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Sensitive detection of albuminuria by graphene oxide-mediated fluorescence quenching aptasensor
Journal Pre-proof Sensitive detection of albuminuria by graphene oxide-mediated fluorescence quenching aptasensor
Wireeya Chawjiraphan, Chayachon Apiwat, Khoonsake Segkhoonthod, Kiatnida Treerattrakoon, Preedee Pinpradup, Nuankanya Sathirapongsasuti, Prapasiri Pongprayoon, Patraporn Luksirikul, Patcharee Isarankura-Na-Ayudhya, Deanpen Japrung PII:
S1386-1425(20)30105-0
DOI:
https://doi.org/10.1016/j.saa.2020.118128
Reference:
SAA 118128
To appear in:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Received date:
30 September 2019
Revised date:
12 January 2020
Accepted date:
3 February 2020
Please cite this article as: W. Chawjiraphan, C. Apiwat, K. Segkhoonthod, et al., Sensitive detection of albuminuria by graphene oxide-mediated fluorescence quenching aptasensor, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy(2020), https://doi.org/10.1016/j.saa.2020.118128
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© 2020 Published by Elsevier.
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Submitted to Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy Sensitive detection of albuminuria by graphene oxide-mediated fluorescence quenching aptasensor Wireeya Chawjiraphana, Chayachon Apiwata,c, Khoonsake Segkhoonthoda, Kiatnida Treerattrakoona, Preedee Pinpradupa, Nuankanya Sathirapongsasutib, Prapasiri Pongprayoonc,d, Patraporn Luksirikulc,d, Patcharee Isarankura-Na-Ayudhyae, Deanpen Japrunga,* a
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*Corresponding author: [email protected] Phone number: +66 2564 6985
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National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), Thailand Science Park, Pathumthani, Thailand b Section for Translational Medicine, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand c Department of Chemistry, Faculty of Science, Kasetsart University, Bangkok, Thailand d Center for Advanced Studies in Nanotechnology for Chemical, Food and Agricultural Industries, KU Institute for Advanced Studies, Kasetsart University, Bangkok, Thailand. e Department of Medical Technology, Faculty of Allied Health Science, Thammasat University, Pathumthani, Thailand
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Abstract
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Albuminuria is a pathological condition wherein the human serum albumin (HSA) protein is present in abnormally excess amounts in the urine. A simple and sensitive graphene oxide-mediated fluorescence quenching aptasensor is developed to quantify albumin in urine samples and HSA in serum samples. The aptamer-bound HSA used in this aptasensor has hairpin structures, which are characteristic of the aptamer binding site. The limit of detection of the developed platform is 0.05 μg.mL-1 and the detection range is 0.1–14.0 μg.mL-1, which covers the albuminuria concentration range present in normal human urine and the urine of the patient with kidney diseases. This approach can be modified to measure HSA using a high-throughput quantification platform and portable point of care testing. In addition, the production cost for one reaction is cheaper than those for other standard automated methods. Therefore, this aptasensor has significant potential for commercialization and wide-scale public use.
Keywords Albuminuria; Human serum albumin; kidney disease; Aptasensor; Graphene oxide; Fluorescence quenching 1. Introduction Albuminuria refers to an abnormal level of human serum albumin (HSA) protein in urine, and is the most common protein involved in blood circulation. It acts as a carrier and helps in maintaining the blood volume and pressure. The glomerulus network inside the kidney acts as a filter to remove the waste products and prevent the passage of HSA. The most common causes of kidney malfunction are diabetes and high blood pressure as these can damage the glomerulus network [1-5]. When the filter network is impaired, HSA passes from the blood into the urine, which is called albuminuria. As the kidney function declines, the amount of albumin in urine increases [2-5]. Therefore, albuminuria detection can be used for the screening and monitoring of kidney malfunction [6].
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The most common method for albuminuria screening includes the use of albumin dipstick employing sulfonephthalein dye that binds to the albumin protein or gold-labeled antibody that binds specifically to the albumin protein and a color is observed on the test line compared to the control line. Although the dipstick platform is simple because of one-step assay and no requirement of a washing step, it has some limitations such as low sensitivity and obtainment of only qualitative or semi-quantitative results. For the quantitative analysis of albuminuria, traditional detection platforms include immunological-based assays such as immunonephelometry, immunoturbidimetry, and radioimmunoassay. These three platforms require anti-human albumin antibody to produce an albumin-antibody binding reaction, followed by the measurements of product scattering, aggregation, and radioactivity [7]. Although the immunological assays are highly sensitive, they involve the use of expensive automated instruments.
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In this study, a novel sensitive detection platform is developed to quantify albuminuria using a graphene oxide (GO)-mediated fluorescence quenching aptasensor. This developed platform used the specific target binding property of the selected aptamer, which is short single stranded DNA or RNA (<100 bases), combining with the fluorescence quenching property of the graphene oxide.
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The limit of detection (LOD) of this aptasensor is 120-fold lower than that of the standard immunoturbidimetry assay. The detection of albuminuria in clinical urine samples is successfully demonstrated using a portable fluorometer, which can potentially be used as a commercial point of care testing (POCT) device in local hospitals and remote areas.
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2. Materials and methods 2.1 Materials
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The albumin binding aptamer used in this study was the 87-nucleotide ssDNA, which was selected based on our previous work [8], and was an HPLC grade material synthesized by Integrated DNA Technologies, Singapore. The sequence of the DNA aptamer was 5’/Cy5/ATA CCA GCT TAT TCA ATT CCC CCG GCT TTG GTT TAG AGG TAG TTG CTC ATT ACT TGT ACG CTC CGG ATG AGA TAG TAA GTG CAA TCT/3’. The fluorescence-labeled aptamers were dissolved in sterile water to make a stock solution of 100 μM, which was aliquoted and kept at -20 °C until future use. The purified human albumin (A9731) was purchased from Sigma-Aldrich (St. Louis, Missouri, USA). The GO monolayer powder was prepared using Hummers method and dissolved in sterile water to obtain 5-mg.mL-1 solution as described in our previous study [9]. The phosphate buffered saline (PBS) consisted of 137 mmol.L-1 NaCl, 2.7 mmol.L-1 KCL, 10 mmol.L-1 Na2HPO4, and 1.8 mmol.L-1 KH2PO4 (pH 7.4), and was autoclaved at 121 °C for 15 min and kept at room temperature until use.
