Portable glucose meter-based label-free strategy for target DNA detection

Journal Pre-proof Portable glucose meter-based label-free strategy for target DNA detection Hyo Yong Kim, Jun Ki Ahn, Ki Soo Park, Hyun Gyu Park

PII:

S0925-4005(20)30155-6

DOI:

https://doi.org/10.1016/j.snb.2020.127808

Reference:

SNB 127808

To appear in:

Sensors and Actuators: B. Chemical

Received Date:

28 September 2019

Revised Date:

13 January 2020

Accepted Date:

1 February 2020

Please cite this article as: Kim HY, Ahn JK, Park KS, Park HG, Portable glucose meter-based label-free strategy for target DNA detection, Sensors and Actuators: B. Chemical (2020), doi: https://doi.org/10.1016/j.snb.2020.127808

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier.

Portable glucose meter-based label-free strategy for target DNA detection

a

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

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Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea. b

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

Human Convergence Technology Group, Korea Institute of Industrial Technology

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

To whom correspondence should be addressed.

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*

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05029, Republic of Korea.

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c

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(KITECH), 143 Hanggaul-ro, Sangnok-gu, Ansan 15588, Republic of Korea.

E-mail: [email protected] (H.G. Park); Phone: +82-42-350-3932; Fax: +82-42-350-3910.

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E-mail: [email protected] (K.S. Park); Phone: +82-2-350-3742; Fax: +82-2-350-3742.

Highlights  This is the first report to develop a PGM-based label-free strategy for target DNA detection.



 PCR amplification that consumes the dNTP can be linked to the glucose level.

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This method does not require any tedious steps (e.g. purification step after PCR) that typically make the detection procedure complex, time-consuming, and labor-intensive.

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his method could enable the facile on-site prescreening of target DNA and even other biological markers with the ingenious designs.

Abstract

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We herein developed a portable glucose meter (PGM)-based method for label-free target DNA detection, which relies on the cascade enzymatic reaction (CER) promoted by hexokinase and

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pyruvate kinase to link the amount of deoxynucleoside triphosphate (dNTP) with the glucose

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level. In the absence of target DNA or the presence of non-target DNA, the initial high concentration of dNTP, a phosphate source for hexokinase, is maintained because polymerase

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chain reaction (PCR) amplification is not executed, which results in the significant reduction

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of glucose level by the effective CER process. On the other hand, the PCR amplification works in the presence of target DNA by consuming dNTP and thus CER process cannot be effectively

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executed, leading to the maintenance of initial high glucose level. Finally, the resulting glucose

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level is simply measured by PGM without any tedious and labor-intensive steps. Utilizing the proposed strategy, we successfully analyzed the target genomic DNA derived from Hepatitis B virus (HBV) down to 102 copies with the high selectivity.

Keywords: DNA detection, Portable glucose meter, Hexokinase, Pyruvate kinase, Biosensor

1. Introduction DNA detection has been of great importance because of its broad utility in the areas of molecular genetics, forensic science, and disease diagnostics [1-3]. The current gold standard method for the detection of target DNA is the quantitative polymerase chain reaction (qPCR) that can monitor DNA amplification in real-time [4-9]. However, despite its great sensitivity

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and selectivity, qPCR method has been deployed only in centralized hospitals or research centers due to the requirement of bulky and sophisticated instrument and thus hardly applied

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to the point-of-care (POC) settings [10-15]. Therefore, in the field of molecular diagnostics, there has been a huge demand for a simple prescreening test that enables on-site detection of

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target DNA.

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As one of the most successful POC devices, a portable glucose meter (PGM) with the meritorious features such as portability, cost-effectiveness, and simplicity has been widely

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utilized as the home-kit for self-monitoring of the blood glucose [16-21]. Since the pioneering

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work to utilize PGM for the biomolecule detection by Xiang et al. in 2011, many researchers have focused on adopting PGM for the detection of non-glucose biomarkers including nucleic

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acids, small molecules, and proteins by transducing the selective target binding into the glucose level [22, 23]. The representative example is the PGM-based strategy to detect target DNA by using the invertase-conjugated DNA probe [24]. Only in the presence of target DNA, the invertase-conjugated DNA probe is captured onto the magnetic beads. After the magnetic

separation, the captured invertase on the magnetic beads catalyzes the hydrolysis of sucrose into glucose that is finally measured by PGM. This study was quite meaningful since it was the first report to utilize PGM for the detection of target DNA. However, this method still has some drawbacks that need to be improved. First, it requires the modification of DNA probe on the invertase, which not only makes the assay complicated and tedious but also negatively affects

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the function of the enzymes [25]. Second, system operation was complex and not suitable for POC testing since it involves the isolation of the bound invertase-conjugated DNA probe by magnetic separation, making it difficult to accomplish a simple “mix-and-read” system [26].

