Multiplex real-time loop-mediated isothermal amplification using an

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Multiplex real-time loop-mediated isothermal amplification using an electrochemical DNA chip consisting single liquid flow channel Koji Hashimoto, Mika Inada, and Keiko Ito Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05284 • Publication Date (Web): 08 Feb 2019 Downloaded from http://pubs.acs.org on February 9, 2019

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Analytical Chemistry

Multiplex real-time loop-mediated isothermal amplification using an electrochemical DNA chip consisting single liquid flow channel Koji Hashimoto*, Mika Inada, Keiko Ito Corporate Research & Development Center, Research & Development Division, Toshiba Corporation, 1 Komukai-Toshibacho, Saiwai-ku, Kawasaki 212-8582, Japan ABSTRACT: We developed a multiplex system capable of simultaneously quantifying different target sequences by applying an electrochemical DNA chip that consists of single liquid flow channel with primers designed for loop-mediated isothermal amplification (LAMP). We applied this system for detecting mature microRNAs (miRNAs). miRNAs extracted from serum were enzymatically lengthened to about 100 base pairs by reverse transcription and elongation reactions. The LAMP primers for amplifying the lengthened miRNAs were adsorbed and immobilized on the surface of the liquid flow channel at five different positions. A LAMP solution containing the lengthened miRNAs, Tin DNA polymerase, and ruthenium hexaamine (RuHex) as a redox compound was injected into the DNA chip. The electrochemical reaction of RuHex in the LAMP solution was then measured continuously via linear sweep voltammetry at 65 °C. The LAMP reaction of the positive control revealed that the cathodic peak current of RuHex increased. Additionally, the initial number of miRNA copies was correlated with the time when the cathodic current began to increase. Five miRNAs were simultaneously detected at 103–106 copies/50 µL within 2 h. We expect these results will be useful for developing a simple and stable electrochemical-based method for the real-time monitoring of miRNAs, while also facilitating the implementation of electrochemical DNA chips for molecular analyses.

techniques, including PCR,12,22-24 rolling circle amplification,18 and LAMP,15-17, 19-22 to achieve a highly sensitive detection and a relatively wide dynamic range. Nevertheless, multiplex assays developed with these techniques still require a complex system involving multi-well plates13,14 and microchamber16 that are used to maintain individual reaction solutions. For multiplex detection, the use of universal primers is well known for PCR25 and LAMP26 however these methods usually has a risk for nonspecific amplification. The new DNA chip described herein was designed to enable a multiplex LAMP reaction in a simple device that is easy to operate. The primers are adsorbed at different positions on the surface of a liquid flow channel on a DNA chip. After a LAMP solution containing polymerase, dNTPs and lengthened (preamplified) products followed by reverse transcription (RT) and elongation (EL) reaction of miRNA is injected into the DNA chip, the primers gradually dissociate from their binding site and diffuse in the liquid flow channel, after which a specific LAMP reaction occurs only around the positions where the primers were spotted. Thus, a multi-well plate and microchamber are unnecessary for a multiplex assay. Moreover, the use of a single liquid flow channel simplifies and stabilizes this method. In this study, we developed a multiplex assay involving a new electrochemical DNA chip for the simultaneous quantification of multiple mature miRNAs extracted from serum. EXPERIMENTAL SECTION Materials and Reagents. Commercially available human sera were purchased from Kohji Bio Co., Ltd. and Kishida Chemical Co., Ltd. miRNAs were extracted from serum using the

Circulating microRNAs (miRNAs) in serum are promising cancer biomarkers. Additionally, quantitative real-time polymerase chain reaction (qRT-PCR) assays,1,2 microarrays,3,4 and next-generation sequencing5,6 have been used to analyze the expression levels of miRNAs extracted from serum. However, these technologies are time-consuming, complicated, and require expensive equipment. Thus, loop-mediated isothermal amplification (LAMP) has been developed as a viable alternative for detecting miRNAs. The target miRNA-initiated LAMP reaction,7 the base stacking LAMP,8 and the targettriggered LAMP9 quantify miRNAs in a simple one-step operation. However, the optical detection system necessary for monitoring the LAMP reaction is relatively expensive because of the cost of the fluorescent dye used for the highly sensitive detection. There has been increasing interest in the application of electrochemical methods for detecting nucleic acids to overcome the drawbacks associated with existing methods involving optical detection. These methods are expected to enable portable and inexpensive multiplex testing. In the mid1990s, electrochemical nucleic acid detection methods were based on the hybridization between a probe-immobilized electrode and the target nucleic acid.10,11 However, the required probe immobilization was complex and the dynamic concentration range was narrow. Recently developed real-time electrochemical nucleic acid detection methods involve a redox compound [osmium compound,12,13 methylene blue,14-16 methylene blue derivatives13,17 and ruthenium hexaamine (RuHex)18-21] without the need for an immobilized probe. These methods involve enzyme-mediated DNA amplification

