar range. Owing to its high sensitivity, the detection can be conducted with only few microliter of sample. The technique can be performed on disposable screen printed device for fast and contamination-free assay 30,31. In this study, ECL system for GA detection was developed using GA in blood and substrate spiked saliva sample as a model for diabetes patient. This study is for the first time, the GA measured by enzymatic method linked to ECL (Scheme 1). The enzyme used was fructosamine oxidase, and the substrate was FZK (N-fructosyl-N-Z-lysine) as a model for glycated albumin. Once the detection of FZK converted products were measured by ECL method, HSA containing GA was tested with protein digestion. Additionally, this work provides two main features. Firstly, the volume of the assay is less in the proposed method. By using sensitive detection method, the amount of albumin required for the assay is reduced to few microliter order, which saves the amount of protease and sample used. The reduction of sample volume aims to provide less invasive sampling procedure. The sensitivity has increased by 70 times for the detection of GA, which is an improvement that can measure concentration range of GA in saliva sample. The use of ECL method offers advantage in simplification of experimental procedure, where less step is involved in mixing and transferring the reagent and detection can be conducted

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using portable device. This work also suggests the future plan for the development of non-invasive GA detection method. EXPERIMENTAL SECTION Reagents All materials used are of analytical grade. Luminol was purchased from Sigma-Aldrich (MO, USA). FZK, fructosyl amino acid oxidase (FAOx), albumin specific protease, and HSA containing GA were kindly donated by Asahi Kasei Pharma. Saliva sample was obtained by authors with informed consent from the individual. Tris (hydroxymethol) aminomethane buffer (Tris) adjusted to pH8.0 with HCl was used. Luminol was prepared by dissolving in 0.1 mM NaOH solution and stirred until completely dissolved and was stored in aliquot in freezer at -20ºC until use. Instrumentation The ECL monitoring system (BDTeCLP100, Biodevice technology Ltd., Kanazawa, Japan) comprises of a photon detection unit (C9692-12; Hamamatsu Photonics K.K., Hamamatsu, Japan) and an USB-powered hand-held potentiostat (Biodevice technology, Ltd., Kanazawa, Japan). This all-inone ECL system was installed with trigger system which sends trigger signal to the photon detection unit for coordinated electrochemical and photon count measurement. The counting time of the photo detection unit was set to 500 msec. Five basic electroanalytical techniques were installed in the potentiostat which are linear sweep voltammetry (LSV), cyclic voltammetry (CV), chronoamperometry (CA), differential pulse voltammetry (DPV), and square wave voltammetry (SWV). The parameter for LSV measurement was set to scanning rate of 50 mV/s from 0 to 0.7 V. Data analysis was conducted by taking the peak height of the ECL peak signal. Disposable screen printed electrode chips were used for the electrochemical measurements. These electrode chips consist of carbon working electrode, carbon counter electrode, and Ag/AgCl reference electrode. The electrode chip used with the ECL system was a chip with size of 12.5 x 4 mm and the working electrode area was 2.64 mm2. Fructosamine oxidase (FAOx) assay reaction The FAOx enzyme reaction was conducted by first mixing 2 µL of 10 mM luminol and 10 µL FAOx and 88 µL buffer solutions, then followed by addition of equal volume of stated FZK concentrations. Enzyme concentrations were stated in initial concentrations unless specified. The timing of enzyme reaction was timed using digital timer and the timing of loading sample on the electrode chip was carefully controlled for obtaining reproducible performance. The overall GA assay was completed in less than 3 minutes detection time. Albumin digestion assay The albumin sample digestion was conducted by mixing 100 µL of 2570 U/mL protease with 350 µL serum albumin containing GA in 50 µL of 1 M Tris pH8 with incubation time. The digested samples were then used for the FAOx enzyme reaction instead of using FZK. For digestion of human serum albumin, protease was diluted with 1 M Tris to ensure the reaction is buffered near pH8 for optimal condition of the enzyme. Various concentrations of glycated albumin were tested

