screen-printed polyaniline-based electrodes for the real-time

challenge, the use of screen-printed electrochemical sensor is reported. To achieve the detection of the DNA amplification reaction, a real-time monit...
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Screen-printed polyaniline-based electrodes for the real-time monitoring of LAMP reactions David Gosselin, Maxime Gougis, Mélissa Baque, Fabrice P Navarro, Mohamed Naceur Belgacem, Didier Chaussy, Anne-Gaelle Bourdat, Pascal Mailley, and Jean Berthier Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02394 • Publication Date (Web): 12 Sep 2017 Downloaded from http://pubs.acs.org on September 12, 2017

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Analytical Chemistry

SCREEN-PRINTED POLYANILINE-BASED ELECTRODES FOR THE REAL-TIME MONITORING OF LAMP REACTIONS David Gosselina,b,c*, Maxime Gougisa,b, Mélissa Baquea,b, Fabrice P. Navarroa,b, Mohamed N. Belgacemc, Didier Chaussyc, Anne-Gaëlle Bourdata,b, Pascal Mailleya,b, and Jean Berthiera,b a

Univ. Grenoble Alpes F-38000 Grenoble, France.

b

CEA, LETI, MINATEC Campus, F-38054, Grenoble, France.

c

Univ. Grenoble Alpes, CNRS, Grenoble INP, LGP2, F-38000 Grenoble, France

*Corresponding author: [email protected]

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ABSTRACT: Nucleic acid amplification testing is a very powerful method to perform efficient and early diagnostics. However, the integration of a DNA amplification reaction with its associated detection in a low-cost, portable and autonomous device remains challenging. Addressing this challenge, the use of screen-printed electrochemical sensor is reported. To achieve the detection of the DNA amplification reaction, a real-time monitoring of the hydronium ions concentration, a by-product of this reaction, is performed. Such measurements are done by potentiometry using PolyAniline (PAni)-based working electrodes and Silver/Silver Chloride reference electrodes. The developed potentiometric sensor is shown to enable the real-time monitoring of a LAMP reaction with an initial number of DNA strands as low as 10 copies. In addition, the performance of this PAni-based sensor is compared to fluorescence measurements and it is shown that similar results are obtained for both methods.

KEYWORDS: PolyAniline; Potentiometry; LAMP; Pointof-care diagnostics; NAAT; Screen-printing To lead an efficient fight against infectious diseases and epidemics, reliable and sensitive diagnostics are of utmost importance. Diagnostic is the first step toward treatment and without it, broad-spectrum antibiotic are generally used, 1 which may result in an increase microbial resistance . Nucleic Acid Amplification Tests (NAAT) are powerful tools to perform such diagnostic. In fact, due to the amplification step, a very small amount of DNA copies can be detected, allowing for early diagnostics. Presently, such diagnostics are routinely performed in medical laboratories but their availability at the Point-of-Care is still limited. This raises an issue when medical environments are not easily accessible, in 2 particular in developing countries . In fact the World Health Organization has established a criteria for diagnostic systems which are aimed to be used at the Point-of-Care: they must fulfill the ASSURED (Affordable, Sensitive, Specific, Userfriendly, Rapid and Robust, Equipment-free and Delivered)

requirements . In the case of NAAT diagnostics, while the development of isothermal amplification techniques has simplified the thermal management of the device, the detection remains challenging to integrate in a portable device. Numerous detection techniques have been used for NAAT. They can be classified according to two categories: the endpoint measurements and the real-time monitoring techniques. The first methods are on growing trend because they can offer simple and visual readouts. For example, lateral 4–6 flow assays have been used to perform such a detection . However because they must be used after the amplification step, their use require valves or manual operations, which either make more complex the fabrication of the system or make them less user-friendly. Other techniques based on 7 8 turbimetry or pH sensitive dyes allow for naked-eye, visual detection. Both of them detect by-products of the DNA amplification instead of the DNA strands. Indeed, it is established that each time the DNA polymerase adds a dNTP to a new DNA strand, a pyrophosphate ion and a hydronium ion 9 are released . Thus by detecting one of these two byproducts, one can detect the DNA amplification. While the turbimetry method detects the precipitation of the pyrophosphate ion with a magnesium ion, the second one detects the pH change of the solution with a pH sensitive dye. However the detection by naked eye only gives a Yes/No result. Among the real-time techniques, if the fluorescent detec10–13 tion is the most used , other techniques based on voltammetry have been developed to better comply with requirements, such as low-cost and integration, needed for 14 point-of-care devices . Although electrochemical measurements during a DNA amplification have been reported since 15–17 2003 , the first real-time monitoring of a PCR reaction 18 have been demonstrated in 2009 through the monitoring of the consumption of free electroactive deoxynucleotide triphosphates (dNTPs). Afterwards methods based on DNA 14,19–21 intercalating redox probes have been reported , allowing 22 the detection of a single copy of double-stranded DNA . These latter offer the possibility for DNA quantification through the real-time monitoring of the amplification. The approach used in this article is to continuously monitor the pH of the solution during the DNA amplification, to

