A Personal Glucose Meter for Label-Free and Washing-Free

Subscriber access provided by University of South Dakota

Article

A personal glucose meter for label-free and washing-free biomolecular detection Jun Ki Ahn, Hyo Yong Kim, Ki Soo Park, and Hyun Gyu Park Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02014 • Publication Date (Web): 28 Aug 2018 Downloaded from http://pubs.acs.org on August 29, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

A personal glucose meter for label-free and washing-free biomolecular detection

Jun Ki Ahn,a Hyo Yong Kim,a Ki Soo Park* b and Hyun Gyu Park* a

a

Department of Chemical and Biomolecular Engineering (BK21+ Program), KAIST, 291

Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea. b

Department of Biological Engineering, College of Engineering, Konkuk University, Seoul

05029, Republic of Korea

*To whom correspondence should be addressed.

Correspondence: Hyun Gyu Park Ph.D., Department of Chemical and Biomolecular Engineering (BK21+ Program), KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea, Tel.: +82 42-350-3932, Fax: +82 42-350-3910, E-mail: [email protected] Ki Soo Park Ph.D., Department of Biological Engineering, College of Engineering, Konkuk University, Seoul 05029, Republic of Korea, Tel.: +82-2-450-3742, Fax: +82-2-450-3742, E-mail: [email protected]

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Page 2 of 20

Page 3 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ABSTRACT We developed a label-free and washing-free method for biomolecular detection using a personal glucose meter (PGM). As a model target, ATP was selected and cascade enzymatic reactions promoted by hexokinase and pyruvate kinase were adopted to link the amount of ATP to glucose that is detectable by a hand-held PGM. In principle, the presence of target ATP enables hexokinase to catalyze the conversion of glucose to glucose-6phosphate by providing a phosphate group to glucose and thus the amount of glucose is decreased in proportion to the amount of ATP. In addition, the adenosine 5′-diphosphate (ADP), which is generated after hexokinase-catalyzed enzymatic reaction, is recovered to ATP by a pyruvate kinase enzyme. The regenerated ATP is again supplemented to catalyze multiple rounds of cascade enzymatic reactions, leading to the signal amplification. As a result, the change of glucose amount that is inversely proportional to ATP amount is simply measured by a hand-held PGM. By employing this strategy, we successfully determined ATP down to 49 nM with high selectivity even in real samples such as tap water, human serum, and bovine urine. Importantly, the developed system does not require the expensive modification and washing steps, but is conveniently operated with a commercially available PGM, which would pave the way for the development of a simple and cost-effective sensing platform.

Keywords: Adenosine 5’-triphosphate, Personal glucose meter, Glucose, Hexokinase, Pyruvate kinase, Biosensor



1. INTRODUCTION Numerous biological or chemical substances have posed a serious threat to human beings and the demand for decentralized, point-of-care (POC) diagnostic systems has accordingly increased. Among many POC devices that are commercially available on the market, a personal glucose meter (PGM) with high portability, low cost, simple operation, and reliable quantitativity has been regarded as one of the most successful.1 In 2011, Lu’s group proposed a novel strategy to utilize the PGM for the detection of non-glucose targets.2 The method relies on the invertase-conjugated DNA that is released in the presence of analytical targets and function to convert sucrose into glucose that is detectable by a PGM. By adopting suitable functional DNA partners, the authors demonstrated the detection of various non-glucose targets.3,4 However, it inevitably requires DNA modification onto invertase enzyme, which could not only have a negative impact on the structure and activity of the enzyme, but also hinder its universal application because a new invertase-conjugated DNA should be prepared for each target.5 In addition, this strategy involves magnetic separation steps, which could increase hands-on time and reduce reproducibility, consequently preventing widespread applications in facility-limited environments. Since then, many groups tried to utilize a PGM for simply sensing the various target molecules.6-10 However, the most techniques could not solve the problems that modification and separation steps are required. To solve this problem and ensure the versatile utilization of the PGM, we herein proposed a simple method that does not require both tedious modification and separation steps. As a model target to demonstrate this idea, we selected adenosine-5’-triphosphate (ATP), a multifunctional nucleotide that plays important roles in various biological processes including intracellular energy transfer, muscle contraction, biosynthesis, DNA replication,


Page 4 of 20

Page 5 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and membrane

transport and also has clinical importance as the diagnostic markers for

Alzheimer’s diseases and cancers.11-16 For a label-free and washing-free detection in a PGM, the cascade enzymatic reactions promoted by hexokinase and pyruvate kinase were rationally adopted to link the amount of ATP to glucose that is detectable by a PGM. Using this novel strategy, we successfully analyzed ATP with the high selectivity even in tap water, human serum, and bovine urine.