2.2 Secondary structure prediction The secondary structure of albumin-binding aptamer was predicted using the Mfold web Server [10-11]. The folding temperature was set at 25 °C and other parameters included (1) [Na+] concentration of 1 M, (2) linear DNA sequence, (3) percent sub-optimality of 5, and (4) an upper limit on the number of computed folding of 50. 2.3 Optimization of aptamer concentration As the aptamer used in this study was different from our previous report [9], the concentration of aptamer used in the system was first optimized. For optimization, 15 μL of 5-mg.mL-1 GO was mixed with 15 μL of the H8 aptamer of various concentrations (0–10 μM) and incubated at room
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temperature (25 °C) for 5 min in the dark. Thereafter, 2 μL of 3-mg.mL-1 HSA was added into the mixed complex, the volume was adjusted to 200 μL, and the fluorescence was measured. The optimal aptamer concentration was the concentration that completely quenched the fluorescence intensity of the system. 2.4 Binding affinity study
𝐹𝑜𝑏−𝐹𝑚𝑖𝑛
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The binding affinity of the aptamer to the HSA protein was determined using the modified version of the protocol described in our previous work [9]. In brief, 15 μL of 5-mg.mL-1 GO was incubated with 15 μL of 5-μM fluorescence-labeled aptamer (H8) at room temperature in the dark for 5 min to form the GO–aptamer complex. Then, 2 μL of HSA with concentration between 5 mg.mL-1 and 70 mg.mL-1 (stock concentration) was added to the GO–aptamer complex and PBS was immediately added to obtain a final volume of 200 μL. After incubation at room temperature in the dark for 30 min, the fluorescence intensity was measured with excitation at 630 nm and emission at 670 nm using a Quantus portable fluorometer (Promega Corp., Madison, Wisconsin, USA). The percentage of fluorescence response (F) was calculated using equation 1.
𝐹𝑚𝑎𝑥−𝐹𝑚𝑖𝑛
Eq. 1
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Where Fob is the fluorescence intensity at various HSA concentrations (5–70 mg.mL-1), Fmin is the fluorescence intensity of the aptamer-bound GO (GO–aptamer complex), which is the negative control, and Fmax is the maximum fluorescence intensity. The percentage of fluorescence response is plotted against the HSA concentrations, and the equilibrium dissociation constant (Kd) is obtained by fitting the data directly using nonlinear least squares analysis [12]. [𝐻𝑆𝐴]
𝐾𝑑+[𝐻𝑆𝐴]
Eq. 2
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Fraction saturation of HSA =
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Equation 2 reveals that the concentration of HSA required to bind half of the aptamer (fractional saturation or percentage of fluorescence response) is equal to Kd, which can be read directly from a binding curve.
2.5 Aptasensor performance for HSA measurement The GO-mediated fluorescence quenching aptasensor was modified compared to our previous study [9]. In brief, the fluorescence-labeled aptamer was bound to GO and the fluorescence signal was quenched. When the target molecule was added to the system, the fluorescence-labeled aptamer detached the GO to bind to the target molecule and the fluorescence signal was recovered (Figure 1).
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Figure 1: Schematic of graphene oxide-mediated fluorescence quenching aptasensor for the detection of albuminuria in urine and HSA in human serum.
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ΔF = 𝐹𝑜𝑏 − 𝐹𝑚𝑖𝑛
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In this study, the target molecule was HSA and the aptamer was the 87-nucleotide DNA aptamer (H8). The optimized protocol was similar to that of the binding affinity study and the subtracted fluorescence intensity (ΔF) was obtained (Eq. 3). Eq. 3
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The sensitivity of the developed aptasensor was determined by obtaining the ΔF values upon adding various concentrations of the purified HSA protein (0.01–14.0 g.mL-1) in the system. The ΔF values were plotted against the concentrations of HSA, linear regression equation was applied, and the LOD (3.3 × SD/Slope) was calculated.
2.6 Preparation of clinical samples
Urine samples: Thirty-nine urine samples were collected from the Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand, under the Research Network of NANOTEC (RNN) program with the MTA agreement (P1851893, ethical approval no. COA. MURA2019/796). The random spot urine samples were collected in sterile screw cap tubes, aliquoted, and stored at −80 °C until these were used further. Serum samples: Sixty-seven blood samples (leftover specimens) were collected from the Department of Medical Technology, Faculty of Allied Health Science, Thammasat University, Pathumthani, Thailand (Ethical approval no. COA. MURA2019/796). The samples were drawn into the red topped tubes with no anticoagulant (Vacuette, LabNet International, Inc., Edison, New Jersey, USA) and centrifuged at 5000 g for 10 min to obtain a clear supernatant serum. Then, the serum samples were aliquoted and stored at −80 °C, until further use. 2.7 Evaluation of the aptasensor for HSA detection in clinical samples The HSA concentrations in the serum and urine samples were analyzed using the standard method used in the hospital and compared with the corresponding results of the aptasensor developed in this study.
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In brief, the HSA concentrations in the serum samples were measured using the automated chemiluminescence method (Architech i200sr, Abbot Laboratories, Chicago, Illinois, USA) and the developed aptasensor using the same protocol as described in section 2.4. Considering the albuminuria or HSA concentrations in the urine samples, the HSA concentrations were measured using the immunoturbidimetry method (Architect i2000SR, Abbott Laboratories) and the results were compared with those obtained by the aptasensor platform using the same protocol as described in section 2.4.