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miRCURY™ RNA Extraction Kit (Exiqon, MA, USA). The five miRNAs used as targets were hsa-miR-16-5p, hsa-miR191-5p, and hsa-miR-423-5p, which are highly expressed in serum,27 and hsa-miR-1246 and hsa-miR-1307-3p, which are breast cancer biomarkers.28 Details regarding the primers and miRNA used for the reverse transcription (RT), elongation (EL), PCR, and LAMP reaction are listed in Table S-1. All oligonucleotides were synthesized by Life technologies Japan and FASMAC (Kanagawa, Japan). A qRT-PCR was performed with the miRCURY LNA Universal RT microRNA PCR system (Exiqon) for hsa-miR-1246 and the TaqMan Advanced miRNA assay kit (Thermo Fisher, MA, USA) for the other miRNAs. All other chemicals used were of analytical grade. The electrochemical DNA chip, which comprised the electrode substrate, a liquid flow channel (1 mm × 1 mm × 60 mm) made from a silicone module, and a plastic cassette (Fig. S-1), was purchased from Toshiba Hokuto Electronics, Co. Ltd. (Hokkaido, Japan).29 The electrode substrate was consisted 30 regions with working electrodes (Φ = 200 µm), counter electrodes and reference electrodes patterned on a Pyrex glass substrate (22 mm × 21 mm × 0.8 mm). All electrodes were made of gold. Extraction and amplification of miRNAs. The miRNAs were extracted from 300 µL serum samples according to the manufacturer’s protocol, ultimately resulting in 30 µL miRNA solutions. The principle underlying the amplification of miRNAs based on RT, EL, and the LAMP reaction is presented in Figure 1. First, miRNAs were lengthened by the RT and EL reactions. The RT primer contained a sequence that is complementary to part of the 3’ end of the miRNA sequence and the B2 region of the backward inner primer (BIP) for the LAMP reaction. The RT reaction was completed in a 20 µL solution containing 2 µL extracted miRNA, 67 U MultiScribe™ reverse transcriptase (Thermo Fisher), and 5 nM RT primer.

