This document is the Accepted Manuscript version of a Published Work that appeared in final form in Analytical Chemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see: https://dx.doi.org/10.1021/acs.analchem.9b02560. Experimental Comparison in Sensing Breast Cancer Mutations by Signal ON and Signal OFF Paper-Based Electroanalytical Strips Stefano Cinti,*§ Giulia Cinotti,† Claudio Parolo,α Emily P. Nguyen,α Veronica Caratelli,† Danila Mo- scone,† Fabiana Arduini†, and Arben Merkoci*α,\ § Department of Pharmacy, University of Naples “Federico II”, Via D. Montesano 49, 80131 Naples, Italy. α Nanobioelectronics & Biosensors Group, Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC and BIST, Campus UAB, Bellaterra, 08193 Barcelona, Spain. † Department of Chemical Science and Technologies, University of Rome “Tor Vergata”, Via della Ricerca Scientifica 1, 00133 Rome, Italy. \ ICREA, Institució Catalana de Recerca i Estudis Avançats, Pg. Lluís Companys 23, 08010 Barcelona, Spain. *Corresponding authors: Stefano Cinti, E-mail: Stefano.cinti@unina.it; Arben Merkoci, E-mail: arben.merkoci@icn2.cat. ABSTRACT: The development of paper-based electroanalytical strips as powerful diagnostic tools has gained a lot of attention within the sensor community. In particular, the detection of nucleic acids in complex matrices represents a trending topic, especially when focused towards the development of emerging technologies, such as liquid biopsy. The DNA-based biosensors have been largely applied in this direction and, currently, there are two main approaches based on target/probe hybridization reported in litera- ture, namely Signal ON and Signal OFF. In this technical note, the two approaches are evaluated in combination with paper-based electrodes, using a single strand DNA relative to H1047R (A3140G) missense mutation in exon 20 in breast cancer as the model target. A detailed comparison among the analytical performances, detection protocol, and cost associated with the two systems is provided, highlighting the advantages and drawbacks depending on the application. The present work is aimed to a wide audience, particularly for those in the field of point-of-care, and it is intended to provide the know-how to manage with the design and devel- opment stages, and to optimize the platform for the sensing of nucleic acids using a paper-based detection method. KEYWORDS: Paper-based, DNA detection, Signal ON, Signal OFF, Redox probe, Breast cancer The determination of nucleic acid sequences is widely reported clear advantages, including the capture of specific nucleic acids, for applications in various analytical fields, as it offers clear ad- also avoiding the use of costly and painful tissue-biopsies. It has vantages in term of specificity. Detection of bacterial infections, been successfully applied to different cancers, 8 e.g. lung, pan- food allergens, environmental pollution, and early disease diag- creas, colon, breast. nosis represents few of the enormous possibilities around the Thus far, the most sensitive approach used in liquid biopsy di- world of nucleic acids.1-3 Although the gold standard is repre- agnostics is BEAMing (Beads, Emulsion, Amplification, and sented by PCR-based approaches, the need of specialized users, Magnetics).9 However, sophisticated equipment and workflows dedicated equipment, and time-consuming procedures reduces limit its routine implementation. On the other hand, electroanal- the implementation of such technology outside the realms of the ysis is able to provide several advantages towards the develop- laboratory, i.e. in situ.4 Thus, the need for rapid and portable ment of hand-held tests, easily interrogated with portable instru- methods for analysis of real matrices, whilst also avoiding la- mentation, for analysis of complex samples, such as river water boratory-bound processes, are of high demand. In particular, and blood.10 In addition, the introduction of novel paper-based healthcare represents a field amenable to decrease the gap be- platforms obtained with easy-to-develop fabrication routes,11 tween patients and analysis; the request for point-of-care de- i.e. screen-printing and wax printing, leads to a novel concept vices are growing in parallel with the rising of precision medi- of sustainable electroanalysis, wherein the use of paper lowers cine. The detection of both DNA and RNA, found within bodily the production costs, the operative tasks, and the management fluids, to diagnose diseases and infections at early-stages, and of waste.12-13 to evaluate for eventual therapeutic treatments, represents