Ultimate Single-Copy DNA Detection Using Real-Time

Jun 6, 2016 - PCR Technologies for Point of Care Testing: Progress and Perspectives. Salvatore Petralia and Sabrina Conoci. ACS Sensors 2017 2 (7), 87...
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Ultimate single-copy DNA detection using real-time electrochemical LAMP Alexandra Martin, Kathryn Betty Grant, Franziska Stressmann, Jean-Marc Ghigo, Damien Marchal, and Benoit Limoges ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.6b00125 • Publication Date (Web): 06 Jun 2016 Downloaded from http://pubs.acs.org on June 11, 2016

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ACS Sensors

Ultimate single-copy DNA detection using real-time electrochemical LAMP Alexandra Martin,a Kathryn B. Grant,b Franziska Stressmann,c Jean-Marc Ghigo,c Damien Marchal,a,* Benoît Limogesa,* a

Laboratoire d’Electrochimie Moléculaire, UMR 7591 CNRS, Université Paris Diderot, Sorbonne Paris Cité, 15 rue Jean-Antoine de Baïf, F-75205 Paris Cedex 13, France b Department of Chemistry, Georgia State University, Atlanta, Georgia 30302-3965, United States c Institut Pasteur, Unité de Génétique des Biofilms, Département de Microbiologie, 25-28 rue du Dr. Roux F75015 Paris, France ABSTRACT: Herein, we report the first successful detection of single DNA copies using real-time electrochemical loopmediated isothermal amplification (LAMP). Towards this end, real-time electrochemical LAMP amplifications of bacteriophage M13mp18 DNA and of a genomic DNA sequence from the pathogen Flavobacterium columnare were systematically run each with four different redox reporters (i.e., three intercalating reporters with different ds-DNA binding strengths and one non-intercalating probe able to sense the pyrophosphate ion concentration produced during LAMP). In order to provide a reliable, rapid assessment of the LAMP performances, the experiments were carried out using a prototype high-throughput house-built automatized electrochemical read-out device, optimized for parallel monitoring of up to 48 samples. The electrochemical results were then compared to those achieved with conventional real-time fluorescence LAMP and PCR methods (using commercially available fluorescent dyes and standard bench-top equipment). The best electrochemical performances were obtained with the two strongest intercalating redox reporters, affording analytical sensitivity and detection limits comparable to real-time fluorescence LAMP. These electrochemical reporters enabled us to assemble highly accurate, log-linear calibration curves in only 30 min, over a six-decade dynamic concentration range with detection limits as low as two DNA copies of genomic target in 50 µL. These are to our knowledge the best results so far reported for electrochemical-based real-time LAMP, rivaling those achieved with fluorescencebased real-time LAMPs. The major advantages of our electrochemical LAMP approach over the fluorescence-based LAMP method include miniaturization, portability, robustness, low cost, and the ability to work with non-transparent reaction mixtures. Keywords: DNA amplification, nucleic acid, point-of-care, DNA testing, electrochemical readout, electrochemical microplate, screen-printed electrodes. The development of ultrasensitive and quantitative point-of-care (POC) analytical tools for genetic detection of infectious diseases at limited resource settings (e.g., local clinics, small hospitals or in place where the infection originates) is an important step forward in worldwide public health, food safety testing, forensic analysis, homeland security and environmental monitoring.1-3 To be suitable for limited resource settings, genetic POC detection devices should ideally be affordable, sensitive, specific, portable, robust, userfriendly and capable of screening a large number of unprocessed samples in a short time.4 In order to enable the number of DNA copies per sample to be accurately determined, it should also support real-time, quantitative analysis across a wider dynamic range than conventional end-point nucleic acid detection methods are able to afford. Toward this end, ap-

proaches based on real-time monitoring of isothermal DNA amplification using simple read-out systems are the most desirable.1,5,6 This is particularly true in the case of electrochemical detection methods used for monitoring, in real-time, isothermal nucleic acid amplifications.7-11 These emerging techniques offer the combined advantages of exponential DNA (or RNA) target amplification at a constant temperature (therefore eliminating the need for accurate temperature control such as thermocycling in PCR) with that of in situ electrochemical detection (offering low-cost, easy integration in a miniaturized format, high sensitivity and robustness, and ability to operate in non-transparent reaction mixtures and reaction vessels with complex biological samples).7-11 The combination of advantages unique to the real-time electrochemical isothermal approach is expected to make possible the development of low-