2. EXPERIMENTAL SECTION 2.1 Materials D-glucose, magnesium chloride (MgCl2), Tris(hydroxymethyl) aminomethane hydrochloride (Tris-HCl), β-nicotinamide adenine dinucleotide phosphate hydrate (β-NADP), phosphoenolpyruvic acid (PEP), adenosine 5′-triphosphate disodium salt hydrate (ATP), cytidine 5′-triphosphate disodium salt (CTP), guanosine 5′-triphosphate sodium salt hydrate (GTP), uridine 5′-triphosphate trisodium salt dehydrate (UTP), hexokinase, glucose-6phosphate dehydrogenase (G6PD), pyruvate kinase, human serum, and bovine urine were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were of analytical grade and used without further purification. Aqueous solutions were prepared using ultrapure DNase/RNase-free distilled water (D.W.) purchased from Bioneer®. The brand names of the personal glucose meter (PGM) used in this work were Accu-Chek Aviva (Roche, Basel, Swiss), Green Doctor (Green Cross, Korea), and Acura Plus (i-SENS, Korea). Numerous biological or chemical substances have posed a serious threat to human

2.2. Cascade enzymatic reactions and PGM-based signal detection



18 µL of enzyme mixture containing hexokinase (5 U), pyruvate kinase (5 U), and G6PD (0.4 U) was mixed with 5 µL of D-glucose (50 mM), 1 µL of β-NADP (50 mM), 1 µL of PEP (100 mM), and 5 µL of 10X reaction buffer (1 M Tris-HCl, 100 mM MgCl2, pH 7.4), which was then incubated at 30 °C for 30 min with 20 µL of target ATP at different concentrations or CTP, GTP, and UTP (10 µM). Finally, the resulting glucose level in the mixture was measured by a PGM.

2.3. Electrochemical measurement of a PGM strip The electrode in glucose meter strip (Acura Plus) was used as a working electrode with Ag/AgCl reference electrode and platinum counter electrode. Cyclic voltammetry (CV) was performed using a reference 600 electrochemical analyzer (GAMRY, Warminster, PA, USA) in the range from -0.4 V to 0.1 V using 200 mV s−1 scan rates.

2.4. Recovery test Target ATP at different concentrations was spiked into real samples such as tap water, human serum, and bovine urine, which were then analyzed by following the same detection procedure used in the reaction buffer (2.2.). Only in the case of human serum, the sample diluted 10 times with 1X reaction buffer (100 mM Tris-HCl, 10 mM MgCl2, pH 7.4) was filtered through a centrifugal filter device (MWCO = 30 kDa, Millipore) and the filtrate was used for the recovery test.17 To determine the amount of spiked ATP, the calibration curve was first drawn with a set of standards containing a known amount of ATP in the real samples and the unknown amount of ATP was determined based on the calibration curve.


Page 6 of 20

Page 7 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3. RESULTS AND DISCUSSION 3.1 Detection principle The conceptual design of the PGM-based label-free and washing-free system to detect adenosine 5′-triphosphate (ATP) is illustrated in Scheme 1. The proposed system utilizes cascade enzymatic reactions promoted by hexokinase and pyruvate kinase that link the level of ATP to glucose. In principle, the presence of target ATP allows hexokinase enzyme to catalyze the conversion of glucose to glucose-6-phosphate by providing a phosphate group to glucose and thus the amount of glucose is decreased in proportion to the amount of ATP. In addition, the adenosine 5′-diphosphate (ADP), which is generated after hexokinase-catalyzed enzymatic reaction, is recovered to ATP by a pyruvate kinase enzyme that catalyzes the conversion of phosphoenolpyruvic acid (PEP) to Pyruvate. The regenerated ATP is again supplemented to catalyze multiple rounds of cascade enzymatic reactions, leading to the signal amplification that is manifested by the significantly reduced glucose amount. On the contrary, in the absence of ATP, hexokinase enzyme does not initiate the cascade enzymatic reactions and thus the initial, high glucose amount is retained. As a result, the change of glucose amount is simply measured by a hand-held PGM and used for the quantitative analysis of ATP. First, we tested several PGM devices commercially available on the market to find the best one for biomolecular detection. As shown in Table S1, the PGM device from company Accu-Chek exhibited the highest reproducibility and precision that is evidenced by the lowest standard deviation and coefficient of variation, and thus this device was selected for further experiments. Next, the optimal conditions for the efficient detection of ATP were investigated. The results of experiments in which the concentration of Tris-HCl, MgCl2, and glucose and cascade enzymatic reaction times were varied, demonstrate that 100 mM of Tris-