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In this study, we developed a PGM-based label-free strategy for target DNA detection,

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which relies on our new finding that PCR amplification that consumes the deoxynucleoside

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triphosphate (dNTP) can be linked to the glucose level. Specifically, our recent work that reports the relationship between the amount of ATP and glucose level by employing hexokinase

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and pyruvate kinase-catalyzed cascade enzymatic reaction (CER) forms the solid basis for this work [27]. With the proposed strategy that overcomes the drawbacks in the previous PGM-

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based DNA detection methods, we successfully determined the target DNA by simply

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measuring the glucose signal utilizing the hand-held PGM without any cumbersome procedures.

2. Material and methods 2.1 Materials All DNA oligonucleotides used in this study were synthesized and purified with high performance

liquid

chromatography

Tris(hydroxymethyl)aminomethane

(HPLC)

(Tris),

by

magnesium

Bioneer® chloride

(Daejeon, (MgCl2),

Korea).

D-glucose,

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phosphoenolpyruvic acid (PEP), hexokinase, pyruvate kinase, and ethidium bromide (EtBr) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The deoxynucleoside triphosphate

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(dNTP) and i-TaqTM DNA polymerase were purchased from New England Biolabs Inc. (Beverly, MA, USA) and Intron Biotechnology Inc. (Daejeon, Korea), respectively. The

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portable glucose meter (PGM) was purchased from Accu-Chek (Roche, Basel, Switzerland).

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Ultrapure DNase/RNase-free distilled water (DW) purchased from Bioneer® was used in all

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experiments. All chemicals used in this study were of analytical grade.

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2.2 Quantitative analysis of PCR product PCR amplification of P gene encoding polymerase in Hepatitis B virus (HBV) genomic DNA

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(gDNA) was first carried out on S1000TM thermal cycler (Bio-Rad, CA, USA) in a total reaction volume of 25 μL containing 100 μM each dNTP, 0.4 μM each primer (forward primer: 5’-CTC GCC AAC TTA CAA GGC-3’ and reverse primer: 5’-CAG AGG TGA AGC GAA GTG-3’), 2.5 U i-TaqTM DNA polymerase, 104 copies target DNA, and 1X PCR buffer (10 mM Tris-HCl

(pH 8.3), 50 mM KCl, and 2 mM MgCl2). The PCR solution was heated up to 95 ˚C for 5 min, followed by different numbers (32 - 40) of PCR cycle consisting of 95 ˚C for 30 s, 55 ˚C for 30 s, and 72 ˚C for 60 s, and finally incubated at 72 ˚C for 5 min. Next, the PCR solution was added to CER solution (25 μL) containing 4 mM D-glucose, 4 mM PEP, 0.2 U/μL hexokinase, 0.2 U/μL pyruvate kinase, and 2X CER buffer (100 mM Tris-HCl (pH 7.4) and 20 mM MgCl2).

measured by PGM.

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2.3 Sensitivity and selectivity for target DNA detection

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After the incubation of the mixture at 30 ˚C for 30 min, the resulting glucose level was

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PCR solution (25 μL) was first prepared by mixing 100 μM each dNTP, 0.4 μM each primer,

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2.5 U i-TaqTM DNA polymerase, varying amounts of target DNA (1 - 104 copies), and 1X PCR buffer (10 mM Tris-HCl (pH 8.3), 50 mM KCl, and 2 mM MgCl2) and then analyzed based on

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the same manner described in ‘2.2 Quantitative analysis of PCR product’ except that PCR cycle

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number was fixed at 40.

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2.4 Gel electrophoresis analysis of PCR product The mixture of 5 μL PCR solution and 1 μL gel electrophoresis loading buffer (6X; Bioneer®) was loaded on 2% agarose gel and the gel electrophoresis was conducted at 135 V for 40 min by using 1X TBE as the running buffer. After staining with EtBr, a gel image was taken and

the band intensities were analyzed for the quantification of PCR products by utilizing Gel

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DocTM EZ Imager (Bio-Rad, CA, USA).