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cost, simpler and smaller battery-operated POC devices, ideal for nucleic acid analysis. Among the isothermal amplification methods that have been so far developed, only two have been successfully coupled to real-time electrochemical detection: helicase-dependent amplification (HDA)12 and loopmediated isothermal amplification (LAMP).13-18 In these approaches, monitoring amplicon formation in real-time is almost always based on the time-course electrochemical (or electrical) measurement of the interactions occurring between a redox-active reporter added to the reaction solution and the DNA products or byproducts generated during the isothermal amplification. Depending on the nature of these interactions, two categories of redox reporters have been proposed, i.e., those that can intercalate into double-strand DNA amplicons and probes that bind to DNA primarily by electrostatic interactions. In the case of redox-active DNA intercalators, amplicon detection takes advantage of the reduced diffusivity that occurs when a low molecular weight intercalating redox reporter binds to high molecular weight ds-DNA, reducing thus the efficiency with which the redox reporter is electrochemically detected. This strategy was first demonstrated for real-time electrochemical PCR19,20 and thereafter extended to isothermal amplification methods (i.e., HDA12 and LAMP13-17). To date, two redox-active DNA intercalators have been studied, i.e. [Os(bpy)2dppz]2+ (with dppz = dipyrido[3,2-a:2’,3’c]phenazine) and methylene blue (MB). The osmium polypyridyl complex was demonstrated effective for monitoring electrochemically, in real-time, isothermal HDA reactions.12 However, due to the occurrence of nonspecific amplification of primer-dimers, analytical performances were poor (the lower limit of detection was restricted to only 105 DNA copies).12 The second DNA intercalator, MB, was revealed to be efficient in real-time electrochemical LAMP, with detection limits in the range of 16 to 400 target DNA copies in 20 µL and a dynamic range spanning 3 to 4 orders of magnitude.14,17 In the case of redox reporters that interact electrostatically, real-time electrochemical isothermal DNA amplification involves ion-pairing between positively charged redox reporters and negatively charged DNA products and/or pyrophosphate ions generated during the amplification reaction. For the reporter crystal violet, such interactions have been shown to generate an increase in the electrical resistance of the LAMP reaction mixes.16 In LAMP reactions that employ the reversible oneelectron redox probe Ru(NH3)63+, a decrease of the faradaic current response is observed.21 For the latter, it was initially assumed that the electrochemical signal decrease resulted from the electrostatic interaction between Ru(NH3)63+ and the anionic DNA backbone of amplicons,21 but we have very recently demonstrated that this was instead the consequence of a coprecipitation of Ru(NH3)63+ with the pyrophosphate anions generated during LAMP.22 With this nonintercalating redox probe, a detection limit of 20-30

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target DNA copies/µL (i.e., 1000-1500 copies per tube) was claimed, but in our opinion, the high data scattering and poor temporal resolution of the published realtime plots do not allow LAMP performances to be accurately evaluated.21 The analytical performances of the real-time electrochemical isothermal DNA amplifications so far developed appear promising,13-17,21 but it is difficult to directly compare them, each being carried out under different conditions, using various DNA targets and redox probes. Furthermore, many of the reported electrochemical real-time devices have not been optimally designed for fully automated high-throughput point-ofcare analysis in disposable arrays of electrochemical closed-cells. Instead, most of the previous studies have been based on open electrode-in-tube setups13,15,21 or on single electrochemical microchambers integrated in a microfluidic system,14 restricting these approaches to use in low-throughput laboratory settings. Additionally, the dynamic concentration ranges so far reported do not rival those achieved using fluorescence LAMP or PCR-based detection. In the most favorable case, loglinear calibration curves spanning 3 to 4 orders of magnitude were obtained,13-17 which is significantly less than the 6 to 8 order of magnitude reached by benchtop fluorescence methods.23-25 Finally yet importantly, none has demonstrated the possibility of reaching the ultimate level of single-copy DNA detection per assay (usually in 20-50 µL), performances that are attainable in real-time fluorescent PCR and LAMP.23-25 While the electrochemical isothermal systems are superior with respect to portability and cost effectiveness, even permitting analysis of non-transparent biological samples, this apparent lack of analytical performance is clearly a limitation for their widespread adoption in POC nucleic acid analysis. For all of these reasons, we report here an evaluation of two real-time electrochemical LAMPs in a high throughput custom-made electrochemical read-out device. In so doing, we have amplified bacteriophage M13mp18 DNA and Flavobacterium columnare (F. columnare) DNA each in the presence of the four structurally distinct, intercalating or electrostatic redox reporters (Scheme 1). The electrochemical results were then compared to those achieved with conventional real-time fluorescence LAMP and PCR procedures (both performed using commercialized benchtop equipment and an intercalating fluorescent dye). In the present report, with the objectives of accessing and optimizing LAMP analytical performances, we have successfully extended the detection limit of realtime electrochemical loop-mediated isothermal amplification from ~ 16 to 400 target DNA14,17 to single DNA molecules.

MATERIALS AND METHODS Reagents. All oligonucleotides were obtained from Eurogentec. The primer sequences BIP, FIP, B3 and F3 used to specifically amplify a 274-bp DNA target in M13mp18 were the same as previously described.26 LB (5’-CGA GCT CGA ATT CGT AAT CA-3’) and LF (5’-

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ACS Sensors Real-time fluorescence monitoring of LAMP. The optical experiments were performed with an ABI 7500 real-time PCR system (Applied Biosystem Instruments) in MicroAmp FAST optical 96-wells reaction plates. Each well was filled with 50 µL of LAMP master mixture containing 400 µM of each of the dNTPs (Qiagen), 10 µL 10× Thermopol buffer, 1.6 µM of each of the BIP and FIP primers, 0.8 µM of each of the LB and LF primers, 0.4 µM of each of the B3 and F3 primers, 1 mM betaine, 0.32 U of Bst 2 WarmStart™ DNA polymerase, a suitable amount of the EvaGreen® (Biotium) intercalating fluorescent dye (0.1×) and 2 µL target DNA (at a concentration ranging from 0.2 to 1010 copies/mL). LAMP reactions were carried out at 65°C for 80 min and the fluorescence response of each microwell was monitored every 90 s. Fluorescence-based real-time PCR. Fluorescence PCR of the F. columnare target was performed with an ABI 7500 real-time PCR system (Applied Biosystem Instruments) in the presence of the SYBR Green I® DNA intercalating fluorescent dye. Each well of the optical microplate was filled with 20 µL of PCR master mix containing all the reagents necessary for PCR of F. columnare: 1x power SYBR Green I® PCR master mix (Applied Biosystem), 500 nM of each of the F3 and B3 primers and different concentrations of target DNA (ranging from 0.1 to 106 copies per microtube). The temperature cycle program consisted of an initial polymerase activation step at 95 °C for 10 min, followed by 40 cycles of two-step temperature (i.e., 95 °C for 15 s followed by 60 °C for 1 min).