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obtain quantitative measurements. Although such a monitoring has been demonstrated with ion sensitive field effect 23,24 , these latter are based on semitransistors (ISFET) conductor technologies which are expensive and therefore less suitable for Point-of-Care systems. In the present study, the development of an electrochemical detection based on printed electrodes can ensure a low-cost and high throughput production of the detection system using screen-printing technologies. Besides, in comparison to voltammetric measurements, the potentiometric approach allows for “on-thefly” measurements. In fact the potentiometric response of the system directly provides the meaningful information, i.e. the potential difference between the working and the reference electrode, and thus does not require sequential operations (in voltammetry first a potential sweep is performed and then the peak current value has to be extracted). To achieve such a detection, PolyAniline (PAni) is used to build a pH potentiometric sensor. The pH-sensitivity of PAni 25–28 has been largely discussed in the literature . It is established that PAni is subject to both RedOx and protonationdeprotonation mechanisms. PAni has three levels of oxidation, namely leucoemeraldine (LE), emeraldine salt (ES), and pernigraniline (PE). Because the RedOx reaction between the ES and the PE forms implies protons, the equilibrium poten27,29 tial of this reaction is pH-dependent , and PAni can be used for potentiometric measurement of pH. Although PAni 30,31 has already been use for potentiometric biosensors , its use for the monitoring of a DNA amplification reaction has not been reported yet. It is demonstrated in this paper that the use of PAni-based potentiometric sensor is a promising approach to allow an integrated real-time monitoring at lowcost and low energy consumption. In fact, the sensor presented in this article is shown to be easily integrated in a microfluidic system and is envisioned to be a basic unit of a 32 more complex system which would integrate Joule heating 33 and hot embossing .

EXPERIMENTAL SECTION Screen-printed electrodes The reported potentiometric measurement is based on a PAni-based working electrode and a silver/silver chloride reference electrode. The latter is made with the DuPont 5874 (DuPont, USA) silver/silver chloride paste. Its potential is fixed by the chloride ions concentration, and thus does not depend on the pH of the solution. The working electrode is coated with PolyAniline (purchased at Rescoll, France), 26,28,29 which equilibrium potential depends on the pH . In the designs used for this study, both the reference and the working electrodes have a disk shape. Designs with either two working electrodes of 1mm of radius (Figure 1A), or four working electrodes of 0.75mm of radius have been printed. For this study, a 175µm-thick foil of PolyCarbonate (PC) is used as a substrate for the screen-printing. All the screenprinting steps, except the deposition of the PolyAniline, are performed by Séribase Industrie (Chateau-Gontier, France). The PolyAniline coating is made by drop casting. Although done by drop casting for simplicity, the PAni layer will also be screen-printed for the industrial device.

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micro-machined microfluidic chamber (Figure 1B). This chamber is machined in a 1.2mm-height PMMA sheet with a Datron M7HP equipment (DATRON, Germany). It has a height of 500µm, a length of 10mm and a width of 4mm. Double-sided tape is used to bond the machined chamber to the screen-printed PC foil.

Figure 1. Pictures of the screen-printed electrodes (A) and of its integration in a microfluidic chamber (B).