the Concerning the detection of nucleic acids at electrochemical building block for making the therapy more effective by reduc- surfaces, the most adopted approach is by designing a recogni- ing time and under/over drug dosage. Among plenty of applica- tion probe able to specifically bind with the target of interest. tions, the detection of circulating nucleic acids, also known as The use of an electrochemical mediator is able to provide a liquid biopsy, opens up a revolutionary opportunity in cancer change in electrical signal upon recognition or interaction with detection and therapy monitoring,5-6 and was recently listed the target. Typically, two different experimental set-ups for sig- within the top 10 of emerging technologies by the World Eco- nomic Forum.7 Direct access of the bloodstream offers several nal visualization are mainly proposed, where i) the electro- attachment onto the AuNP-modified strips. A synthetic comple- chemical mediator is pre-attached at the recognition probe, or mentary 19-base single stranded DNA (5’- ii) it is added once the target has been recognized. Regarding GATGCACGTCATGGTGGCT-3’) relative to H1047R the first set-up, an electrochemical platform has been produced (A3140G) missense mutation in exon 20 in breast cancer was for single and double stranded DNA detection by designing used as the model target for developing both the assays. For se- methylene (MB) blue-tagged recognition probes and demon- lectivity study, the following sequences have been used: 1-MM strated the detection of a HIV-1 sequence.14 The presence of the (5’-GATGCACGTCTTGGTGGCT-3’), 3 -MM (5’- target was consistent with the generation of a rigid structure GATGCACGTGTAGGTGGCT-3’) and random (5′- which limited the electron transfer between MB and the elec- CCCCCCTTTTCTTTT-3′). trode. This outcome was observed as a decrease of current, i.e. Preparation of the DNA-paper based strips. Filter paper- signal OFF.14-16 The other possibility recently described by Kel- based electrodes were fabricated as described elsewhere.19-20 ley et al., was the first electrochemical assay to detect circulat- Briefly, to define the working area and to isolate it from the ing tumor DNA, associated with lung cancer,17 by means of a electrical connection, wax templates were printed onto filter pa- novel visualization principle. In this case, a positively charged per (67 g/m2, 135 mm caliper, Cordenons, Italy) using a wax electrochemical mediator (ruthenium hexamine chloride) was printer (ColorQube 8580, Xerox, USA). A three-electrode sys- added after the target was recognized by the probe. As the tem was then screen-printed by using Ag/AgCl ink (Electrodag probe-target adduct resulted with a higher density of negative 477 SS, Acheson, Italy) for the reference electrode and graphite charges, compared to the unhybridized probe, the amount of at- ink (Electrodag 421, Acheson, Italy) for the working and coun- tracted ruthenium-based mediator was elevated and thus lead to ter electrodes. Subsequently, 8 L of aqueous gold nanoparti- an increase of electron transfer between the redox mediator and cles dispersion were drop casted onto the working electrode the electrode, i.e. signal ON. area. DNA probes were then immobilized onto the paper-based In this technical note, a direct comparison between the signal strips by following a reported protocol.21 20 μL drop of the cho- ON and signal OFF approaches in paper-based platforms is pro- sen DNA probe (50, 100, 200 nM) was casted on top of the vided. As a model study, the two systems are applied to detect working electrode area and left to incubate for 60 min at room traces of H1047R (A3140G), a missense mutation in exon 20 of temperature. The empty spaces were then passivated by drop the PIK3CA gene in breast cancer, which has been reported to casting a solution of 10 mM mercapto-hexanol, left for 90 min. be present in circulating blood samples of patients.18 The de- The DNA modified strips were rinsed with distilled water and vices are fabricated using filter paper-based strips and utilize stored overnight at 4°C in the working buffer solution (50 mM gold nanoparticles to anchor the recognition probes to the sur- phosphate buffer containing 150 mM NaCl, pH=7). All the in- face of the sensor. A comparison between the two analytical cubation steps were conducted in a humid chamber to avoid sol- platforms, in terms of fabrication, operation and analytical per- vent evaporation. formance, aims to provide readers with comprehensive insight Experimental setup 1: Signal ON platform. All measure- into the capabilities