CTT GCA GCA CAT CCC CCT TT-3’) sequences were designed manually. The M13mp18 DNA was purchased from New England Biolabs Inc. Quantified samples of extracted genomic DNA of F. columnare (ALG00530) were provided by the team of Dr. J-M. Ghigo (Institut Pasteur, Paris). The primer sequences used for the LAMP and PCR amplifications of a 311-bp genomic DNA sequence of F. columnare were designed using the software Premier Biosoft (Tables S1 and S2 in Supporting Information, SI). Bovine serum albumin (BSA), polyvinylpyrrolidone (PVP, MW 40 kDa) and betaine solution were supplied by Sigma Aldrich. The Bst 2 WarmStart™ DNA polymerase and buffer were from New England Biolabs Inc. For the electrochemical LAMP experiments, a 10× homemade detergent-free Tris buffer (10× TB, pH 8.8: 0.2 M Tris-HCl, 500 mM KCl, 0.1 M (NH4)2SO4 and 0.04 M MgSO4) was used. The complex [Os(bpy)2dppz](PF6)2 and methylene blue derivative PhP were synthetized according to published procedures.27,28 Methylene blue (MB) and Ru(NH3)6Cl3 were supplied by Sigma-Aldrich. Real-time electrochemical LAMP. Real-time electrochemical monitoring of LAMP reactions was performed with a custom-made instrument12 (co-developed with Easy Life Science company) in which a disposable 48well electrochemical microplate is fastened flat over a Peltier-heating block and then electrically wired to a multiplexed potentiostat. (See main text for further information.) Wells of the electrochemical microplate were each filled with 50 µL of LAMP reaction mixture containing 400 µM of each of the dNTPs (Qiagen), 5 µL 10× TB, 1.6 µM of each of the BIP and FIP primers, 0.8 µM of each of the LB and LF primers, 0.4 µM of each of the B3 and F3 primers, 1 mM betaine, 0.025 % wt BSA, 1 % wt PVP, 0.32 U of Bst 2 WarmStart™ DNA polymerase, a suitable amount of the redox reporter (see text), and 2 µL of target DNA (concentrations ranging from 0 to 1010 copies/mL). After the microplate was sealed with an optical adhesive film (MicroAmp®, Thermo Fisher Scientific), LAMP reaction mixes were maintained at 65 °C while the square wave voltammetric (SWV) responses of each well were recorded at a suitable sampling rate (every 40 s) to get a sufficiently well-resolved evolution of the signal. (During the LAMP reactions, the lid of the thermocycler was held tightly closed over the electrochemical microplate to insure good contact with both the underlying heating block and the electrical connections.) Melt curve analysis was performed immediately following each LAMP reaction by applying a linear ramp of temperature to the 48well electrochemical microplate, starting from a cooling step at 50 °C for 1 min followed by a continuous rise of temperature up to 91 °C at a ramp rate of a 1.1 °C min1 . During the continuous increase of temperature, SWV signals were acquired every 13 s. Each SWV peak was automatically baseline-corrected and integrated, and the resulting peak charge then reported as a function of either time or temperature.

RESULTS AND DISCUSSION In this study, we have focused on LAMP for the reasons that it has a higher specificity and superior amplification yields compared to other isothermal DNA amplification methods.26,29-31 For instance, LAMP can produce more than 0.5 µg amplicon/µL, which is more than 100-fold greater than PCR or HDA. Likewise, because LAMP requires four to six different primers to recognize six to eight different regions within the target, its specificity is significantly greater than other DNA amplification methods. The LAMP approach also offers in some circumstances greater tolerance to well-known inhibitors contained in biological matrices such as blood, serum and food ingredients, allowing one to work directly with crude biological samples (thereby eliminating the need for a DNA extraction step, which is particularly useful for POC applications).32-34 The first LAMP reaction we have investigated in this report corresponds to the amplification of a model DNA sequence from the bacteriophage M13mp18.26 The second LAMP is related to the amplification of a more biologically relevant genomic DNA sequence from Flavobacterium columnare, a fish pathogen causing columnaris disease, the detection of which is a major concern in the catfish and warm water fish industries. The M13mp18 and Flavobacterium columnare LAMP assays were performed with each of four different redox reporters (Scheme 1), which were selected on the

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ACS Sensors basis of our recent screening study of DNA redox probes in real-time electrochemical LAMP.22 Accordingly, the three DNA-intercalating

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Scheme 2. Schematic diagram of the real-time electrochemical LAMP.

High-throughput real-time electrochemical LAMP device. To carry out a large number of experiments under well-defined conditions and in a reproducible and reliable manner, temperature controlled LAMP reactions were run directly in an automatized electrochemical read-out device.12,36 Consisting of closedelectrochemical cells of 50 µL working volume, the house-built instrument was designed for monitoring in parallel and in real-time up to 48 separate DNA samples in a single run (Figure 1). It consists, on one side, of a conventional PCR thermocycler (Figure 1A) wherein the heating block has been replaced by a flat Peltier-heating block, integrating laterally two arrays of 75-pins connectors (Figure 1B), and, on the other side, a disposable 48-well electrochemical microplate (Figure 1C) that can be fastened flat on the heating block and wired electrically (through the two 75-pins connectors) to a PCB interface containing the electronic circuitry of the multiplexed potentiostat (Figure 1D, the PCB interface is placed just behind the thermocycler). Using this device, the content of each of the 48electrochemical wells (50 µL) was held at a constant temperature of 65°C for LAMP while being scanned by SWV at a maximal sampling rate of 0.3 Hz. With the help of a dedicated Matlab program (Figure 1E), SWV peak current integrations were automatically extracted from raw signals after baseline correction and then plotted as a function of the LAMP amplification time.