HCl, 10 mM of MgCl2, and 5 mM of glucose, and 30 min are optimal for the efficient analysis of ATP (Figure S1 and S2). Under the optimal conditions, we verified the detection feasibility of the developed strategy. As envisioned in the schematic illustration (Scheme 1), the presence of target ATP, which initiates the cascade enzymatic reactions promoted by hexokinase and pyruvate kinase, produced the significant PGM signal change (∆P = P0 – P, where P0 and P indicate the PGM signal in the absence and presence of ATP, respectively.) (Figure 1, a). In contrast, when there were no enzymes, the PGM signal was not changed even in the presence of ATP (Figure 1, b). In addition, when hexokinase enzyme was solely present, the PGM signal was changed in the presence of ATP (Figure 1, c), but the signal change was much smaller than that of the sample containing both enzymes (Figure 1, a). These results confirm that the cascade enzymatic reactions by both enzymes are critical for the sensitive detection of ATP. To further support the results obtained on the PGM, the electrochemical experiments were executed to examine cyclic voltammetry responses on the PGM strip. As shown in Figure S3, both anodic and cathodic peak currents that are proportional to the amount of glucose were decreased by the presence of ATP when both hexokinase and pyruvate kinase enzymes were present (a and d). However, when there were either no enzymes (b) or hexokinase only (c), the peak currents were not decreased by the presence of ATP as significantly as the one in the presence of both enzymes (a) (Figure S3). These electrochemical results are consistent with the results obtained on a PGM, clearly demonstrating that target ATP can be sensitively detected by a PGM on the basis of the cascade enzymatic reactions promoted by hexokinase and pyruvate kinase.

3.2. Sensitivity test


Page 8 of 20

Page 9 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The detection sensitivity of the new ATP detection method utilizing a PGM was determined by measuring the PGM signal as a function of ATP concentration. The results in Figure 2 show that the PGM signal change (∆P) increases with increasing concentration of ATP up to 2 µM, and reaches a plateau at concentration over 2 µM. An excellent linear relationship (R2=0.9965) exists in the range from 0.05 to 0.4 µM and the limit of detection (LOD) (3σ/ slope) was ca. 49 nM, which is comparable or higher than those reported for other ATP detection methods (Table S2).18-25 However, it should be noted that the LOD value of our strategy is much lower than that of commercial ATP detection kit (1 µM) and is good enough to detect ATP in the real samples.26 In addition, the developed method based on a hand-held PGM is simply operated without the requirement of complicated modifications and/or magnetic separation steps.

3.3. Selectivity test Next, the detection selectivity of the new ATP detection method was investigated by employing ATP analogs such as UTP, GTP, and CTP and comparing their abilities to induce the PGM signal change (∆P) with that of ATP. As presented in Figure 3, the high PGM signal change (∆P) was obtained from the sample containing ATP. On the other hand, the negligible signal change was observed even though ATP analogs were present at ten times higher than that of ATP. In addition, the PGM signal change in the presence of ATP at the LOD (49 nM) was measured and compared with that of three ATP analogs at the concentration of 10 µM. The observed PGM signal change in the presence of ATP at the concentration of 49 nM was statistically higher than that of ATP analogs (p < 0.0462; unpaired two-tailed t-test), confirming the high selectivity of our ATP detection strategy.