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bringing the total digestion volume to 500 µL. At this point, the dilution factor of the buffer, protease, and GA were set to be 10, 5, and 1.43, respectively. Sample tubes were placed on block heater set at 37°C and incubated for the duration of 1 hour. To compare the protein digestion, another set of sample tubes were incubated for 3 hours. Following the incubation, sample tubes were cooled to room temperature and 100 µL of the solution was used for FAOx reaction. Saliva sample GA detection The saliva sample was obtained from an individual with informed consent. The total albumin content of serum was quantified using commercial kit (Autowako Micro albumin, Wako pure chemical industries, Osaka, Japan). The preparation of real saliva samples included filtration through several filters (removal of charged molecule and by size) and the sample was labeled as either sample A or AF according to this step. First, the saliva samples were pre-filtered using Salivette (Sarstedt, Numbrecht, Germany) for removing charged molecules and designated as sample A. Then, sample labeled AF was further filtered with size cutoff of 10 K Da to ensure that all large molecules were removed. Subsequently, AF filtrate were spiked with HSA containing known amount of GA. The spiked samples contained 7.5 % (AF 7.5), 15 % (AF 15), 30 % (AF 30) of GA (w/w with respect to HSA), which approximately contain 0.057 µM, 0.11 µM, and 0.23 µM of GA concentration, respectively. Samples A and AF including spiked samples were then undergone protease digestion with 2.5 KU/mL protease at 37°C for 1 hour. After digestion, all the samples (A, AF 7.5, 15, 30) were then filtered through size selective filter with cutoff of 10 K Da. The blank solution contained water instead of saliva using the filter condition of sample A. For spiking experiment, the sample was spiked with initial FZK concentrations of 0.5, 0.1 and 0.05 mM. The final concentrations of FZK in the sample were 20 times dilution of the initial concentration and were calculated to be 25, 5, and 2.5 µM. RESULTS & DISCUSSION ECL profile of GA assay The FAOx enzyme reaction with observed with final concentration of 0.3 mM FZK solution after 1 min incubation time at ambient temperature (Fig. 1). Two ECL peaks were observed at 0 V and 0.4 V applied potential. The first peak resulted from the cathodic ECL reaction and the second peak was the anodic ECL peak representing the luminol oxidation reaction at the working electrode. The anodic ECL peak presented show similar peak to that observed with H2O2 based luminol ECL indicating that the enzyme reaction successfully yielded H2O2 molecules from the FZK substrate. The enzyme reaction is dependent on incubation time and the rate of reaction depends on the amount of FAOx concentrations used. The chronicle change of ECL response to initial concentration of 2 µM FZK was observed in figure 2A where the FAOx enzyme reaction of 0.02 U/mL to 20 U/mL was measured over time. Each measurement was taken with new electrode chip to ensure a contaminant free measurement. The results show that the reaction is reaching near plateau at 20 U/mL and 0.02 U/mL. At 0.2 and 1 U/mL FAOx concentration, the reaction rate is in linear relation with time, whereas

the reaction gradually reach plateau at 2 U/mL concentration. The amount of FAOx concentration was varied from 0.02 to 2000 U/mL to see the reaction dependence to enzyme concentration (Fig. 2B). The inlet shows concentrations represented in log scale to cover wide range of data. The initial amount of FZK tested here was 1 µM. The circle point represents the average ECL response of sample measured after 1 min of reaction obtained in triplet and the error bars show the standard deviation of ECL response obtained in such manner. The incubation time of 1 min was chosen because sufficient ECL signal can be obtained at this incubation time and the assay time is short. The result shown in figure 2B is the enzyme activity profile of FZK conversion in 1 min at different FAOx concentrations. The gradual increase of ECL response showed dependence to FAOx concentration between 0.2 U/mL to 200 U/mL range. At 0.02 U/mL, the ECL response was low likely because the reaction was proceeding very slowly and not enough H2O2 was produced over the 1 min reaction time. The ECL response peaks at 200 U/mL showing all or most FZK were converted to H2O2 at 1 min. However, the ECL response beyond this point drops by 20% probably due to high enzyme protein content in the sample, because high amino acids content is known to quench ECL reaction 32. The luminol based ECL response to FAOx concentration and time was optimized with 200 U/mL and 1 min and the ECL response to various FZK concentrations was tested. The standard curve for FZK was constructed between 0.1 to 2 µM in Tris-HCl buffer pH 8 (Fig. 3). The values represent average ECL response of three individual measurements and the error bars show the standard deviation of three trials. The concentration dependence of FZK showed positive correlation to the ECL peak intensity in the tested range. The lowest detection made was 0.1 µM FZK. The limit of detection defined as 3 times the standard deviation of luminol background was estimated to be 0.1 µM FZK which was about 70 times better than the lower detection range of the conventional colorimetric method. The linear regression line of best fit was y= 1560x + 96.0 with square of the correlation coefficient (R2) value of 0.99. Protease digestion assay As demonstrated by earlier observation of quenched ECL by enzyme, large amount of protein content in sample solution result in the quenching of ECL. Thus, for the determination of protein digestion procedure, the protease concentration had to be considered carefully not to influence the subsequent FAOx reaction and the ECL reaction. In the result presented in figure 4A, various protease concentrations were tested with FZK spiked with 0.3 mM. The protease should not act on FZK and remain intact during the 5 min digestion procedure, and then this sample was added to the mixture of luminol and FAOx and incubated for 1 min before ECL measurement. The effect of protease to the ECL reaction was demonstrated with protease concentrations from 257 KU/mL to 2.57 U/mL. The result shows absolute quenching effect by 257 KU/mL protease where luminol background signal also diminished. Protease concentration of 2570 U/mL showed about 50% quenching and lower concentrations beyond this showed even less quenching effect (Fig. 4B). This result suggests that further dilution of protein content can suppress the quenching effect by protease. Digestion of protein using enzyme reaction is,