Scheme 1. List of redox probes used for real-time electrochemical monitoring of LAMP. The reported formal poten0’ tials (E ) are at pH 8.8 and vs. Ag/AgCl.

compounds we selected for the present study, i.e., [Os(bpy)2dppz]2+, PhP and MB, have ds-DNA binding strengths ranging over two orders of magnitude (i.e., from ca. 106 M-1 for the osmium complex to ca. 104 M-1 for MB), which is important for gauging the effect of this parameter on the analytical performances of real-time electrochemical LAMPs. The methylene blue derivative baptized PhP, which according to its DNA anchor arm (a piperazinexylene chain) has a 10-fold higher affinity binding for ds-DNA(Kb∼ 105 M-1) than MB.35 The Ru(NH3)63+ was added to the list of reporters because it is a good illustrative example of non-intercalating redox probe.21 The principle of the LAMP detection method employed here is briefly illustrated in Scheme 2. It relies on the in situ and real-time electrochemical monitoring of the faradaic current response (recorded in square wave voltammetry, SWV) generated from a free-to-diffuse redox probe added to the LAMP reaction mixture. As the reaction progresses, an increasing amount of the redox reporter binds to the exponentially amplified double-stranded amplicons (or in the case of a nonintercalating species as Ru(NH3)63+, to the exponentially released byproduct pyrophosphate anion). The consequence is a lowering of the apparent mass transport of the redox indicator to the electrode surface (because of the lower diffusion coefficient of the bound redoxactive probe compared to the free one). This in turn translates into an exponential decrease of the faradaic current response as a function of the amplification time. The onset-signal decrease occurring at a time-tothreshold value, tt, is indirectly related to the initial DNA target copy number.

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ACS Sensors the amplification curves obtained with each of the four redox probes reveals several marked differences not only in the baseline shape of the plots, but also in the time-to-threshold values and signal decrease amplitudes. With the osmium complex, the strongest DNA binder, the total disappearance of the electrochemical signal for positive samples (Figure 2A) indicates that the intercalating probe is entirely captured by the exponentially grown amplicon. This is in contrast to the two other intercalating MB and PhP molecules, which lead to average maximal signal decreases of ∼60% and ∼70%, respectively (Figures 2B and 2C). Such behavior can be easily understood by considering on one hand the much lower starting amount of [Os(bpy)2dppz]2+ per well (0.025 nmol) compared to MB and PhP (0.75 nmol), and on the other hand the higher affinity of the osmium complex for ds-DNA. Consequently, when a sufficiently large excess of ds-DNA binding sites is produced by LAMP, almost all of the osmium molecules intercalate into the double-stranded amplicons. In contrast, for MB and PhP, at the end of the LAMP, a large fraction of these probes remains free-to-diffuse in solution. It is interesting to note that the relatively large amplitude signal decrease observed with PhP is in line with its 10-fold higher affinity for dsDNA than MB. In the case of the non-intercalating compound Ru(NH3)63+, the signal decrease is remarkably greater than for MB and PhP (Figure 2D), reaching nearly 100% diminution. However, the onset-signal decrease occurs after a longer amplification time, revealing significantly lower assay sensitivity. This distinctive behavior highlights a different way by which the electrochemical signal can be decreased in LAMP. The sudden reduction in signal we observed with positive samples is related to the co-precipitation of Ru(NH3)63+ with the rising concentration of pyrophosphate anions released during reactions. This process has been shown to occur significantly only when millimolar levels of pyrophosphate are present in Ru(NH3)63+ solutions.22 It is thus necessary to wait until nearly the end of the LAMP reaction before reaching a sufficiently high pyrophosphate concentration, the consequence of which is a lowering of the detection sensitivity of Ru(NH3)63+ LAMP compared to the intercalating redox probes. Concerning the shape of amplification curves, the DNA intercalator [Os(bpy)2dppz]2+ produces a continuous and approximately linear baseline increase with time, while for MB, PhP, and Ru(NH3)63+ baselines rapidly stabilize in a few minutes. The continuous baseline drift observed with the

Figure 1. (A) Overview of the real-time electrochemical prototype that monitors in parallel up to 48 LAMP reactions. Shown are selected views of its various components: (B) flat Peltier-heating block and its two 72-pins connectors mounted in a standard PCR thermocycler, (C) 48-well electrochemical microplate, (D) PCB interface containing the electronic circuitry of the multiplexed potentiostat, (E) dedicated software for piloting the PCB interface and extracting the electrochemical response from each microwell.