3.4. Real sample test The practical applicability of the new assay system was finally demonstrated by analyzing ATP present in real samples such as tap water, human serum, and bovine urine. As shown in Figure S4, the patterns of PGM signal change (∆P) at different ATP concentrations in the real samples were nearly the same with that obtained in the buffer solution (Figure 2). In addition, the PGM signal change increased with increasing concentration of ATP in real samples and a good linear correlation existed in the range from 0 to 0.4 µM (Figure S4). The excellent reproducibility and precision of our strategy was confirmed by a coefficient of variation (CV) less than 8% and a recovery ratio between 98 and 105% (Table 1). Overall, these results prove that the developed ATP detection system has the potential to reliably determine the amount of ATP present in real samples.22,27-32


Page 10 of 20

Page 11 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


4. CONCLUSIONS We herein developed a label-free and washing-free strategy for biomolecular detection based on a hand-held PGM. As a model target, ATP was selected and the cascade enzymatic reactions promoted by hexokinase and pyruvate kinase were rationally employed for the sensitive detection of ATP on a PGM. This design principle was successfully demonstrated by determining target ATP with high selectivity even in the tap water, human serum, and bovine urine. Importantly, the developed system was simply operated with a PGM, while overcoming the drawbacks in the previous ATP detection methods that require the expensive modification, tedious separation steps, and bulky operation systems. In addition, since a PGM is commercially available on the market at the low cost, it is expected that the developed strategy for ATP detection would be readily adopted in a variety of POC settings. We believe that the developed system could be universally applied to the detection of different target analytes such as small molecules, nucleic acids, and proteins by choosing appropriate enzymatic reactions that link the amount of target analyte to glucose. For example, this system can be applied to the determination of enzyme activity such as alkaline phosphatase that scavenges on ATP, a phosphate source for hexokinase-pyruvate kinase coupling reactions. Finally, it would be a new platform for the convenient and cost-effective biomolecular detection.

ACKNOWLEDGMENTS Financial support was provided by Center for BioNano Health-Guard funded by the Ministry of Science and ICT(MSIT) of Korea as Global Frontier Project (Grant HGUARD_2013M3A6B2078964) and Basic Science Research Program through the National



Research

Foundation

(NRF)

funded

by

the

Page 12 of 20

Ministry

of

Education

(No.

2015R1A2A1A01005393).

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publication website at DOI: x.xxxx/

REFERENCES (1) Carroll, A. E.; Marrero, D. G.; Downs, S. M. Diabetes Technol. Ther. 2007, 9, 158-164. (2) Xiang, Y.; Lu,Y. Nat. Chem. 2011, 3, 697-703. (3) Xiang, Y.; Lu, Y. Anal. Chem. 2012, 84, 4174-4178. (4) Xiang, Y.; Lu, Y. Anal. Chem. 2012, 84, 1975-1980. (5) Park, K. S.; Lee, C. Y.; Park, H. G. ChemComm 2015, 51, 9942-9945. (6) Wang, Q.; Wang, H.; Yang, X.; Wang, K.; Liu, F.; Zhao, Q.; Liu, P.; Liu, R.; ChemComm 2014, 50, 3824-3826. (7) Wang, Q.; Liu, F.; Yang, X.; Wang, K.; Wang, H.; Deng, X. Biosens. Bioelectron. 2015, 64, 161-164. (8) Xue-tao, X.; Kai-yi L.; Jia-ying, Zeng. Biosens. Bioelectron. 2015, 64, 671-675. (9) Huang, H.; Zhao, G.; Dou W. Biosens. Bioelectron. 2018, 107, 266-271. (10) Bai, J.; Liu, L.; Han, Y.; Jia, C.; Liang, C. Anal. Methods. 2018, 10, 2075-2080. (11) Abraham, E. H.; Okunieff, P.; Scala, S.; Vos, P.; Oosterveld, M. J.; Chen, A. Y.; Shrivastav, B.; Guidotti, G. Science 1997, 275, 1324-1326. (12) Gruenhagen, J. A.; Lovell, P.; Moroz, L. L.; Yeung, E. S. J. Neurosci. Methods 2004, 139, 145-152. (13) Patriarca, A.; Eliseo, T.; Sinibaldi, F.; Piro, M. C.; Melis, R.; Paci, M.; Cicero, D. O.; Polticelli, F.; Santucci, R.; Fiorucci, L.; Biochemistry 2009, 48, 3279-3287. (14) Erecińska, M.; Wilson, D. F. J. Membr. Biol. 1982, 70, 1-14. (15) Zhang, C.; Rissman, R. A.; Feng, J. J. Alzheimer's Dis. 2015, 44, 375-378. (16) Singer, S.; Souza, K.; Thilly, W. G. Cancer Res. 1995, 55 (17) Lee, C. Y.; Park, K. S.; Park, H. G. Biosens. Bioelectron. 2017, 98, 210-214. (18) Wang, Y.; He, X.; Wang, K.; Ni, X.; Biosens. Bioelectron. 2010, 25, 2101-2106.