2.8 Statistical analysis
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The HSA concentrations detected by the standard automated methods were compared with the values obtained from the developed aptasensor. The Pearson correlation coefficients (r) and P values were determined using the SPSS Statistics version 20.0 software and Origin version 6.0 software. The correlation of two data sets was determined based on the P value. If the P values were < 0.01, these two data sets were considered to be statistically significantly correlated. Contrarily, if the P values were ≥ 0.01, these data sets were not considered to be correlated.
3.1 Characteristics of aptamer-bound HSA protein
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3. Results and Discussion
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The aptamer used in this study was the 87-nucleotide ssDNA aptamer (H8), which was selected based on our previous studies [8, 13]. The MFold program [10-11] was used to predict the secondary structure of the selected aptamer. The results show the formation of 3-stem-loop structures or hairpin structures (Figure 2a), which is one of the characteristics of the aptamer [14]. The stemloop formation stabilizes the ssDNA and RNA structures and assists in the binding to the target molecule. The energy required (ΔG) to form this secondary structure is calculated as -8.23 kcal.mol-1; the minus value of ΔG indicates that this structure can be formed at room temperature.
Figure 2: Characteristics of H8 aptamer-bound HSA. (a) Secondary structure of the H8 aptamer predicted by the Mfold program showing the formation of the 3-hairpin structure. (b) Binding affinity of the H8 aptamer to HSA.
Considering the binding affinity of the H8 aptamer to the HSA protein, the percentage of fluorescence response was plotted against the concentration of the purified HSA protein, and Kd was calculated using equation 2. The Kd value of the aptamer-bound HSA is 0.9 μmol (60 μg), which is in the same range as those for other aptamers that are bound to their target molecules, detected using UV-vis spectroscopy [15].
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These results confirm that the H8 aptamer is bound to the HSA protein with the Kd value similar to those obtained in other studies of the aptamer. In addition, the H8 aptamer forms a secondary structure, which is a characteristic aptamer property. 3.2 Performance of the GO-mediated fluorescence quenching aptasensor
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The results of the optimization study show that 15 μL of 5-μM aptamer concentration completely quenches the fluorescence intensity (Figure S1). Therefore, all experiments in this study are conducted using 5-μM aptamer concentration (stock solution). Simulation studies indicate that the aptamer prefers to bind to the subdomain IIIB of the HSA protein (unpublished results) owing to the positive charge of this subdomain. Therefore, it is assumed that one molecule of the aptamer binds one HSA molecule on this subdomain. Based on the calculation of the maximum binding capacity of the aptamer, the result indicates that the aptasensor can analyze the HSA protein up to a maximum amount of 75 μmol (5 mg). Thus, it can be used for the detection of HSA in urine and serum without sample dilution because the albuminuria concentration in normal urine is usually lower than 30 ng.μL1 [7] and the HSA protein in normal serum is 30–50 μg.μL-1 [7]. For the incubation time, the saturation of fluorescence intensity starts at 25 min and is kept constant until 90 min. Therefore, 30min incubation times are used in all experiments. As the pH of human urine is 4–10, the pH values of the sensor system were measured after the addition of 2 μL of samples with pH of 4, 7, and 10 in the system and it was determined that the pH values of the present system were not significantly different (Table S1). This indicates that the pH of 4–10 does not significantly affect the aptasensor system. Considering the sensitivity and specificity of the developed aptasensor, the calibration curve (Figure 3) using the purified HSA protein shows that the fluorescence intensity is linearly correlated with HSA concentration between 0.1–14 μg.mL-1 (R2 = 0.989) with an LOD of 0.05 μg.mL-1 (3.3 × SD/Slope) [16], which is 120-fold lower than the LOD of the standard immunoturbidimetry method (LOD of 6 μg.mL-1) [7]. These results indicate that this sensor has a high sensitivity and noble capability for HSA measurement in urine samples, which usually contain very low concentrations of HSA (<30 μg.mL-1) [17]. The specificity of the H8 aptamer has been determined and described in our previous work [13], which indicates high specific binding with HSA and insignificant response to small molecules in blood and urine (glucose, glycine, folic acid, and ampicillin) samples. HSA and bovine serum albumin (BSA) have similar structures with 76% sequence similarity and BSA has been used as a blocking buffer to prevent the nonspecific binding in several detection platforms [18]. Therefore, the specificity of the developed aptasensor is evaluated for the detection of HSA and BSA. The aptasensor described in this study is five times more specific to HSA than BSA at 150-μg.mL-1 protein concentration (supplementary information, Figure S2). Although the results show very low response of BSA in the aptasensor platform (<20%), BSA-containing buffer is excluded to prevent interference in the system.
Figure 3: Calibration curve of HSA (0.0001–0.014 mg.mL-1) derived from graphene oxide-mediated fluorescence quenching aptasensor (R2=0.989).