Evaluation of the real-time electrochemical LAMP of M13mp18 phage DNA target using different redox probes. Analytical performances were first evaluated through the establishment of calibration plots of M13mp18 plasmid target. For this purpose, the realtime electrochemical amplification curves generated from serial dilutions of the M13mp18 phage DNA template (ranging from 102 to 108 copies per electrochemical well) were determined for each of the four selected redox probes (Figure 2). Depending on whether or not there was a propensity to inhibit the LAMP reaction,22 the concentration of each probe was suitably adjusted. For instance, in the case of strongly intercalating [Os(bpy)2dppz]2+, a concentration as low as 500 nM was selected to avoid inhibition of LAMP at higher probe concentrations. For the MB, PhP, Ru(NH3)63+, redox reporters showing lower inhibitory effects, a concentration of 15 µM was found to be optimal. As shown in Figure 2, whatever the redox probe, a typical exponential signal decrease from a characteristic time-to-threshold value was systematically observed for all of the positive samples but not for the negative template control reaction (NC, no target). In addition, the onset-signal decrease occurred sooner when the starting amount of target was higher. A comparison of

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osmium complex is analogous to our previously reported real-time electrochemical HDA experiments, in which baseline drift was attributed to a slow adsorption of metal complex on the working carbon electrode surface.12,22 The higher baseline stabilities observed in the case of MB, PhP and Ru(NH3)63+ are advantageous for more reliably discriminating the onset-signal decreases of positive samples from the unchanged signal responses of negative controls. Baseline correction by subtraction of an extrapolated least-square fitted line across the early amplification time (considering only the early linear part of the plot) allowed us to recover the tt value for all of the positive amplification curves (tt was defined as the time required for the signal to reach a threshold decrease that corresponds to 5-fold the noise determined for the early steady-state response, i.e. ca. 10% signal decrease). Shown in Figure 3 are the resulting semilogarithmic calibration plots of tt as a function of initial DNA copy number for each of the four redox probes. Also overlaid is the calibration plot determined with real-time fluorescence LAMP using EvaGreen® as an intercalating reporter dye. (See Figure S1 for the amplification curves.) Whatever the intercalating probes (i.e., [Os(bpy)2dppz]2+, MB, PhP or EvaGreen®), all calibration curve converge was within the same time range window, showing no significant difference in the assay sensitivities. This result indicates that the intercalating binding strength of the redox reporter is not the determining factor for LAMP assay sensitivity (at least for intercalating probes having a DNA binding constant within the range 106 to 104 M-1). The plots also reveal that the LAMP assay sensitivities obtained with the intercalating redox probes are comparable to those obtained in fluorescence LAMP using an intercalating dye. This allowed us to conclude that the detection mode (i.e., electrochemical vs. fluorescence) is not critical for real-time LAMP performance. (It is also interesting to note that the assay sensitivity appears not to depend on the detection being based in an “off-” vs. in an “on-signal” measurement.) The LAMP assay sensitivity is however significantly lower in the presence of the non-intercalating probe Ru(NH3)63+. This is clearly illustrated by the shift of the calibration plot at much longer amplification times (Figure 3). As mentioned previously, loss of sensitivity results from the need to produce large amount of pyrophosphate anions for initiating a substantial signal decrease through Ru(NH3)63+ co-precipitation. This result is in agreement with the lower sensitivities and detection limits generally observed with real-time LAMPs based on the turbidimetric or colorimetric detection of released pyrophosphate anions compared to those based on the fluorescence detection by an intercalating dye.31 It also clearly demonstrates the importance of preferentially using ds-DNA intercalating redox reporters rather than electrostatic redox indicators for improving sensitivity in real-time electrochemical LAMP. It is worth noting that all of the calibration plots we have obtained with M13mp18 DNA target are roughly linear when the number of DNA copies is higher than 103, but

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Time /min Figure 2. Real-time electrochemical LAMP curves for duplicate (except in D) serial dilutions of the M13mp18 phage DNA target (the starting number of target copies per well is reported for each plot in the figure) and in the 2+ presence of (A) 0.5 µM [Os(bpy)2dppz] , (B) 15 µM MB, (C) 15 µM PhP, and (D) 15 µM Ru(NH3)63+ reporter. All of the curves were normalized to the SWV peak charge recorded after 7 min of LAMP reaction.

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at lower DNA copies deviate from linearity as a function of amplification time (Figure 3). We have attributed this behavior to a lack of optimization of LAMP primer design and/or LAMP experimental conditions.37

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ing data reveal a semi-logarithmic linear relationship between the initial target copy number and the tt values across the entire dynamic range we investigated with one exception. For MB, the lowest DNA concentration (i.e., 2 copies per well)

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Characterization of the analytical performances of real-time electrochemical LAMP with a relevant biological target. In order to highlight the benefits of real-time electrochemical LAMP for quantitative detection in authentic biological samples, it was necessary to switch from an M13mp18 DNA to a more relevant genomic target. For this reason, we designed a new LAMP reaction for the quantitative detection of the gram-negative pathogenic bacterium F. columnare. Based on previously published PCR experiments,38 species-specific regions in the 16S F. columnare rRNA gene were used to design a set of LAMP primers (Tables S1 and S2 in SI). A series of electrochemical LAMP curves was then generated from 10-fold serial dilutions of a quantified biological extract of F. columnare (genomic DNA concentration of 5 × 107 copies/µL) in such a way as to have a range of concentrations from 0.2 to 2 × 106 copies per electrochemical microwell (i.e., in 50 µL). The same DNA concentration range was then independently analyzed with the gold standard technique of real-time fluorescence PCR, using SYBR Green I® as a reporter dye (Figure S2), and then by fluorescence LAMP with EvaGreen® as a DNA intercalating dye. Electrochemical LAMP amplification curves obtained with [Os(bpy)2dppz]2+, MB and PhP are shown in Figures 4A to 4C. (For clarity, only one curve per concentration is reported.) The fluorescent LAMP amplification curves performed on the same serial dilutions of DNA target are shown in Figure 4D. The strong correlation between the initial target copy number and the tt values recovered from the amplification curves enabled us to construct the F. columnare calibration plots reported in Figure 5. The result-

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Figure 4. (A-C) Real-time electrochemical LAMP curves and their corresponding (A’-C’) melting curves obtained for serial dilutions of the F. columnare DNA target (the starting number of target copies per well is reported for each plot on the graph) and using: (A, A’) 0.5 µM 2+ [Os(bpy)2dppz] , (B, B’) 15 µM MB, and (C, C’) 15 µM PhP reporter. (D) Real-time fluorescence LAMP curves with their corresponding (D’) melting curves recorded in ® the presence of EvaGreen and for serial dilutions of the F. columnare target. All real-time kinetic curves were normalized to the SWV peak charge or fluorescence intensity recorded after 7 min of LAMP reaction.