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The RT program was as follows: 16 °C for 10 min and then 42°C for 5 min. The RT reaction synthesized the complementary DNA sequence for each miRNA with the BIP sequence. Meanwhile, the EL primer included part of the 5’ end of the miRNA sequence, the B1c region of the BIP, and the F1 and F2 regions of the forward inner primer (FIP). The EL reaction required the addition of a 5 µL solution containing 0.5 U Deep Vent DNA polymerase (New England Biolabs, MA, USA) and 5 nM EL primer to the completed RT reaction solution. The mixture was heated at 95 °C for 2 min followed by 20 cycles of a pre-asymmetric amplification step (95 °C for 20 s, 55°C for 30 s, and 72°C for 10 s). The resulting lengthened sequence was in a double-stranded form, and included the F2, F1, B1c, miRNA, and B2c sequences (from the 5’ to 3’ direction). Finally, the LAMP reaction was completed using a 25 µL solution containing 1 µL lengthened sequence, 1.6 µM FIP and BIP, 0.8 µM loop primer (Lp), 1.4 mM dNTPs, 20 mM Tris-HCl (pH 8.0), 60 mM KCl, 10 mM (NH4)2SO4, 8 mM MgSO4, 0.1% Tween 20, 0.8 M betaine, and 0.4 U/µL Tin DNA polymerase (Optigene, UK). The solution was heated at 65 °C for 60 min. A real-time turbidity curve was constructed based on the data from the LT-16 turbidity meter (Nippon Gene Co., Ltd., Toyama, Japan). The amplification time (Tt) was defined as the time when the signal crossed the turbidity threshold of 1,000 [d(turbidity)/dt]. PCR was carried out using a 20 µL solution containing 1.5 µL lengthened sequence, 1× Premix Ex Taq™ (Takara Bio Inc., Shiga, Japan), and 0.3 µM forward and reverse primers. The PCR was completed using the SimpliAmp™ Thermal Cycler (Thermo Fisher), with the following program: 98 °C for 2 min; 35 cycles of 98 °C for 10 s, 59 °C for 30 s, and 72 °C for 15 s. The PCR products were analyzed with the E-Gel Electrophoresis system (Thermo Fisher). Preparation of an electrochemical DNA chip for multiplex real-time LAMP. The FIP and BIP (16 µM each) and the Lp (8 µM) were dissolved in 10 mM Tris-HCl (pH 7.5) containing 0.002% xylene cyanol, after which 0.2 µL was spotted on the surface of the liquid flow channel and dried at room temperature. The primers were immobilized by adsorption at five different positions. The silicone module and the glass substrate, which contained the electrode, were assembled with the plastic cassette. The completed DNA chip was vacuum sealed and stored at −20 °C before use. Fluorescence imaging of the LAMP reaction. The fluorescence image of the LAMP reaction in the DNA chip was examined using the Typhoon 8620 image analyzer (GE Healthcare, IL, USA). The transparent glass substrate was used for the imaging experiment instead of the electrode substrate covered with resin. A 50-µL LAMP solution containing the lengthened products, 1.4 mM dNTPs, 20 mM Tris-HCl (pH 8.0), 60 mM KCl, 10 mM (NH4)2SO4, 8 mM MgSO4, 0.1% Tween 20, 0.8 M betaine, 0.4 U/µL Tin DNA polymerase, and EvaGreen® fluorescent dye (Biotium Inc., CA, USA) was diluted 200 times and then injected into the DNA chip using a manual pipet. Fluorescence was measured with an excitation wavelength of 532 nm and a 526 SP emission filter. The LAMP reaction was completed at 65 °C using a customized holder (Corning® LSE™ digital dry bath heater) that was separate from the Typhoon 8620 image analyzer. One fluorescence image was captured every 15 min.

Figure 1. The principle of the amplification of miRNAs based on RT, EL and LAMP reaction. MicroRNA was lengthened by RT and EL reaction to construct the template of LAMP reaction. Then the lengthened product was amplified LAMP reaction at 65°C for 60 min.

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1307-3p concentrations, and the Tt value gradually decreased with increasing miR-1307-3p concentrations. The semilogarithmic calibration plots of Tt as a function of the initial miR-1307-3p copy number are presented in Figure S-2B. The linear relationship between 103 and 106 copies revealed that the square of the correlation coefficient was 0.99 and the slope was −2.33. The standard deviation (SD) of each point was 0.3, 2.3, 0.4, and 0.0 min (n = 2). We also analyzed the lengthened products by a conventional PCR, and visualized the amplicons using gel electrophoresis (Fig. S-3). The amplicons were the same size as the positive control, and a very weak non-specific amplification was observed for the negative control. Furthermore, Tt was correlated with the initial copy number for miR-16-5p, miR-191-5p, miR-423-5p, and miR-1246 (data not shown). These results implied that the lengthened product was synthesized by the reaction presented in Figure 1. The newly developed LAMP-based miRNA amplification method resulted in specific and sensitive measurements. Real-time LAMP reaction in the DNA chip liquid flow channel. To confirm the utility of an electrochemical DNA chip for a multiplex quantification, we first analyzed the behavior of the adsorbed primers in the liquid flow channel using a fluorescent dye. The primers for the LAMP reaction were immobilized on the liquid flow channel at five different positions, with an interval of approximately 10 mm between positions (Fig. 2A). The primers were adsorbed and dried on the edge of the flow channel because of the hydrophobic nature of the silicone module. The region containing the adsorbed primers was about 0.5 mm long. Additionally, the primers were not easily removed, even when exposed to flowing air. We also confirmed that the DNA chip with adsorbed primers was stable for at least 6 months at 4 °C. Images of the fluorescence from the primers after the LAMP solution was injected are presented in Figure 2B, C, and D. Before heating the DNA chip, the primers diffused approximately 1 mm from the center of the spotted position in an anteroposterior direction (Fig. 2B). The primers did not diffuse downstream following the injection of the LAMP solution, and remained around the position where they were spotted. The primers were moderately adsorbed on the surface of the silicone module, which suggested this material is appropriate for the immobilization of primers in our DNA chip system. The primers gradually diffused approximately 2 mm in an anteroposterior direction following the LAMP reaction at 65 °C for 60 min without a template (Fig. 2C). The expected diffusion distance of primer (16-mer) was calculated to be 1.1 mm (Eq. S-1).17 We supposed that the surface temperature distribution of the chip affected a difference between the measured value and calculated value. When 106 copies of target molecules were included in the LAMP solution, the fluorescence intensity gradually increased at each adsorbed primer position, likely because of the amplified product resulting from the LAMP reaction. These results indicated that the multiplex LAMP reaction occurred in the single liquid flow channel (Fig. 2D). Because the primers diffused over time, separate reaction areas were needed in the flow channel to ensure precise analyses. On the basis of our experiment, we speculate that an 8 mm interval between primer positions is required for the complete separation of reactions to prevent a cross contamination.