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3.3 Evaluation of GO-mediated fluorescence quenching aptasensor in clinical samples
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The concentration of HSA in normal urine is lower than 30 ng.μL-1, which is 1000 times lower than HSA present in the human serum (30–50 μg.μL-1) [7]. The high concentration of albuminuria is usually because of the HSA leakage from blood to urine, which can be caused by a chronic kidney disease. The results show that the developed aptasensor has a high sensitivity (LOD of 0.05 μg.mL-1) and specificity for albuminuria detection without requiring sample dilution. Herein, the sensor performance was compared with that of the standard immunoturbidimetry automated method used in hospitals by testing 39 urine samples. The results show that albuminuria concentrations detected by the standard automated method are 0.003–1.42 mg.mL-1, whereas those detected by the developed aptasensor are 0.01–1.06 mg.mL-1 (Figure 4a). Two groups of data are observed, which include the albuminuria concentrations detected by the standard automated method at ≥0.1 and <0.1 mg.mL-1. Considering the HSA concentrations of ≥0.1 mg.mL-1, and using the SPSS and Origin software to analyze the Pearson correlation coefficients (r) and P values, the results show that the albuminuria concentrations determined by this developed aptasensor are significantly correlated with the data obtained by the standard automated method with P<0.01 (r=0.95). For the HSA concentrations of <0.1 mg.mL-1, the albuminuria concentrations obtained by the method proposed in this study are better differentiated than the values derived from the standard automated methods due to the lower LOD of the aptasensor. These results are in agreement with the results of the previous studies [7, 19-22], in which different detection platforms afford different values but are significantly correlated at certain HSA concentrations. The aptasensor capability to afford the differentiation of the HSA concentrations lower than 0.1 mg.mL-1, which are critical values in normal control, can be applied to monitor albuminuria before it progresses to the chronic kidney disease stage.
Figure 4: Correlation of the HSA concentrations measured by graphene oxide-mediated fluorescence quenching aptasensor with those obtained by the standard methods. (a) Correlation of HSA in urine and (b) serum samples analyzed by the developed method (y-axis) to the results obtained by the standard automated method (x-axis). The optimized sensor was also evaluated by measuring HSA in 67 serum samples and these were compared with the values obtained by the standard automated chemiluminescence method. The HSA concentrations analyzed by the aptasensor are 26.0–52.1 mg.mL-1, which significantly correlate
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with the HSA concentrations obtained by the standard automated method (12.0–51.0 mg.mL-1) with P<0.01 (r=0.745) (Figure 4b). These results indicate that this platform can enable the measurement of HSA in both serum and urine samples without sample dilution. As this aptasensor system requires small amounts of both GO (15 μL of 5 mg.mL-1) and aptamer (15 μL of 5 μM), the production cost, which includes direct material, direct labor, and manufacturing overhead, is calculated to evaluate the potential for commercialization. The cost to produce one reaction is $0.3, which is 6-10 times cheaper than those for automated methods. In addition, this platform can be applied to measure HSA using both 96- or 365-well plate fluorometer and portable fluorometer (Figure S3). These results indicate that the aptasensor has considerable potential for commercialization and POCT. 4. Conclusions
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In summary, a simple, cheap, and sensitive aptasensor is developed to quantify albumin in urine and serum. The sensitivity of our platform is sufficiently high to detect albumin in urine and serum without sample dilution. In addition, it can be applied for high-throughput HSA or albuminuria detection and as a portable POCT device. As the production cost is low, it has a significant potential for commercialization. A portable fluorometer with mobile application for the analysis of albuminuria and HSA is in development and will be reported in future publications.
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Declaration of competing interest
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The authors declare no competing financial interests or personal relationships that influence the work reported in this paper.
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Acknowledgements
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References
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This work was supported by the National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), Thailand [grant number P1751330]. We also acknowledge the Research Network NANOTEC program (RNN-Ramathibodi hospital and RNNKasersart University).
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Submitted to Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Supporting Information Sensitive detection of albuminuria by graphene oxide-mediated fluorescence quenching aptasensor Wireeya Chawjiraphana, Chayachon Apiwata,c, Khoonsake Segkhoonthoda, Kiatnida Treerattrakoona, Preedee Pinpradupa, Nuankanya Sathirapongsasutib, Prapasiri Pongprayoon c,d, Patraporn Luksirikulc.d, Patcharee Isarankura-Na-Ayudhyae, Deanpen Japrunga,* a
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*Corresponding author: [email protected] Phone number: +66 2564 6985
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National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), Thailand Science Park, Pathumthani, Thailand b Section for Translational Medicine, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand c Department of Chemistry, Faculty of Science, Kasetsart University, Bangkok, Thailand d Center for Advanced Studies in Nanotechnology for Chemical, Food and Agricultural Industries, KU Institute for Advanced Studies, Kasetsart University, Bangkok, Thailand. e Department of Medical Technology, Faculty of Allied Health Science, Thammasat University, Pathumthani, Thailand
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Table 1: pH values of the developed aptasensor in comparison with the pH values of the standard solution Aptasensor system pH 7.28 7.36 7.50
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Standard solution pH 4.0 7.0 10.0
Figure S1: Effect of aptamer concentration on the fluorescence intensity of the graphene-aptamer complex when 15 μL of 5 mg.mL-1 GO is incubated with 15 μL of 0–10-μM aptamer at room temperature for 5 min.
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Figure S2: Specificity of the graphene oxide-mediated fluorescence quenching aptasensor toward HSA in comparison with BSA.
Figure S3: Portable fluorometer and solutions (aptamer, graphene, and PBS buffer) used in the graphene oxide-mediated fluorescence quenching aptasensor.