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ACS Sensors could not be consistently detected (absence of signal decrease), and when detectable, resulted in a tt value that was delayed at longer time points. In all other cases, the log-linear relationship we observed was found to be in complete agreement with the exponential reaction kinetics of LAMP during its initial phase.30,37 The small standard error bars on the real-time electrochemical LAMP calibration plots (obtained from triplicated or duplicated experiments) attest to good reproducibility, comparable to the small standard deviation bars associated with the fluorescence LAMP data (Figure 5). The wide dynamic range of electrochemical LAMP target quantification, over at least six orders of magnitude, is also comparable to that achieved with the bench-top fluorescence real-time PCR system (Figure S2). The overall good correlation between the gold standard calibration curves of fluorescence-based real-time PCR and those of real-time electrochemical LAMP clearly demonstrates the strong agreement between the two methods. In the case of the redox reporters [Os(bpy)2dppz]2+ and PhP, a detection limit as low as 2 DNA copies of the F. columnare genomic target was achieved in a reaction time less than 30 min (red lines in Figure 4A and 4C, red and green lines in Figure 5). (This corresponds to ∼7 × 10-21 mol/L of DNA in the 50 µL LAMP mixture or 103 copies/mL in the 2 µL target DNA sample added to the 50 µL LAMP mix.) To our knowledge, this detection of single DNA copies constitutes the best performance so far reported for the electrochemical-based real-time detection of LAMP products, a performance that finally rivals that achieved by real-time fluorescence LAMP (Figure 5). [Os(bpy)2dppz]2+ and

SWV peak current) show, for all the three intercalating redox probes and at all DNA target concentrations greater than 0.2 copies, a well-defined and highly reproducible sharp signal increase at ∼85-87°C (Figure 4A’ to 4C’). Significant melting temperature changes were not observed in the case of DNA negative template controls (Figure 4A’ to 4C’) as well as with the non-intercalating redox probe Ru(NH3)63+ (data not shown).22 The sudden signal increase produced by the intercalating dyes is in good agreement with that previously reported22 and is characteristic of the melting temperature (Tm) at which the double-stranded DNA amplicons denature into single-stranded DNA, thereby leading to an abrupt increase in the SWV response due to the release of the DNA intercalated redox probe into solution. These data also correlate well with the melting fluorescent curves recorded at the end of the real-time fluorescence LAMP (Figure 4D’). Whatever the readout system, the absence of signal transition in the melt curves of all negative template controls attests clearly to the remarkably high specificity of LAMP, a distinction that can explain why it is possible to reach here the ultimate detection level of single-copies of DNA target per well. The real-time electrochemical LAMP results reported here indeed constitute a decisive advantage over our previously published real-time electrochemical isothermal HDA data,12 for which the detection limit was markedly limited by the nonspecific primer-dimer amplification.

CONCLUSION In the present report, we have shown that it is possible to perform real-time electrochemical LAMP with analytical performances comparable to those achieved in real-time fluorescence LAMP and real-time fluorescence PCR. This was made possible through the combined use of an entirely automatized multiplexed electrochemical read-out device with high-throughput capability and the selection of optimal DNA intercalating redox reporters. Among the four redox probes we investigated, the best results were obtained with the dsDNA intercalating agents. The strongest intercalator [Os(bpy)2dppz]2+ was found to be very effective for monitoring LAMP, but had the drawback of baseline drift which can complicate the extraction of signal threshold. Robust intercalative binding also led to significant LAMP inhibition at high probe concentrations, requiring that the LAMP assay be run with a barely detectable amount of probe. The non-intercalating complex Ru(NH3)63+ was also confirmed as a valuable redox reporter, but it led to a significantly lower LAMP assay sensitivity. All in all, the intercalating probe PhP was shown by far to be the most attractive for the reasons that: (i) in the absence of target amplification it provides a stable baseline response throughout the entire duration of LAMP; (ii) with DNA positive samples it furnishes large signal amplitude decreases; (iii) it is not as strongly intercalating as [Os(bpy)2dppz]2+ and as a result, the LAMP reaction is not significantly inhibited by micromolar concentrations of PhP, allowing one to perform LAMP with an easily measurable probe con-

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Log. initial copy number Figure 5. Linear regression calibration plots of tt vs. logarithmic input of the F. columnare DNA target. (For MB, the lowest concentration was not considered in the regression analysis since it was out of the linear range.) Error bars are the standard deviations of duplicate or triplicate runs.