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however, a procedure that should be conducted in timely manner and is required to compromise between time and effectiveness. In this study, the protease concentration of 2570 U/mL was chosen in order to maintain a fast albumin digestion step while the ECL quenching effect was within the acceptable range. The digestion of serum albumin with digestion times of 1 hour and 3 hours were compared in figure 5. The initial concentrations of GA tested were ranging from 0.3 µM to 30 µM which were diluted by about 3 times after protease digestion and FAOx reaction, thus final GA concentrations were plotted in figure 5. The ECL response showed concentration dependence of GA and the result indicate successful digestion of albumin and subsequent FAOx reaction. The lowest GA detected was 0.1 µM and the ECL intensity of the tested GA concentration up to 2 µM was the same between 1 hour and 3 hour digestion time meaning that digestion reaction was completed within 1 hour by using 2570 U/mL protease. The ECL intensity of 10 µM GA was higher at 3 hour digestion, this maybe because the incubation time of 1 hour was not enough to digest 10 µM GA which could be considered a substantially large amount compared for the 2570 U/mL of protease used. As for the limit of detection, GA concentrations were measured in the 0 to 10 µM range. The result showed lowest GA measureable at 0.1 µM (6.6 µg/mL), which was lower than the 0.47 mg/mL reported in the colorimetric assay33 or the 50 µg/mL aptamer based fluorescence sensor34 by factor of 70 and 10 times, respectively. Table 1 compared the detection limit of each method. Specificity and interference by intrinsic molecules There are some antioxidant species present in blood plasma known to interfere with the glucose sensor. The specificity and interference by uric acid, ascorbic acid, and glucose to the ECL response of enzymatic reaction was tested. The tested concentrations were determined from the range of healthy individual level. For the specificity test, sample mixture of FAOx, luminol, and tested substrate was prepared. The presence of 0.01 mM uric acid, 7 mM glucose, and 0.08 mM ascorbic acid in the solution showed ECL response of 0.83, 1.40, and 6.70 when the background signal of ECL was assigned as 1, respectively (Table 2). The result suggests that uric acid and glucose does not affect enzyme reaction and ECL intensity, but ascorbic acid increased the background ECL intensity by about 7 times. The interference study, which was prepared with 0.3 mM FZK as the reference ECL intensity, showed the relative ECL intensity of 0.81, 0.93, and 0.83 for uric acid, glucose, and ascorbic acid, respectively (Table 3). The uric acid and ascorbic acid decreased the ECL signal by about 20 %. Diluted samples are often used for assays, and thus the interference effect after thousand times dilution was also tested. The effect of uric acid and ascorbic acid was shown to be suppressed by dilution of the substrate. Presence of glucose in micro molar range did not have negative effect on the outcome of both the enzymatic reaction and the ECL reaction. GA detection in real saliva sample The ECL method of GA detection has been applied to test spiked FZK in real saliva sample. The total albumin content of saliva was determined to be 49.3 µg/mL. The value was used to calculate the amount of GA to be added. Figure 6 shows the