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Journal Pre-proof Credit author statement Deanpen Japrung: Supervision, Conceptualization, Funding acquisition, Writing - Review & Editing Wireeya Chawjiraphan: Methodology, Writing - Original Draft, Writing - Review & Editing Chayachon Apiwat: Methodology, Resources Khoonsake Segkhoonthod: Resources, Formal analysis Kiatnida Treerattrakoon: Resources, Formal analysis Preedee Pinpradup: Resources, Formal analysis Nuankanya Sathirapongsasuti: Resources, Formal analysis, Original Draft
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Prapasiri Pongprayoon: Resources, Formal analysis, Original Draft Patraporn Luksirikul: Resources, Formal analysis, Original Draft
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Patcharee Isarankura-Na-Ayudhya: Resources, Original Draft
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Journal Pre-proof Graphical abstract
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Highlights Albuminuria and HSA are quantitatively analyzed by simple and sensitive aptasensor Aptasensor KidneyLOD is 1200-fold lower than that of standard immunoturbidimetry method Analysis results correlate well with those obtained by standard automated methods malfunction Aptasensor has considerable potential for POCT and commercialization
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Urine
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Wireeya Chawjiraphan, Chayachon Apiwat, Khoonsake Segkhoonthod, Kiatnida Treerattrakoon, Preedee Pinpradup, Nuankanya Sathirapongsasuti, Prapasiri Pongprayoon, Patraporn Luksirikul, Patcharee Isarankura-Na-Ayudhya, Deanpen Japrung PII:
S1386-1425(20)30105-0
DOI:
https://doi.org/10.1016/j.saa.2020.118128
Reference:
SAA 118128
To appear in:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Received date:
30 September 2019
Revised date:
12 January 2020
Accepted date:
3 February 2020
Please cite this article as: W. Chawjiraphan, C. Apiwat, K. Segkhoonthod, et al., Sensitive detection of albuminuria by graphene oxide-mediated fluorescence quenching aptasensor, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy(2020), https://doi.org/10.1016/j.saa.2020.118128
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Submitted to Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy Sensitive detection of albuminuria by graphene oxide-mediated fluorescence quenching aptasensor Wireeya Chawjiraphana, Chayachon Apiwata,c, Khoonsake Segkhoonthoda, Kiatnida Treerattrakoona, Preedee Pinpradupa, Nuankanya Sathirapongsasutib, Prapasiri Pongprayoonc,d, Patraporn Luksirikulc,d, Patcharee Isarankura-Na-Ayudhyae, Deanpen Japrunga,* a
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*Corresponding author: [email protected] Phone number: +66 2564 6985
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National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), Thailand Science Park, Pathumthani, Thailand b Section for Translational Medicine, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand c Department of Chemistry, Faculty of Science, Kasetsart University, Bangkok, Thailand d Center for Advanced Studies in Nanotechnology for Chemical, Food and Agricultural Industries, KU Institute for Advanced Studies, Kasetsart University, Bangkok, Thailand. e Department of Medical Technology, Faculty of Allied Health Science, Thammasat University, Pathumthani, Thailand
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Abstract
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Albuminuria is a pathological condition wherein the human serum albumin (HSA) protein is present in abnormally excess amounts in the urine. A simple and sensitive graphene oxide-mediated fluorescence quenching aptasensor is developed to quantify albumin in urine samples and HSA in serum samples. The aptamer-bound HSA used in this aptasensor has hairpin structures, which are characteristic of the aptamer binding site. The limit of detection of the developed platform is 0.05 μg.mL-1 and the detection range is 0.1–14.0 μg.mL-1, which covers the albuminuria concentration range present in normal human urine and the urine of the patient with kidney diseases. This approach can be modified to measure HSA using a high-throughput quantification platform and portable point of care testing. In addition, the production cost for one reaction is cheaper than those for other standard automated methods. Therefore, this aptasensor has significant potential for commercialization and wide-scale public use.
Keywords Albuminuria; Human serum albumin; kidney disease; Aptasensor; Graphene oxide; Fluorescence quenching 1. Introduction Albuminuria refers to an abnormal level of human serum albumin (HSA) protein in urine, and is the most common protein involved in blood circulation. It acts as a carrier and helps in maintaining the blood volume and pressure. The glomerulus network inside the kidney acts as a filter to remove the waste products and prevent the passage of HSA. The most common causes of kidney malfunction are diabetes and high blood pressure as these can damage the glomerulus network [1-5]. When the filter network is impaired, HSA passes from the blood into the urine, which is called albuminuria. As the kidney function declines, the amount of albumin in urine increases [2-5]. Therefore, albuminuria detection can be used for the screening and monitoring of kidney malfunction [6].
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The most common method for albuminuria screening includes the use of albumin dipstick employing sulfonephthalein dye that binds to the albumin protein or gold-labeled antibody that binds specifically to the albumin protein and a color is observed on the test line compared to the control line. Although the dipstick platform is simple because of one-step assay and no requirement of a washing step, it has some limitations such as low sensitivity and obtainment of only qualitative or semi-quantitative results. For the quantitative analysis of albuminuria, traditional detection platforms include immunological-based assays such as immunonephelometry, immunoturbidimetry, and radioimmunoassay. These three platforms require anti-human albumin antibody to produce an albumin-antibody binding reaction, followed by the measurements of product scattering, aggregation, and radioactivity [7]. Although the immunological assays are highly sensitive, they involve the use of expensive automated instruments.
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In this study, a novel sensitive detection platform is developed to quantify albuminuria using a graphene oxide (GO)-mediated fluorescence quenching aptasensor. This developed platform used the specific target binding property of the selected aptamer, which is short single stranded DNA or RNA (<100 bases), combining with the fluorescence quenching property of the graphene oxide.
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The limit of detection (LOD) of this aptasensor is 120-fold lower than that of the standard immunoturbidimetry assay. The detection of albuminuria in clinical urine samples is successfully demonstrated using a portable fluorometer, which can potentially be used as a commercial point of care testing (POCT) device in local hospitals and remote areas.
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2. Materials and methods 2.1 Materials
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The albumin binding aptamer used in this study was the 87-nucleotide ssDNA, which was selected based on our previous work [8], and was an HPLC grade material synthesized by Integrated DNA Technologies, Singapore. The sequence of the DNA aptamer was 5’/Cy5/ATA CCA GCT TAT TCA ATT CCC CCG GCT TTG GTT TAG AGG TAG TTG CTC ATT ACT TGT ACG CTC CGG ATG AGA TAG TAA GTG CAA TCT/3’. The fluorescence-labeled aptamers were dissolved in sterile water to make a stock solution of 100 μM, which was aliquoted and kept at -20 °C until future use. The purified human albumin (A9731) was purchased from Sigma-Aldrich (St. Louis, Missouri, USA). The GO monolayer powder was prepared using Hummers method and dissolved in sterile water to obtain 5-mg.mL-1 solution as described in our previous study [9]. The phosphate buffered saline (PBS) consisted of 137 mmol.L-1 NaCl, 2.7 mmol.L-1 KCL, 10 mmol.L-1 Na2HPO4, and 1.8 mmol.L-1 KH2PO4 (pH 7.4), and was autoclaved at 121 °C for 15 min and kept at room temperature until use.