Electrochemical melting curves recorded at the end of LAMP (i.e., by applying a linear ramp of temperature to the overall samples and concurrently monitoring the

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ACS Sensors (4) Peeling, R. W.; Holmes, K. K.; Mabey, D.; Ronald, A. Rapid Testsfor Sexually Transmitted Infections (STIs): The Way Forward. Sex. Transm. Infect. 2006, 82, 1−6. (5) Craw, P.; Balachandran, W. Isothermal nucleic acid amplification technologies for point-of-care diagnostics: a critical review. Lab Chip 2012, 12, 2469–86. (6) Asiello, P.J.; Baeumner, A.J. Miniaturized isothermal nucleic acid amplification, a review. Lab Chip 2011, 11, 1420–30. (7) Lee, T.M.; Hsing, I.M. DNA-based bioanalytical microsystems for handheld device applications. Anal. Chim. Acta 2006, 556, 26–37. (8) Patterson, A.S.; Hsieh, K.; Soh, H.T.; Plaxco, K.W. Electrochemical real-time nucleic acid amplification: towards point-of-care quantification of pathogens. Trends Biotechnol. 2013, 31, 704–12. (9) Zhang, X.; Lowe, S.B.; Gooding, J.J. Brief review of monitoring methods for loop-mediated isothermal amplification (LAMP). Biosens. Bioelectron. 2014, 61, 491–9. (10) Goda, T.; Tabata, M.; Miyahara, Y. Electrical and Electrochemical Monitoring of Nucleic Acid Amplification.Front. Bioeng. Biotechnol. 2015, 3, 1–15. (11) Hsieh, K.; Ferguson, B.S.; Eisenstein, M.; Plaxco, K.W.; Soh, H.T. Integrated Electrochemical Microsystems for Genetic Detection of Pathogens at the Point of Care. Acc. Chem. Res. 2015, 911–20. (12) Kivlehan, F.; Mavre, F.; Talini, L.; Limoges, B.; Marchal, D. Real-time electrochemical monitoring of isothermal helicase-dependent amplification of nucleic acids. Analyst 2011, 136, 3635–42. (13) Nagatani, N.; Yamanaka, K.; Saito, M.; Koketsu, R.; Sasaki, T.; Ikuta, K.; Miyahara, T.; Tamiya, E. Semi-real time electrochemical monitoring for influenza virus RNA by reverse transcription loop-mediated isothermal amplification using a USB powered portable potentiostat. Analyst 2011, 136, 5143–50. (14) Hsieh, K.; Patterson, A.S.; Ferguson, B.S.; Plaxco, K.W.; Soh, H.T. Rapid, Sensitive, and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic Electrochemical Quantitative Loop-Mediated Isothermal Amplification. Angew. Chem. Int. 2012, 51, 4896–900. (15) Jiang, D.; Xiang, G.; Wu，J.; Liu, C.; Liu, F.; Pu, X. A Real-time Resistance Measurement for DNA Amplification and Detection. Int. J. Electrochem. Sci., 2012, 7, 5273–85. (16) Xiang, G.; Pu, X.; Jiang, D.; Liu, L.; Liu, C.; Liu, X. Development of a real-time resistance measurement for Vibrio parahaemolyticus detection by the lecithindependent hemolysin gene. PLoS One 2013, 8, e72342. (17) Luo, J.; Fang, X.; Ye, D.; Li, H.; Chen, H.; Zhang, S.; Kong, J. A real-time microfluidic multiplex electrochemical loop-mediated isothermal amplification chip for differentiating bacteria. Biosens. Bioelectron. 2014, 60, 84–91. (18) Zhang, X.;Li, Jin, Q.; X.; Jiang, C.; Lu, Y.; Tavallaie, R.; Gooding, J. J. Quantitative determination of target gene with electrical sensor. Sci. Report 2015, 5, 2045– 2322. (19) Deféver, T.; Druet, M.; Evrard, D.; Marchal, D.; Limoges, B. Real-time electrochemical PCR with a DNA intercalating redox probe. Anal. Chem. 2011, 83, 1815– 21. (20) Limoges, B.; Deféver, T.; Marchal, D. Method for electrochemically identifying target nucleotide sequences.

centration; (iv) the probe leads to an excellent LAMP detection limit, reaching the ultimate low level of a few DNA copies per electrochemical microwell. In closing, the analytical performances of LAMP achieved in the present work provide a better and more realistic assessment of the potentialities offered by real-time electrochemical LAMP. On account of the numerous advantages conveyed by LAMP (exquisite specificity, sensitivity, low running costs, short turnaround times and higher tolerance to well-known PCR inhibitors) together with the benefits of electrochemical over optical detection (integration, miniaturization, portability, robustness, low cost, insensitivity to nontransparent reaction mixtures, etc.), real-time electrochemical LAMP holds great promise for the development of inexpensive, simple, fast, robust and highly sensitive point-of-care DNA-based devices, especially those devoted to early detection and identification of microbial pathogens.

ASSOCIATED CONTENT Supporting Information Available The following files are available free of charge: Tables S1 and S2 in which are reported the primer sequences used for LAMP and PCR, including their characteristic properties, and Figures S1 and S2 showing respectively the realtime fluorescence LAMP curves of the M13mp18 phage DNA target and the real-time fluorescence PCR calibration of the F. columnare genomic DNA target.

AUTHOR INFORMATION Corresponding Authors [email protected] ; [email protected]

Author Contributions The manuscript was written through contributions of A.M., D.M. and B.L., and edited by K.B.G. The LAMPs were designed by A.M. with the contributions of F.S. and J-M.G. for the F. columnare target. A.M. has performed the experiments and K.B.G. contributed to the synthesis of PhP. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT This work was supported by the Ile-de-France region via the DIM Analytics. A. M. thanks the DIM Analytics for its Ph.D. fellowship.