3. Results and discussion 3.1 Principle of PGM-based label-free target DNA detection The working principle of PGM-based label-free target DNA detection is illustrated in Fig. 1, in which CER that is promoted by hexokinase and pyruvate kinase is employed to link the concentration of dNTP with glucose level [27]. In principle, when the target DNA is not present,

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PCR amplification is not executed and the initial high concentration of dNTP is maintained (step 1). Thus, the hexokinase effectively catalyzes the conversion of glucose to glucose-6-

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phosphate (G6P) by consuming dNTP as a phosphate source. In addition, the pyruvate kinase catalyzes the phosphorylation of deoxynucleoside diphosphate (dNDP) that is generated after

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the hexokinase-catalyzed reaction, to regenerate dNTP by converting phosphoenolpyruvic acid

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(PEP) into pyruvate. The regenerated dNTP is again utilized to trigger multiple cycles of CER, resulting in the significant reduction of the glucose level (step 2). On the other hand, in the

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presence of target DNA, dNTP is consumed during PCR amplification and thus CER is not

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effectively executed, leading to the maintenance of initial high glucose level. Finally, the

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resulting glucose level is simply measured by hand-held PGM.

3.2 Feasibility of PGM-based label-free target DNA detection We first characterized the employed PGM by measuring the response at varying concentrations of glucose in the reaction buffer. As evidenced by the results in Fig. S1, the glucose even in the

reaction buffer for the target DNA detection was successfully determined with high reproducibility in the range from 10 to 600 mg/dL with excellent linear relationship (R2 = 0.9985), demonstrating that the buffer condition does not have any interference on the measurement of the glucose level utilizing PGM. Then, we first examined the effect of dNTP to trigger CER by measuring the glucose level from the reaction solutions at different

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conditions. As shown in Fig. 2(a), when the hexokinase was only added into the reaction solution (2), the glucose level was slightly reduced compared to that from the reaction solution without enzymes (1), which could be attributed to the hexokinase-catalyzed conversion of

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glucose to G6P. However, when the hexokinase and pyruvate kinase were simultaneously

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present (3), the glucose level was significantly decreased. These results clearly confirm that

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CER promoted by hexokinase and pyruvate kinase induced the substantial reduction of glucose level. In addition, it was demonstrated that the glucose level was not reduced when dNTP was

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excluded in the reaction solution despite the presence of hexokinase and pyruvate kinase (4), verifying that CER can be triggered only in the presence of dNTP. Importantly, the glucose

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level in the presence of dNTP was changed in the similar degree compared to that of the

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reaction solution with ATP (5), a representative substrate for hexokinase, indicating that CER also can be efficiently promoted by dNTP (p-value = 0.1548 based on unpaired two-tailed ttest). After demonstrating that CER can link dNTP to the glucose level, we next evaluated the

feasibility of PGM-based target DNA detection by measuring the glucose level from the reaction solutions in the absence and presence of target DNA. As a model system, genomic DNA (gDNA) derived from Hepatitis B virus (HBV) was determined with the proposed strategy. The results in Fig. 2(b) show that the difference in the absence and presence of target DNA was negligible when neither hexokinase nor pyruvate kinase were added (1). In addition,

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when the hexokinase was solely added (2), the glucose level in the absence of target DNA was slightly decreased as compared to the one in the presence of target DNA. However, this signal difference was not large enough to detect target DNA. On the other hand, when both hexokinase

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and pyruvate kinase were added (3), the glucose levels in the absence and presence of target

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DNA were clearly distinguished, demonstrating the detection feasibility of this strategy.

3.3 PGM-based quantitative analysis of PCR product

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To achieve the best results for target DNA detection, reaction conditions were optimized by examining the degree of glucose level change defined as L/L0 where L0 and L are glucose levels

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from reaction solutions in the absence and presence of PCR product, respectively. The results

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in Fig. S2, S3, and S4 demonstrate that 50 μM each dNTP, 2 mM glucose, 2 mM PEP, 0.1 U/μL hexokinase, 0.1 U/μL pyruvate kinase, 30 ˚C CER temperature, and 30 min CER time were the optimal conditions for the effective analysis of PCR product. Under the optimized conditions, the capability to quantitatively analyze PCR product

utilizing PGM was validated by measuring glucose level from reaction solutions containing PCR products at different concentrations. To prepare PCR products at different concentrations, the PCR cycle numbers were varied, and their concentrations were estimated by comparing their gel band intensities with that of DNA ladder at known concentration (Fig. S5). The results in Fig. 3 show that the glucose level was proportional to the concentration of PCR products in

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the range from 0 to 75 nM with excellent linear relationship (R2 = 0.9972). It should be noted that the coefficient of variation (CV) values of the glucose levels measured from the reaction solutions containing PCR products at varying concentrations were all less than 10%,

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confirming high reproducibility of this strategy (Table S1) [28]. In addition, importantly, the

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dynamic range of this method is well suited for the analysis of PCR products when considering

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the typical concentration range (10 to 100 nM) after PCR amplification [29, 30].