2.2 Secondary structure prediction The secondary structure of albumin-binding aptamer was predicted using the Mfold web Server [10-11]. The folding temperature was set at 25 °C and other parameters included (1) [Na+] concentration of 1 M, (2) linear DNA sequence, (3) percent sub-optimality of 5, and (4) an upper limit on the number of computed folding of 50. 2.3 Optimization of aptamer concentration As the aptamer used in this study was different from our previous report [9], the concentration of aptamer used in the system was first optimized. For optimization, 15 μL of 5-mg.mL-1 GO was mixed with 15 μL of the H8 aptamer of various concentrations (0–10 μM) and incubated at room
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temperature (25 °C) for 5 min in the dark. Thereafter, 2 μL of 3-mg.mL-1 HSA was added into the mixed complex, the volume was adjusted to 200 μL, and the fluorescence was measured. The optimal aptamer concentration was the concentration that completely quenched the fluorescence intensity of the system. 2.4 Binding affinity study
𝐹𝑜𝑏−𝐹𝑚𝑖𝑛
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The binding affinity of the aptamer to the HSA protein was determined using the modified version of the protocol described in our previous work [9]. In brief, 15 μL of 5-mg.mL-1 GO was incubated with 15 μL of 5-μM fluorescence-labeled aptamer (H8) at room temperature in the dark for 5 min to form the GO–aptamer complex. Then, 2 μL of HSA with concentration between 5 mg.mL-1 and 70 mg.mL-1 (stock concentration) was added to the GO–aptamer complex and PBS was immediately added to obtain a final volume of 200 μL. After incubation at room temperature in the dark for 30 min, the fluorescence intensity was measured with excitation at 630 nm and emission at 670 nm using a Quantus portable fluorometer (Promega Corp., Madison, Wisconsin, USA). The percentage of fluorescence response (F) was calculated using equation 1.
𝐹𝑚𝑎𝑥−𝐹𝑚𝑖𝑛
Eq. 1
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Where Fob is the fluorescence intensity at various HSA concentrations (5–70 mg.mL-1), Fmin is the fluorescence intensity of the aptamer-bound GO (GO–aptamer complex), which is the negative control, and Fmax is the maximum fluorescence intensity. The percentage of fluorescence response is plotted against the HSA concentrations, and the equilibrium dissociation constant (Kd) is obtained by fitting the data directly using nonlinear least squares analysis [12]. [𝐻𝑆𝐴]
𝐾𝑑+[𝐻𝑆𝐴]
Eq. 2
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Fraction saturation of HSA =
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Equation 2 reveals that the concentration of HSA required to bind half of the aptamer (fractional saturation or percentage of fluorescence response) is equal to Kd, which can be read directly from a binding curve.
2.5 Aptasensor performance for HSA measurement The GO-mediated fluorescence quenching aptasensor was modified compared to our previous study [9]. In brief, the fluorescence-labeled aptamer was bound to GO and the fluorescence signal was quenched. When the target molecule was added to the system, the fluorescence-labeled aptamer detached the GO to bind to the target molecule and the fluorescence signal was recovered (Figure 1).
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Figure 1: Schematic of graphene oxide-mediated fluorescence quenching aptasensor for the detection of albuminuria in urine and HSA in human serum.
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ΔF = 𝐹𝑜𝑏 − 𝐹𝑚𝑖𝑛
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In this study, the target molecule was HSA and the aptamer was the 87-nucleotide DNA aptamer (H8). The optimized protocol was similar to that of the binding affinity study and the subtracted fluorescence intensity (ΔF) was obtained (Eq. 3). Eq. 3
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The sensitivity of the developed aptasensor was determined by obtaining the ΔF values upon adding various concentrations of the purified HSA protein (0.01–14.0 g.mL-1) in the system. The ΔF values were plotted against the concentrations of HSA, linear regression equation was applied, and the LOD (3.3 × SD/Slope) was calculated.
2.6 Preparation of clinical samples
Urine samples: Thirty-nine urine samples were collected from the Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand, under the Research Network of NANOTEC (RNN) program with the MTA agreement (P1851893, ethical approval no. COA. MURA2019/796). The random spot urine samples were collected in sterile screw cap tubes, aliquoted, and stored at −80 °C until these were used further. Serum samples: Sixty-seven blood samples (leftover specimens) were collected from the Department of Medical Technology, Faculty of Allied Health Science, Thammasat University, Pathumthani, Thailand (Ethical approval no. COA. MURA2019/796). The samples were drawn into the red topped tubes with no anticoagulant (Vacuette, LabNet International, Inc., Edison, New Jersey, USA) and centrifuged at 5000 g for 10 min to obtain a clear supernatant serum. Then, the serum samples were aliquoted and stored at −80 °C, until further use. 2.7 Evaluation of the aptasensor for HSA detection in clinical samples The HSA concentrations in the serum and urine samples were analyzed using the standard method used in the hospital and compared with the corresponding results of the aptasensor developed in this study.
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In brief, the HSA concentrations in the serum samples were measured using the automated chemiluminescence method (Architech i200sr, Abbot Laboratories, Chicago, Illinois, USA) and the developed aptasensor using the same protocol as described in section 2.4. Considering the albuminuria or HSA concentrations in the urine samples, the HSA concentrations were measured using the immunoturbidimetry method (Architect i2000SR, Abbott Laboratories) and the results were compared with those obtained by the aptasensor platform using the same protocol as described in section 2.4.