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ECL response graph of Sample AF with three FZK spiking conditions with different diluted solutions of Sample AF. Spiked FZK concentrations of 0.05 mM, 0.1 mM and 0.5 mM were tested at saliva sample from 1 to 100 times dilution. Spiked samples were prepared by diluting saliva samples with Tris buffer by dilution factor and mixed with spiked FZK mentioned above. Then the spiked sample was used for the FAOx enzyme reaction. The final concentration of FZK, in this case was 2.5, 5, and 25 µM, respectively. At 50 and 100 times dilution, the ECL intensity reached the same height as the Tris solution mixed with same concentrations of FZK. The effect of dilution on ECL response followed similar pattern for all the three spiking conditions and they show partial ECL response compared to the Tris condition. The ECL response of 1, 5, and 10 times dilution compared to Tris solution were12±2.6, 61±3.8, 78±2.3%, respectively. The dilution of Sample AF seemed to increase the ECL response. It is possible that the quenching effect by small molecules in saliva to be suppressed once the saliva samples were diluted resulting in the increase of the enzymatic ECL response. The ECL quenching effect by saliva impurities were investigated by observing the ECL and LSV of different saliva sample filtering conditions. Figure 7 shows the ECL and LSV response of the saliva sample filtrate A, AF, GA spiked AF, and blank. AF samples labeled AF 7.5, AF 15, and AF 30 were spiked with HSA and they contained approximately 0.057 µM, 0.11 µM, and 0.23 µM of GA concentration, respectively. The ECL of all samples show ECL peak at the luminol oxidation potential, but the peak height of all samples was not significantly higher than that of the blank which contained water instead saliva (Fig. 7A). The highest concentration of GA tested here contained GA concentration within the detection limit of the ECL method but the ECL peak was suppressed. This may be due to small molecules remaining in the saliva sample after filtration. The LSV voltammogram on figure 7B shows presence of oxidized molecules from the peak at 0.2 V and 0.6 V. The peak at 0.6 V can be seen in blank sample showing that the water sample reacted with filter to form a reductive molecule. In contrast, the peak at 0.2 V was absent in blank sample, suggesting the presence of oxidizable small molecules in saliva that passed through 10 K size filtration. The identity of the impurity is unknown but the quenching effect is substantial for GA detection in saliva sample. The effect of the impurities in both ECL and LSV can be suppressed by simply diluting the saliva sample as shown in figure 7C. The filtered sample A had been diluted by dilution factor of 5 to 100 times. The effect of dilution can be seen even at 5 times dilution of the sample indicated by increase of ECL signal without spiking FZK. This ECL intensity increase was the result of suppressing the quenching effect by the present impurities and not by the reaction with the coreactant species. The average ECL intensity was stable beyond 10 times dilution. This ECL result is in agreement with the obtained LSV result (Fig. 7D). The LSV voltammogram of Sample A showing 1 to 100 times dilution demonstrates that the dilution of sample reduces LSV peak current at 0.2 and 0.6 V. At 5 times dilution, a slight peak can be seen at 0.2 V and slightly higher 0.6 V current than other higher dilutions. The peak at 0.2 V disappears in 10 times dilution and the current at 0.6 V is much lower than the blank. Similarly, ECL response of diluted solutions of Sample AF and AF30 were tested (Fig.