REFERENCES

(1) Niemz, A.; Ferguson, T.M.; Boyle, D.S. Point-of-care nucleic acid testing for infectious diseases. Trends Biotechnol. 2011, 29, 240–50. (2) Hartman, M. R.; Ruiz, R. C. H.; Hamada, S.; Xu, C.; Yancey, K. G.; Yu, Y.; Han, W.; Luo, D. Point-of-care nucleic acid detection using nanotechnology. Nanoscale 2013, 5, 10141–54. (3) St John, A.; Price, C. P. Existing and Emerging Technologies for Point-of-Care Testing. ClinBiochem Rev. 2014, 35, 155–167. 9

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Formation. Biochem. Biophys. Res. Commun. 2001, 289, 150–4. (31) Parida, M.; Sannarangaiafh, S.; Dash, P.K.; Rao, P.V.; Morita, K. Loop mediated isothermal amplification (LAMP): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases. Rev. Med. Virol. 2008, 18, 407–21. (32) Kaneko, H.; Kawana, T.; Fukushima, E.; Suzutani, T. Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances. J. Biochem. Biophys. Methods 2007, 70,499–501. (33) Poon, L.L.M.; Wong, B.W.Y.; Ma, E.H.T.; Chan, K.H.; Chow, L.M.C.; Abeyewickreme,W.; Tangpukdee, N.; Yuen, K.Y.; Guan, Y.; Looareesuwan, S.; Peiris, J.S.M. Sensitive and inexpensive molecular test for falciparum malaria: detecting Plasmodium falciparum DNA directly from heat-treated blood by loop-mediated isothermal amplification. Clin. Chem. 2006, 52, 303–6. (34) Dugan, L.; Bearinger, J.; Hinckley, A.; Strout, C.; Souza, B. Detection of Bacillus anthracis from spores and cells by loop-mediated isothermal amplification without sample preparation. J. Microbiol. Methods 2012, 90, 280– 4. (35) Wilson, B.; Fernández, M-J.; Lorente, A.; Grant, K.B. Syntheses and DNA photocleavage by mono- and bisphenothiazinium–piperazinexylene intercalators. Tetrahedron 2008, 64, 3429–36. (36) Miranda-Castro, R.; Marchal, D.; Limoges, B.; Mavré, F. Homogeneous electrochemical monitoring of exonuclease III activity and its application to nucleic acid testing by target recycling. Chem. Commun. 2012, 48, 8772–4. (37) Mori, Y.; Kitao, M.; Tomita, N.; Notomi, T.Real-time turbidimetry of LAMP reaction for quantifying template DNA. J. Biochem. Biophys. Methods 2004, 59, 145–57. (38) Darwish, A.M.; Ismaiel, A.A.; Newton, J.C.; Tang, J. Identification of Flavobacterium columnare by a speciesspecific polymerase chain reaction and renaming of ATCC43622 strain to Flavobacterium johnsoniae. Mol. Cell Probes 2004, 18, 421–7.

Patent FR20080003143, 2008-06-05, WO2009147322 (A1), 2009-12-10. (21) Ahmed, M.U.; Nahar, S.; Safavieh, M.; Zourob, M. Real-time electrochemical detection of pathogen DNA using electrostatic interaction of a redox probe. Analyst 2013,138, 907–15. (22) Martin, A.; Bouffier, L.; Grant, K. B.; Limoges, B.; Marchal, D. Real-time electrochemical LAMP: a rational comparative study of different DNA intercalating and nonintercalating redox probes. Analyst 2016, ASAP (DOI: 10.1039/c6an00867d). (23) Ahmad, F.; Seyrig, G.; Tourlousse, D.M.; Stedtfeld, R.D.; Tiedje, J.M.; Hashsham; S.A. A CCD-based fluorescence imaging system for real-time loop-mediated isothermal amplification-based rapid and sensitive detection of waterborne pathogens on microchips. Biomed. Microdevices 2011, 13, 929–37. (24) Yi, M.Y.; Ling, L.; Neogi, S.B.; Fan, Y.S.; Tang, D.Y.; Yamasaki, S.; Shi, L.; Ye L. Real time loop-mediated isothermal amplification using a portable fluorescence scanner for rapid and simple detection of Vibrio parahaemolyticus. Food Control 2014, 41, 91–5. (25) Real-time PCR, T. Dorak, Ed., 2006, Taylor & Francis Group (ISBN: 0-4153-7734-X). (26) Notomi, T.; Okayama, H.; Masubuchi, H.; Yonekawa, T.; Watanabe, K.; Amino, N.; Hase, T. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000, 28, e63. (27) Maruyama, K.; Mishima, Y.; Minagawa, K.; Motonaka, J. Electrochemical and DNA-binding properties of dipyridophenazine complexes of osmium(II). J. Electroanal. Chem.2 001, 510, 96–102. (28) Wilson, B.; Fernández, M-J.; Lorente, A.; Grant, K.B. Synthesis and DNA interactions of a bis-phenothiazinium photosensitizer. Org. Biomol. Chem. 2008, 6, 4026–35. (29) Nagamine, K.; Watanabe, K.; Ohtsuka, K.; Hase, T.; Notomi, T. Loop-mediated isothermal amplification reaction using a nondenatured template. Clin. Chem. 2001, 47, 1742–3. (30) Mori, Y.; Nagamine, K.; Tomita, N.; Notomi, T. Detection of Loop-Mediated Isothermal Amplification Reaction by Turbidity Derived from Magnesium Pyrophosphate

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