2.8 Statistical analysis
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The HSA concentrations detected by the standard automated methods were compared with the values obtained from the developed aptasensor. The Pearson correlation coefficients (r) and P values were determined using the SPSS Statistics version 20.0 software and Origin version 6.0 software. The correlation of two data sets was determined based on the P value. If the P values were < 0.01, these two data sets were considered to be statistically significantly correlated. Contrarily, if the P values were ≥ 0.01, these data sets were not considered to be correlated.
3.1 Characteristics of aptamer-bound HSA protein
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3. Results and Discussion
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The aptamer used in this study was the 87-nucleotide ssDNA aptamer (H8), which was selected based on our previous studies [8, 13]. The MFold program [10-11] was used to predict the secondary structure of the selected aptamer. The results show the formation of 3-stem-loop structures or hairpin structures (Figure 2a), which is one of the characteristics of the aptamer [14]. The stemloop formation stabilizes the ssDNA and RNA structures and assists in the binding to the target molecule. The energy required (ΔG) to form this secondary structure is calculated as -8.23 kcal.mol-1; the minus value of ΔG indicates that this structure can be formed at room temperature.
Figure 2: Characteristics of H8 aptamer-bound HSA. (a) Secondary structure of the H8 aptamer predicted by the Mfold program showing the formation of the 3-hairpin structure. (b) Binding affinity of the H8 aptamer to HSA.
Considering the binding affinity of the H8 aptamer to the HSA protein, the percentage of fluorescence response was plotted against the concentration of the purified HSA protein, and Kd was calculated using equation 2. The Kd value of the aptamer-bound HSA is 0.9 μmol (60 μg), which is in the same range as those for other aptamers that are bound to their target molecules, detected using UV-vis spectroscopy [15].
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These results confirm that the H8 aptamer is bound to the HSA protein with the Kd value similar to those obtained in other studies of the aptamer. In addition, the H8 aptamer forms a secondary structure, which is a characteristic aptamer property. 3.2 Performance of the GO-mediated fluorescence quenching aptasensor
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The results of the optimization study show that 15 μL of 5-μM aptamer concentration completely quenches the fluorescence intensity (Figure S1). Therefore, all experiments in this study are conducted using 5-μM aptamer concentration (stock solution). Simulation studies indicate that the aptamer prefers to bind to the subdomain IIIB of the HSA protein (unpublished results) owing to the positive charge of this subdomain. Therefore, it is assumed that one molecule of the aptamer binds one HSA molecule on this subdomain. Based on the calculation of the maximum binding capacity of the aptamer, the result indicates that the aptasensor can analyze the HSA protein up to a maximum amount of 75 μmol (5 mg). Thus, it can be used for the detection of HSA in urine and serum without sample dilution because the albuminuria concentration in normal urine is usually lower than 30 ng.μL1 [7] and the HSA protein in normal serum is 30–50 μg.μL-1 [7]. For the incubation time, the saturation of fluorescence intensity starts at 25 min and is kept constant until 90 min. Therefore, 30min incubation times are used in all experiments. As the pH of human urine is 4–10, the pH values of the sensor system were measured after the addition of 2 μL of samples with pH of 4, 7, and 10 in the system and it was determined that the pH values of the present system were not significantly different (Table S1). This indicates that the pH of 4–10 does not significantly affect the aptasensor system. Considering the sensitivity and specificity of the developed aptasensor, the calibration curve (Figure 3) using the purified HSA protein shows that the fluorescence intensity is linearly correlated with HSA concentration between 0.1–14 μg.mL-1 (R2 = 0.989) with an LOD of 0.05 μg.mL-1 (3.3 × SD/Slope) [16], which is 120-fold lower than the LOD of the standard immunoturbidimetry method (LOD of 6 μg.mL-1) [7]. These results indicate that this sensor has a high sensitivity and noble capability for HSA measurement in urine samples, which usually contain very low concentrations of HSA (<30 μg.mL-1) [17]. The specificity of the H8 aptamer has been determined and described in our previous work [13], which indicates high specific binding with HSA and insignificant response to small molecules in blood and urine (glucose, glycine, folic acid, and ampicillin) samples. HSA and bovine serum albumin (BSA) have similar structures with 76% sequence similarity and BSA has been used as a blocking buffer to prevent the nonspecific binding in several detection platforms [18]. Therefore, the specificity of the developed aptasensor is evaluated for the detection of HSA and BSA. The aptasensor described in this study is five times more specific to HSA than BSA at 150-μg.mL-1 protein concentration (supplementary information, Figure S2). Although the results show very low response of BSA in the aptasensor platform (<20%), BSA-containing buffer is excluded to prevent interference in the system.
Figure 3: Calibration curve of HSA (0.0001–0.014 mg.mL-1) derived from graphene oxide-mediated fluorescence quenching aptasensor (R2=0.989).
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3.3 Evaluation of GO-mediated fluorescence quenching aptasensor in clinical samples
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The concentration of HSA in normal urine is lower than 30 ng.μL-1, which is 1000 times lower than HSA present in the human serum (30–50 μg.μL-1) [7]. The high concentration of albuminuria is usually because of the HSA leakage from blood to urine, which can be caused by a chronic kidney disease. The results show that the developed aptasensor has a high sensitivity (LOD of 0.05 μg.mL-1) and specificity for albuminuria detection without requiring sample dilution. Herein, the sensor performance was compared with that of the standard immunoturbidimetry automated method used in hospitals by testing 39 urine samples. The results show that albuminuria concentrations detected by the standard automated method are 0.003–1.42 mg.mL-1, whereas those detected by the developed aptasensor are 0.01–1.06 mg.mL-1 (Figure 4a). Two groups of data are observed, which include the albuminuria concentrations detected by the standard automated method at ≥0.1 and <0.1 mg.mL-1. Considering the HSA concentrations of ≥0.1 mg.mL-1, and using the SPSS and Origin software to analyze the Pearson correlation coefficients (r) and P values, the results show that the albuminuria concentrations determined by this developed aptasensor are significantly correlated with the data obtained by the standard automated method with P<0.01 (r=0.95). For the HSA concentrations of <0.1 mg.mL-1, the albuminuria concentrations obtained by the method proposed in this study are better differentiated than the values derived from the standard automated methods due to the lower LOD of the aptasensor. These results are in agreement with the results of the previous studies [7, 19-22], in which different detection platforms afford different values but are significantly correlated at certain HSA concentrations. The aptasensor capability to afford the differentiation of the HSA concentrations lower than 0.1 mg.mL-1, which are critical values in normal control, can be applied to monitor albuminuria before it progresses to the chronic kidney disease stage.