Page 13 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(19) Zhang, Z.; Sharon, E.; Freeman, R.; Liu, X.; Willner, I. Anal. Chem. 2012, 84, 47894797. (20) Kong, L.; Xu, J.; Xu, Y.; Xiang, Y.; Yuan, R.; Chai, Y. Biosens. Bioelectron. 2013, 42, 193197. (21) Chen, L.; Chen, Z.-N. Talanta 2015, 132, 664-668. (22) Huang, X.; Li, Y.; Zhang, X.; Zhang, X.; Chen, Y.; Gao, W. Analyst 2015, 140, 60156024. (23) Wei, Y.; Chen, Y.; Li, H.; Shuang, S.; Dong, C.; Wang, G. Biosens. Bioelectron. 2015, 63, 311-316. (24) Chen, J.; Liu, Y.; Ji, X.; He, Z.; Biosens. Bioelectron. 2016, 83, 221-228. (25) Ji, X.; Yi, B.; Xu, Y.; Zhao, Y.; Zhong, H.; Ding, C. Talanta 2017, 169, 8-12. (26) http://www.abcam.com/atp-assay-kit-colorimetricfluorometric-ab83355.html (27) Saljooqi, A.; Shamspur, T.; Mostafavi, A. Bioelectrochemistry 2017, 118, 161-167. (28) Li, Y.; Sun, L.; Zhao, Q. Talanta 2017, 174, 7-13. (29) Tedsana, W.; Tuntulani, T.; Ngeontae, W. Anal. Chim. Acta 2013, 783, 65-73. (30) Sanghavi, B. J.; Sitaula, S.; Griep, M. H.; Karna, S. P.; Ali, M. F.; Swami, N. S. Anal. Chem. 2013, 85, 8158-8165. (31) Zhu, C.; Zhao, Y.; Yan, M.; Huang, Y.; Yan, J.; Bai, W.; Chen, A. Anal. Bioanal. Chem. 2016, 408, 4151-4158. (32) Zhu, Y.; Hu, X.-c.; Shi, S.; Gao, R.-r.; Huang, H.-l.; Zhu, Y.-y.; Lv, X.-y.; Yao, T.-m. Biosens. Bioelectron. 2016, 79, 205-212.



Table of Content (TOC)


Page 14 of 20

Page 15 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 1. Schematic illustration of a label-free and washing-free ATP detection based on personal glucose meter. (ATP: adenosine 5’-triphosphate, ADP: adenosine 5’-diphosphate, PEP: phosphoenolpyruvate)



Figure 1. Detection feasibility of ATP sensing platform. The PGM signal change (∆P) is defined as P0 – P, where P0 and P indicate the PGM signal in the absence and presence of ATP, respectively. (a: hexokinase and pyruvate kinase, b: no enzymes, c: hexokinse only)


Page 16 of 20

Page 17 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. Detection sensitivity of ATP sensing platform. The PGM signal change (∆P) as a function of ATP concentration. Inset: Linear range between PGM signal change and ATP concentration (0 - 0.4 µM).



Figure 3. Detection selectivity of ATP sensing platform. The concentrations of ATP are 1 µM (red bar) and 49 nM (orange bar) and ATP analogs (CTP, GTP, and UTP) are 10 µM. P value smaller than 0.05 was considered significant.


Page 18 of 20

Page 19 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1. Determination of ATP spiked into tap water, human serum, and bovine urine. a Real sample

Added ATP (µM) Measured ATP (µM) b

SD c

CV (%) d Recovery (%) e

0.30

0.310

0.011

3.70

103.5

0.08

0.084

0.007

8.61

105.5

0.20

0.203

0.010

5.09

101.5

0.08

0.082

0.006

7.70

102.9

0.30

0.297

0.012

4.13

98.9

0.08

0.082

0.006

7.79

102.7

Tap water

Human serum

Bovine urine

a

To determine the concentration of ATP in real samples, a calibration curve was first created

by using standards having known concentration of ATP in each real sample such as tap water, human serum, and bovine urine (Figure S4). Based on this calibration curve, the PGM signals from unknown samples were used to determine the concentration of ATP in real samples. Mean of three measurements.

c

Standard deviation of three measurements.

variation = SD/mean × 100. e Measured value/added value × 100.


d

b

Coefficient of

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


Page 20 of 20

A Personal Glucose Meter for Label-Free and Washing-Free

Recommend Documents