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3.4 Sensitivity and selectivity of PGM-based label-free target DNA detection The sensitivity for target DNA detection was investigated by measuring the glucose levels from

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the reaction solutions containing target DNA at different copy numbers. As shown in Fig. 4(a),

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the glucose level increased with increasing amount of target DNA. These results are ascribed to the fact that the more target DNA was present, the more dNTP was consumed through the effective PCR amplification (Fig. S6), resulting in the maintenance of initial high glucose level due to the low efficiency of CER. Notably, the glucose level from the reaction solution

containing 102 copies of target DNA was distinguished from the threshold line (red dotted line) defined as M + 3S where M and S are mean and standard deviation of glucose levels obtained from the negative control without target DNA, verifying high sensitivity for target DNA detection of this strategy. The selectivity was then examined by measuring the glucose levels in the presence of

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target and non-target DNAs. The results in Fig. 4(b) show that the high glucose level was maintained only in the presence of target DNA derived from HBV (2) while those of the reaction solutions containing non-target DNAs derived from Escherichia coli (E. coli) (3) and

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Enterococcus faecium (E. faecium) (4) were significantly dropped and thus almost comparable

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to the one in the absence of target DNA (1). Because the PCR primers were designed to be

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specific to the target DNA, the PCR amplification that consumes dNTP occurred only in the presence of target DNA (Fig. S7), and thus the initial high glucose level was maintained due to

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the non-effective execution of CER. These results clearly confirm the high selectivity of this

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strategy.

4. Conclusions In this study, we developed a PGM-based label-free strategy for target DNA detection, which relies on CER promoted by hexokinase and pyruvate kinase. The working principle is quite simple. Only in the presence of target DNA, the PCR amplification is executed by consuming dNTP, and thus CER cannot be effectively executed, resulting in the high glucose level that is

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simply measured by hand-held PGM. It should be noted that this method does not require any tedious steps (e.g. purification step after PCR) that typically make the detection procedure complex, time-consuming, and labor-intensive. With these advantageous features of the

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developed system, the target genomic DNA was determined down to 102 copies with the high

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selectivity. We believe that this method which utilizes a hand-held PGM with simplicity,

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accuracy, cost-effectiveness, and portability as a sensing component could be extensively applied into the facility-limited environment, enabling the facile on-site prescreening of target

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DNA. In addition, we also expect that this technology could be expanded to determine other

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biological markers by simply correlating the concentration of target analyte to the glucose level.

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Declaration of interests

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

Acknowledgements This research was supported by BioNano Health-Guard Research Center funded by the Ministry of Science and ICT (MSIT) of Korea as Global Frontier Project (Grant number HGUARD_2013M3A6B2078964). This research was also supported by the Mid-career

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Researcher Support Program of the National Research Foundation (NRF) funded by the MSIT

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of Korea (NRF-2018R1A2A1A05022355).

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Fig. 1. Schematic illustration of PGM-based label-free strategy for target DNA detection.

Fig. 2. Feasibility of PGM-based label-free target DNA detection. (a) The effect of dNTP on

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the CER process. (1-5) are the reaction solutions containing (1) dNTP only, (2) dNTP and hexokinase, (3) dNTP, hexokinase, and pyruvate kinase, (4) hexokinase and pyruvate kinase,

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and (5) ATP, hexokinase, and pyruvate kinase. The concentrations of glucose, PEP, dNTP, ATP, hexokinase, and pyruvate kinase were 2 mM, 2 mM, 0.1 mM each, 0.4 mM, 0.1 U/μL,

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and 0.1 U/μL, respectively. The CER time was 120 min. The mean and standard deviation

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values were obtained based on the triplicate measurements. (b) The glucose levels in the absence and presence of target DNA. (1-3) are the samples containing (1) no enzymes, (2)

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hexokinase, and (3) hexokinase and pyruvate kinase in the absence (gray color) and presence

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(red color) of target DNA. The concentrations of glucose, PEP, dNTP, hexokinase, and pyruvate kinase were 2 mM, 2 mM, 50 μM each, 0.1 U/μL, and 0.1 U/μL, respectively. The

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CER time was 30 min. The mean and standard deviation values were obtained based on the triplicate measurements.