Real-time electrochemical measurement of the LAMP reaction. The principle of the real-time electrochemical LAMP used in this study was reported in the previous paper.16 The coprecipitation of RuHex with coproduced pyrophosphate according to LAMP reaction cause the increase of redox current by voltammetry. For the electrochemical measurement, 1 mM RuHex was used as a redox indicator instead of the fluorescent dye in the LAMP solution. Electrochemical measurements were completed at 65 °C using the automated electrochemical DNA detection system (Toshiba Hokuto Electronics).22 The associated software was installed for the continuous measurement of the RuHex cathodic signal. The measurement conditions for the linear sweep voltammetry (LSV) were as follows: initial voltage = 0.1 V (vs. gold reference electrode), final voltage = −0.4 V (vs. gold reference electrode), and scan rate = 0.5 V/s. The voltammograms were recorded every minute for 60 min. The cathodic peak current (Ipc) and peak potential were determined based on the differentiation of the currentpotential curve for the RuHex cathodic signal. The data from 10 electrodes near the positions where the primers were immobilized were analyzed. The amplification time for the electrochemical DNA chip (Ti) was defined as the time when the signal crossed a threshold of 2 [d(Ipc)/dt]. RESULTS AND DISCUSSION Amplification of miRNA. The reproducibility of the RT and EL reaction are very important for quantitative analysis of miRNA. We optimized these reaction conditions before LAMP experiment. The reaction time, temperature, primer concentration and cycle numbers of pre-amplification were very critical especially for specificity and sensitivity (data not shown). The lengthened products prepared with different amounts of the synthesized miRNAs (0, 103, 104, 105, and 106 copies) were amplified by a LAMP reaction at 65 °C using a conventional transparency tube and turbidity meter. The realtime turbidity curves of the LAMP reaction for miR-1307-3p (Fig. S-2A) produced a sigmoidal shape for the various miR-

Figure 2. The behaver of the primers in the liquid flow channel of the DNA chip. (A) The silicone module which having primers at five different position in the liquid flow channel of DNA chip. The arrows indicated the position of primers which was dried on the surface of silicone module. The dotted line indicated the inner wall of liquid flow channel. The width of channel was 1 mm. (B) Fluorescence image of primers immediately after injection of LAMP solution. (C) Fluorescence image of primers after LAMP reaction for 30 min at 65°C without template. (D) Fluorescence image of LAMP reacted with 106 copies of template for 30 min at 65°C.

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Figure 4. Correlation plots for quantification results of the miRNAs determined by DNA chip and qRT-PCR (n=5). Unit of horizontal and vertical axis: log (initial copies of miRNA).

Figure 3. Electrochemical detection of RuHex in LAMP solution. (A) LSV without miRNA. (B) LSV with 106 copies of miR-13073p. (C) The real-time Ipc curves of RuHex for LAMP reaction of miR-1307-3p (0, 103, 104, 105, 106 copies). (D) Calibration plot of Ti vs logarithmic input of the initial miR-1307-3p (n=2).

confirmed there were at least 103–106 copies of the tested miRNAs. The SD of the data above 104 copies of miRNA ranged from 0.0–1.5 min for all miRNAs. However, increased SDs were observed for 103 copies of miRNA, especially for miR-423 and miR-1246. We confirmed that the sensitivity varied depending on the miRNA sequences. Because the miR423 and miR-1246 concentrations in serum were relatively high (105 copies according to the qRT-PCR assay), we used calibration curves based on 104–106 copies of synthetic molecules for these miRNAs. These results suggested that the newly developed electrochemical DNA chip enabled quantifiable multiplex measurements.