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7C). The results show similar characteristics to the dilution profile of Sample A. In case of Sample AF30, however, because the GA in saliva was also diluted along with impurities, the content of GA became lower than the detection limit of the ECL method even at 5 times dilution. The calculated GA content at 5 times dilution was approximately 0.046 µM and although the ECL intensity of Sample AF30 at 5 times dilution was higher than the same dilution of Sample AF or Sample A, statistically significant difference between the three samples could not be observed. To summarize the detection using real saliva sample, the FZK spiked solution in real saliva sample was detectable once the filtered saliva samples were diluted by more than 50 times. An inadequate dilution of sample show quenching of ECL response, where this quenching effect can be attributed to small molecules in the saliva sample that passed through the size filtration and dilution was necessary in order to suppress the effect of impurities. Partial ECL response can be observed with 5 times dilution, however the GA concentration was also diluted by 5 times which brought the GA concentration of Sample AF30 below the detection limit of the ECL method. As shown in this study, impurity contained in the saliva sample affect the ECL outcome by considerable amount. For the development of saliva based sensing technology, it is necessary to develop a system that comprise a sensitive detection platform and the removal of impurity, which needs more attention in future investigations. Moreover, ECL was advantageous in terms of instrumentation because portable compact size ECL device (115 mm x 125 mm x 185 mm, 4 kg) was available. Considering that test-strip based point-of-care device for measuring GA in serum albumin required optical analyzer (345 mm x 260 mm x 290 mm, 11 kg) for colorimetric reading 35, ECL method offers less reagent handling and smaller instrumentation as a health monitoring platform. CONCLUSION In conclusion, highly sensitive detection of GA in serum albumin sample was detected using ECL method. The ECL profile was first characterized using the FAOx enzyme reaction and the enzyme concentration and reaction time was optimized. Standard curve of FZK was constructed from 0.1 to 2 µM range showing positive correlation to FZK concentration. The protease used in excess showed ECL quenching effect and thus the concentration of protease was compromised between reaction time and ECL outcome. The digestion of serum albumin with protease was compared with 1 hour and 3 hours incubation, and the result showed that the digestion was complete in 1 hour for GA content of 2 µM, but the reaction required 3 hours for digestion of 10 µM GA. The result showed lowest GA measureable at 0.1 µM which was lower than the limit of detection by colorimetric or fluorescence method by factor of 70 and 10 times, respectively. Furthermore, using the ECL method optimized in serum albumin, the detection of GA in saliva sample was attempted. The filtered saliva sample showed quenching of ECL by the small reducing molecules present. The impurities showed less quenching effect when the saliva sample was diluted up to 100 times. The FZK spiked experiment demonstrated that FZK concentration of 2.5 µM can be detected in 50 to 100 times diluted saliva sample. Although the result of the direct GA detection in saliva sample

shown statistically indifferent result compared to control conditions due to dilution of GA content, we were able to demonstrate the potential of detecting GA in sub-nanomolar range for a detection of GA by saliva samples instead of using invasive blood sample. Once the small impurity molecules are separated from saliva sample, the ECL method provide adequate sensitivity for such use. Together with low reagent use and high sensitivity detection, this method opens door for future collaboration with MEMS system or an integrated device for POCT.

Acknowledgement This work was supported by KAKENHI Grant-in-Aid for Scientific Research(S) 15H05769 and CREST, JST. We thank Asahi Kasei Pharma for providing the necessary reagents and Ms. A. Araki for technical support.

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Scheme 1: Glycated albumin assay

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Figure 1: ECL intensity graph of FAOx enzyme reaction with 0.3 mM FZK. ECL measurement conducted after 1 min incubation time (Red). Baseline measurement of luminol ECL (Blue). LSV scan rate of 50 mV/s from 0 to 0.7 V.

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Figure 2: (A) Time dependence of FAOx against incubation time. FZK used was 0.002 mM. (B) FAOx concentration dependence tested with FAOx concentrations from 0.02 to 2000 U/mL. ECL measurement was taken after 1 min incubation time with 0.001 mM FZK.

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Figure 3: Standard curve of FZK constructed using FZK concentrations from 0.1 to 2 µM. FAOx concentration used was 200 U/mL with 1 min incubation time.

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Figure 4: (A) ECL profile of 0.3 mM FZK spiked mixture of different concentrations of protease. (B) Peak height of A plotted against protease concentrations (initial concentration).

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Figure 5: ECL response of protease digestion of HSA containing different concentrations of GA followed by FAOx reaction. Digestion time of 1 hour and 3 hour were tested. ECL measurement was conducted with 1 min incubation time.

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Table 1: Summary of different GA detection methods.

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Table 2: Specificity of FAOx to different substrates.

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Table 3: Interference of different substrates to ECL response.

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Figure 6: Graph of ECL response with different dilution of saliva sample comparing three different FZK spiking conditions. Tris condition signifies that tris buffer is used instead of saliva.

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Figure 7: (A) ECL response of saliva sample prepared by different filtration treatments. Blank signifies that water is used instead of saliva. (B) Representative LSV voltammogram of samples tested in A (C) peak ECL graph of three saliva samples with different dilution conditions (D) Representative LSV voltammogram of Sample A with different dilution factors.

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For TOC only

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Sensitive Detection of Glycated Albumin in Human Serum Albumin

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