LAMP reaction Bst 2.0 DNA Polymerase, purchased from NEB (New England Biolabs, USA), is used to perform the LAMP reactions. DNA samples consist in bacillus thuringiensis (Bt) DNA with different final concentrations according to the experiment. The associated primers were purchased at Eurofins Genomics (Eurofins Scientific Group, Germany). The LAMP buffer was 8 based on the work reported by Tanner and al. and adjusted to pH 9. All the reagents and the DNA sample are mix together just before to be pipeted inside the microfluidic device. The inlet and outlet of the device are then sealed with a MicroAmp® tape (Thermo Fisher Scientific, USA) to avoid evaporation. During the LAMP reaction, the temperature is maintained constant at 65°C by using a MJ Research PTC-200 Thermal Cycler (Marshall Scientific, USA).

Acquisition of the potentiometric data The acquisition of the potentiometric data is performed using an electronic acquisition card, developed by CEA-LETI 34 . Note that this acquisition card is consistent with a highly portable system because it is as small as a credit card, powered by a battery and can transfer data by Bluetooth.

RESULTS AND DISCUSSION The potentiometric response

Microfluidic integration

Figure 2. Calibration curve of the screen-printed PAni-based potentiometric sensor. Data extracted from six different electrodes.

The microfluidic integration of the printed electrodes is done by using the screen-printed PC foil as a cover for a

First of all, the potentiometric response of the PAni electrodes was investigated. The calibration curve of the PAni-

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based electrodes is depicted in Figure 2 (more details on the experiments leading to this calibration are available in the Supporting Information). A linear calibration is obtained between the pH and the equilibrium potential which is consistent with other calibra25,29–31 . According to the tion curves found in the literature aforementioned references, the slope of these calibration curves range from -55 and -90 mV/pH unit, depending on the fabrication process of the electrodes. In fact, because the second RedOx reaction of the PAni implies two hydronium ions for one electron, it should have a sensitivity of 120mV/pH. However the presence of others protonation/deprotonation processes can lower this dependency, and can explain the value of -83mV/pH unit obtained in the present study. Nevertheless, the sentivities measured for our drop cast PAni pH electrodes are reproducible.

Detection of a LAMP reaction Having established the robustness and the reproducibility of the reported PAni-based electrodes, their use for the detection and the monitoring of a LAMP reaction will now be studied. Such a detection relies on the fact that each time a DNA polymerase adds a nucleotide during DNA elongation, an hydronium ion is released. This lead a large increase of the concentration of hydronium ions, along with the number of DNA strands, during the amplification process. Thus, continuously measuring the pH with the PAni-based electrodes will allow the monitoring of the DNA amplification. Firstly, let us briefly discuss the effect of the temperature on our system. Indeed, while the potentiometric response is analyzed at the lab temperature ( 22°) the LAMP reaction occurs at 65°C. If it is well known that temperature has an impact on the equilibrium potential according to the Nernst law, it is also established that it can affect chemical reaction constants. In particular, the self-dissociation equilibrium of 35 water has been demonstrated to depend on temperature . In other words, the pH of an aqueous solution depends on the temperature. Therefore the temperature dependency of the potentiometric measurement is difficult to establish. However let us recall that the LAMP reaction is an isothermal amplification, which means that there is no change of temperature during the reaction. Thus, once the system is at its equilibrium temperature, the variation of the potentiometric signal reflects the variation of pH. It turns out that this pH variation is the main parameter to monitor during a DNA amplification assay. Let us remind that the important results to measure during DNA amplification-based diagnostic are whether the amplification occurs or not, and if so when does it happen. While the first result gives the qualitative, Yes/No answer, the second one provides quantification. To achieve such measurements, there is no need for pH quantification. The variation of the potentiometric measurement, reflecting the variation of pH, is thus sufficient for the purpose of such a test. Figure 3 shows the normalized variation of the measured potential associated with positive LAMP reactions and with negative LAMP reactions. The positive LAMP reaction, from 5 a sample with 10 copies, has been conducted three times, in three different systems. The three measurements exhibits a highly similar behavior demonstrating the reproducibility of the measurements. Two different negative reactions have been performed: one without DNA copy and Bst 2.0 enzyme (named enzyme-free blank) and one without DNA copy but