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Fig. 3. Quantitative analysis of PCR product by measuring glucose level from the reaction

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solutions containing PCR products at different concentrations (0, 7.5, 15, 30, 45, and 75 nM). The concentrations of glucose, PEP, dNTP, hexokinase, and pyruvate kinase were 2 mM, 2 mM,

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50 μM each, 0.1 U/μL, and 0.1 U/μL, respectively. The CER time was 30 min. The mean and

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standard deviation values were obtained based on the triplicate measurements.

Fig. 4. Sensitivity and selectivity of PGM-based label-free target DNA detection. (a) The

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glucose levels from the reaction solutions containing target DNA at different copy numbers. The red dotted line indicates the threshold line defined as M + 3S where M and S are mean and

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standard deviation of glucose level obtained from the negative control without target DNA. The concentrations of glucose, PEP, dNTP, hexokinase, and pyruvate kinase were 2 mM, 2 mM,

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50 μM each, 0.1 U/μL, and 0.1 U/μL, respectively. The CER time was 30 min. The mean and

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standard deviation values were obtained based on the triplicate measurements. (b) The glucose levels from the reaction solutions containing (1) no target DNA, (2) HBV gDNA, (3) E. coli

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gDNA, and (4) E. faecium gDNA. The concentrations of glucose, PEP, dNTP, hexokinase, and

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pyruvate kinase were 2 mM, 2 mM, 50 μM each, 0.1 U/μL, and 0.1 U/μL, respectively. The copy number of target and non-specific DNA was 104. The CER time was 30 min. The mean

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and standard deviation values were obtained based on the triplicate measurements.

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http://doi.org/10.1021/acs.analchem.8b02014.

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and washing-free biomolecular detection, Anal. Chem. 90 (2018) 11340-3.

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[28] M. Mei, X. Huang, K. Liao, D. Yuan, Sensitive monitoring of benzoylurea insecticides in water and juice samples treated with multiple monolithic fiber solid-phase

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microextraction and liquid chromatographic analysis, Anal. Chim. Acta, 860 (2015) 2936. http://doi.org/10.1016/j.aca.2014.12.047.

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[29] Y.K. Jung, T.W. Kim, J. Kim, J.M. Kim, H.G. Park, Universal colorimetric detection

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of nucleic acids based on polydiacetylene (PDA) liposomes, Adv. Funct. Mater. 18 (2008) 701-8. http://doi.org/10.1002/adfm.200700929.

[30] K.S. Park, M.I. Kim, D.Y. Cho, H.G. Park, Label-free colorimetric detection of nucleic acids based on target-induced shielding against the peroxidase-mimicking activity of magnetic nanoparticles, Small, 7 (2011) 1521-5. http://doi.org/10.1002/smll.201001886

Author Biographies

 Dr. Hyun Gyu Park is a professor at the department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science & Technology (KAIST), Korea. He received his Ph.D. in Chemical Engineering from KAIST in 1996. During the past 15 years

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since he joined the Chemical and Biomolecular Engineering Department at KAIST, he has published approximately 150 international papers in prestigious journals including Angewandte, Nucleic Acids Research, Advanced Functional Materials, Small, Chemical

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Communications, Analytical Chemistry, and Biosensors and Bioelectronics and holds 50

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international or domestic patents. His research interests include nucleic acid

and nanobiotechnology.

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bioengineering, microarray technology, electrochemical technology for genetic analysis,

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 Dr. Ki Soo Park is an assistant professor in the department of biological engineering at Konkuk University, Korea. Dr. Park received his Ph.D. in the department of chemical and

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biomolecular engineering from KAIST, Korea. For his outstanding work, he received the

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prestigious national research foundation scholarship from Korea (2014) and also won MGH ECOR Tosteson postdoctoral fellowship award (2016). The primary focus of his research is to develop highly sensitive, fast, and cost-effective diagnostic systems by

bringing together diverse fields, including nanotechnology, molecular biology, and medicine.  Hyo Yong Kim is currently studying for Ph.D. degree in the department of chemical and biomolecular engineering at KAIST. His current research is focused on the development of personal glucose meter (PGM)-based detection methods for the analysis of various

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biomolecules of clinical significance.  Jun Ki Ahn received his Ph.D. in the department of chemical and biomolecular engineering at KAIST in 2018. His research is focused on the development of biomolecular

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detection methods using kinase and nicking enzyme.