Figure 4: Correlation of the HSA concentrations measured by graphene oxide-mediated fluorescence quenching aptasensor with those obtained by the standard methods. (a) Correlation of HSA in urine and (b) serum samples analyzed by the developed method (y-axis) to the results obtained by the standard automated method (x-axis). The optimized sensor was also evaluated by measuring HSA in 67 serum samples and these were compared with the values obtained by the standard automated chemiluminescence method. The HSA concentrations analyzed by the aptasensor are 26.0–52.1 mg.mL-1, which significantly correlate
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with the HSA concentrations obtained by the standard automated method (12.0–51.0 mg.mL-1) with P<0.01 (r=0.745) (Figure 4b). These results indicate that this platform can enable the measurement of HSA in both serum and urine samples without sample dilution. As this aptasensor system requires small amounts of both GO (15 μL of 5 mg.mL-1) and aptamer (15 μL of 5 μM), the production cost, which includes direct material, direct labor, and manufacturing overhead, is calculated to evaluate the potential for commercialization. The cost to produce one reaction is $0.3, which is 6-10 times cheaper than those for automated methods. In addition, this platform can be applied to measure HSA using both 96- or 365-well plate fluorometer and portable fluorometer (Figure S3). These results indicate that the aptasensor has considerable potential for commercialization and POCT. 4. Conclusions
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In summary, a simple, cheap, and sensitive aptasensor is developed to quantify albumin in urine and serum. The sensitivity of our platform is sufficiently high to detect albumin in urine and serum without sample dilution. In addition, it can be applied for high-throughput HSA or albuminuria detection and as a portable POCT device. As the production cost is low, it has a significant potential for commercialization. A portable fluorometer with mobile application for the analysis of albuminuria and HSA is in development and will be reported in future publications.
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Declaration of competing interest
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The authors declare no competing financial interests or personal relationships that influence the work reported in this paper.
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Acknowledgements
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References
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This work was supported by the National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), Thailand [grant number P1751330]. We also acknowledge the Research Network NANOTEC program (RNN-Ramathibodi hospital and RNNKasersart University).
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Submitted to Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Supporting Information Sensitive detection of albuminuria by graphene oxide-mediated fluorescence quenching aptasensor Wireeya Chawjiraphana, Chayachon Apiwata,c, Khoonsake Segkhoonthoda, Kiatnida Treerattrakoona, Preedee Pinpradupa, Nuankanya Sathirapongsasutib, Prapasiri Pongprayoon c,d, Patraporn Luksirikulc.d, Patcharee Isarankura-Na-Ayudhyae, Deanpen Japrunga,* a
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*Corresponding author: [email protected] Phone number: +66 2564 6985
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National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), Thailand Science Park, Pathumthani, Thailand b Section for Translational Medicine, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand c Department of Chemistry, Faculty of Science, Kasetsart University, Bangkok, Thailand d Center for Advanced Studies in Nanotechnology for Chemical, Food and Agricultural Industries, KU Institute for Advanced Studies, Kasetsart University, Bangkok, Thailand. e Department of Medical Technology, Faculty of Allied Health Science, Thammasat University, Pathumthani, Thailand
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Table 1: pH values of the developed aptasensor in comparison with the pH values of the standard solution Aptasensor system pH 7.28 7.36 7.50
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Standard solution pH 4.0 7.0 10.0
Figure S1: Effect of aptamer concentration on the fluorescence intensity of the graphene-aptamer complex when 15 μL of 5 mg.mL-1 GO is incubated with 15 μL of 0–10-μM aptamer at room temperature for 5 min.
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Figure S2: Specificity of the graphene oxide-mediated fluorescence quenching aptasensor toward HSA in comparison with BSA.
Figure S3: Portable fluorometer and solutions (aptamer, graphene, and PBS buffer) used in the graphene oxide-mediated fluorescence quenching aptasensor.
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Journal Pre-proof Credit author statement Deanpen Japrung: Supervision, Conceptualization, Funding acquisition, Writing - Review & Editing Wireeya Chawjiraphan: Methodology, Writing - Original Draft, Writing - Review & Editing Chayachon Apiwat: Methodology, Resources Khoonsake Segkhoonthod: Resources, Formal analysis Kiatnida Treerattrakoon: Resources, Formal analysis Preedee Pinpradup: Resources, Formal analysis Nuankanya Sathirapongsasuti: Resources, Formal analysis, Original Draft
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Prapasiri Pongprayoon: Resources, Formal analysis, Original Draft Patraporn Luksirikul: Resources, Formal analysis, Original Draft
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Patcharee Isarankura-Na-Ayudhya: Resources, Original Draft
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Journal Pre-proof Graphical abstract
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Highlights Albuminuria and HSA are quantitatively analyzed by simple and sensitive aptasensor Aptasensor KidneyLOD is 1200-fold lower than that of standard immunoturbidimetry method Analysis results correlate well with those obtained by standard automated methods malfunction Aptasensor has considerable potential for POCT and commercialization
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