with the Bst 2.0 enzyme (named reactive blank). While the potentiometric measurement of the first one remains constant, an increase of potential for the second blank is observed after 17 min. This increase may be due to possible non-specific self-amplification of the primers and, more probably, to the adsorption of the enzyme on the PAni electrode. In fact, while the PAni is polycationic, the Bst 2.0 enzyme is polyanionic (Theoretical isoelectric point: 5.32, data provided by NEB), and electrostatic adsorption may 36,37 occur . Nevertheless, the positive LAMP reactions exhibit a clear increase of the potentiometric measurement after ~16min, depicting the pH decrease expected during the LAMP amplification, and are well-distinguishable from the negative reactions. Besides the shape of these positive curves is consistent with the sigmoid curves usually observed with fluorescent monitoring. Further validation of pH shift (using a pH sensitive dye) and of LAMP amplification (through electrophoresis analysis) are presented in Supporting Information.

Figure 3. Comparison of the potentiometric responses be5 tween 3 positive (10 copies) and the negative amplifications. In addition, the potentiometric response of our screenprinted PAni-based electrodes have been studied for different initial DNA quantities. The DNA amplification was successfully detected for an initial number of DNA strands rang5 ing from 10 to 10 (Figure 4a). By doing a baseline correction using the reactive blank, one obtains Figure 4b. In a similar way as for fluorescent measurement, it is shown that the lower the number of DNA strands in the sample, the longer before obtaining a signal. Finally the performance of the presented detection method has been assessed in comparison with fluorescence measurements. To allow for a parallel measurement of pH and fluorescence, the cresol red dye used in the previous experiments has been replaced with EvaGreen dye (Jena Bioscience, Germany). During this experiment, a fluorescent picture of the microfluidic chamber was taken every 30s during the LAMP reaction. The image was next processed with the ImageJ software to extract the mean grey level of the microfluidic chamber. Figure 5 compares the fluorescent monitoring of the LAMP reaction to the potentiometric one. The two curves depict the monitoring of the same LAMP reaction but with two different detection methods. The two results are very similar. Even if the fluorescent signal increases faster than the potentiometric signal at the beginning of the detectable variation, they start to increase around the same time. In fact, the potentiometric measurement shows only a couple of minutes of

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delay. This delay may be explained by the fact that, contrary to fluorescence measurements which are performed in the volume of the chamber, the electrochemical measurement is done at the surface of the chamber, where the electrodes are located. Thereby, the pH potentiometric monitoring appears to be a highly promising method for real-time monitoring of a DNA amplification.

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mV/pH unit (@22°C), has been demonstrated for the reported PAni-based electrodes. Because the pH level changes during a DNA amplification, due to the DNA polymerase activity, the pH sensitive PAni-based electrodes are able to provide with an efficient monitoring of such a reaction. In addition to the fact that the presented detection method exhibits a very similar response to fluorescence measurement, it has been shown to be sensitive to the initial DNA concentration and to be able to detect a quantity as low as 10 copies of DNA. Besides, the fabrication of these electrodes by screen-printing processes allows for a low-cost production with a relative high throughput. Such screen-printed electrodes can, in addition, easily be embedded in a microfluidic device and the potentiometric measurement allows for a compact and portable acquisition system. Therefore, PAnibased electrodes appear to be a convenient way for a simple, integrated, reliable and cheap detection method for nucleic acid amplification diagnostics at the point-of-care.

ACKNOWLEDGEMENTS The authors are thankful to Perrine Viargues and Remco Den Dulk who have provided the instrumentation for the fluorescence experiment.

ASSOCIATED CONTENT Supporting Information Further details on the experiments describing the potentiometric response of the PAni-based electrodes; colorimetric response of the system; and electrophoresis analysis of amplification products.

REFERENCES (1) Figure 4. A) Comparison of the potentiometric responses for 5 4 3 2 different initial DNA amounts (10 , 10 , 10 , 10 , 10 and 0 copies). B) Comparison of the baseline corrected measurements for different initial DNA amounts.

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Figure 5. Comparison between the presented potentiometric detection method and the routinely used fluorescence one.

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CONCLUSION In conclusion, it has been shown in the present study that screen-printed PolyAniline-based electrodes are able to provide with a continuous potentiometric measurement of the pH during a LAMP reaction. A linear calibration of the potentiometric measurement of the pH, with a slope of -83

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