A multiplex real-time LAMP was completed using an electrochemical DNA chip. The primers used for detecting five miRNAs were immobilized at different positions on the liquid flow channel, with 10 mm intervals between positions. The lengthened products prepared with different concentrations of synthesized miRNAs (0, 103, 104, 105, and 106 copies) were amplified at 65 °C in the liquid flow channel of the DNA chip. Electrochemical measurements via LSV were completed for 60 min with the LAMP reaction involving 1 mM RuHex. The continuous LSV curves for the positive control comprising the lengthened product prepared with 105 copies of miR-1307-3p are presented in Figure 3A, while the results for the negative control are provided in Figure 3B. For the negative control, the baseline of the Ipc drifted slightly until the end of the measurement period. Meanwhile, for the positive control, the Ipc suddenly increased starting at 30 min. This increase was likely associated with the specific LAMP reaction.16 The realtime cathodic current of the RuHex in the LAMP solution is presented in Figure 3C. All curves were sigmoidal for the various miR-1307-3p concentrations, and the Ti value gradually decreased with increasing miR-1307-3p concentrations, similar to the changes in turbidity. The positive signal was obtained from only the electrode where the primer sets for miR-1307-3p was immobilized (Fig. S-5). This result showed that the specific amplification was occurred in the liquid flow channel consisting 5 primer sets (miR-16, miR-191, miR-423, miR1246, miR1307). The data for the analysis of miR-1307-3p using the DNA chip are provided in Figure 3D. The semi-logarithmic calibration plots of Ti as a function of the initial miRNA copy number revealed the linear relationship between 103 and 106 miRNA copies. The square of the correlation coefficient was 0.88 and the SD was 0.5–1.0 min. The data for the other miRNAs analyzed with the same DNA chip are presented in Figure S-4. The semi-logarithmic calibration plots of Ti as a function of the initial miRNA copy number confirmed the linear relationship between 103 and 106 miRNA copies. The square of the correlation coefficient was 0.88–0.97. Moreover, the data

Quantitative analysis of miRNAs extracted from serum. We used five different sera to simultaneously detect five miRNAs, including a normal serum sample (pooled sera) as well as sera from breast cancer patients. The miRNAs extracted from each serum were lengthened by RT and EL reactions. The LAMP solution containing the lengthened miRNAs were injected into the DNA chip and electrochemical measurements were completed to monitor the LAMP reaction. The miRNAs were also quantified by a qRT-PCR assay. We used the miRCURY LNA Universal RT microRNA PCR system to analyze miR1246 because the sensitivity of the TaqMan Advanced miRNA assay kit was insufficient for detecting miR-1246. An analysis of the data revealed a good correlation between the results of the DNA chip electrochemical measurements and the qRT-PCR assay for the five miRNAs (R2: 0.86–0.99) (Fig. 4). These results indicate that the newly developed DNA chip may be used for a multiplex real-time LAMP-based quantification of miRNAs extracted from serum in an experiment that can be completed within 2 h.

CONCLUSIONS We developed a novel electrochemical DNA chip for a multiplex real-time detection system involving a LAMP reaction. This system may be used for the simultaneous quantification of five different miRNAs extracted from serum. Highly specific data were obtained for 103–106 copies of the targeted molecules. The newly designed DNA chip described herein enables a multiplex LAMP-based analysis in a structurally simple device that is easy to operate. This device

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only requires a single liquid flow channel, eliminating the need for multiple wells and sample injections for a multiplex detection assay. We expect the medical applications of this simple and stable method will include the detection of specific nucleic acids (i.e., miRNA, mRNA, and genomic DNA) as well as bacteria and viruses.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Sequences of oligonucleotides used in this work, schematic representation of the electrochemical DNA chip, LAMP reaction for miRNA quantification, gel electrophoresis image for PCR products of lengthened miRNA and calibration results of miRNAs using DNA chip (PDF)

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected].

ORCID Koji Hashimoto: 0000-0001-8746-2010

Author Contributions All authors have given approval to the final version of the manuscript.

Notes All authors are employees of Toshiba Corporation and the technologies reported in this paper are patent pending.

ACKNOWLEDGMENT This research was supported by Japan Agency for Medical Research and Development (AMED) under Grant Number JP18ae0101014. The authors thank Ryota Nosaka for sample preparation and RT-PCR experiments.

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Multiplex real-time loop-mediated isothermal amplification using an

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