and performance of the systems, whilst also ments were performed using 100 L of working solution. For placing emphasis on various relevant aspects that should be evaluation of the signal ON platform, DNA probes without re- considered before and after a measurement. dox label were initially immobilized onto the surface of the electrode (we optimized the probe concentration testing three EXPERIMENTAL SECTION different values: 50, 100 and 200 nM). Prior to the analysis of the sample, the electrode was rinsed with distilled water to re- move any remaining storage buffer. The working solution (50 Reagents and Equipment. Chloroauric acid (HAuCl4), sodium mM phosphate buffer containing 150 mM NaCl (pH=7)) was borohydride, sodium citrate, sodium chloride, potassium chlo- then added to the working area and left to incubate for a mini- ride, potassium dihydrogen phosphate (KH2PO4), potassium mum of 20 min, to allow for hybridization of the probe to the monohydrogen phosphate (K2HPO4), ruthenium hexamine target DNA. Subsequently, 2 L of 50 mM ruthenium hexamine chloride (Ru(NH3)6Cl3), potassium ferricyanide (K3Fe(CN)6), chloride were added and gently mixed with a 20 L pipette tip potassium ferrocyanide (K4Fe(CN)6), 6-mercapto-1-hexanol and left for 10 mins to allow for the electrostatic attachment of (HS(CH2)6OH), and tris(2-carboxyethyl)phosphine hydrochlo- Ru(NH3)63+ around the immobilized probe and/or target/probe ride (TCEP) were purchased from Sigma-Aldrich (St.Louis, adduct. The paper-based strip was then gently rinsed with dis- MO, USA). The Methylene Blue (MB)-tagged probe as the re- tilled water to remove excess of the redox mediator. It should dox reporter, was purchased from Biosearch Technologies (No- be highlighted that, the redox mediator (Ru-based) is added to vato, CA, USA) while the un-tagged probe and all the targets the working solution after the target/probe adduct is formed. were purchased from IBA GmbH (Göttingen, Germany). A The sensing principle of this particular platform exploits the multi-eight portable PalmSens Instrument (PalmSens, Nether- electrostatic attraction between the positively charged redox lands) was used for the electrochemical measurements. mediator and the negatively charged backbone of the DNA: tar- get/probe adduct results more negatively charged than probe, DNA sequences. In order to compare the two approaches, thus more Ru-based redox mediator is attracted and the signal screen-printed paper-based electrodes were fabricated and func- increases. To obtain an electrochemical measurement, the elec- tionalized with two probes: 5′-HS-(CH2)6- trode was rinsed with distilled water and covered with 100 L AGCCACCATGACGTGCATC (CH2)7-NH2-MB-3′ and 5′- of 50 mM phosphate buffer containing 150 mM NaCl (pH=7), HS-(CH2)6-AGCCACCATGACGTGCATC-3’ for the signal and the voltammetric detection was performed by DPV, scan- OFF and ON platform, respectively. Both the probes were mod- ning the potential from -0.1 to -0.5 V. The presence of the target ified with a thiol end at the 5′-side, to allow for the covalent 2 was quantified in comparison with the signal recorded in ab- high concentration of Ru-based mediator and high background sence of the target. The measurements were performed follow- signal of ferricyanide, the use of double mediators was not con- ing the optimized protocol reported elsewhere.17 venient. In addition, avoiding the use of ferricyanide means lowering both the reagents and the tasks for end-users involved in the assay. A survey of literature has revealed that the use of Scheme 1. Signal ON and Signal OFF platforms for paper- 200 nM of DNA probes corresponds to a density of ca. 1012 based electrochemical detection of nucleic acids. molecules/cm2 onto the electrode.24 Figure 1. Evaluation of the DNA probe effect for the Signal ON system, using a) 50, b)100, and c) 200 nM of recognizing probe in absence (black line) and in presence (red line) of 30 nM tar- get. Working solution is 50 mM phosphate buffer containing 150 mM NaCl (pH=7). Current increase of 25% and 80% were observed for 100 nM and 200 nM densities, respectively, while the current remained Experimental setup 2: Signal OFF platform. The signal OFF unchanged for the 50 nM probe density. The higher signal sensor fabrication was the same as the one reported for signal change observed for the sensor with more probes on the surface ON, with the only exception that we used the MB-modified depends on two important factors: A) the higher probability of DNA probe (scheme 1). Prior to the analysis of the sample, the binding target molecules for a higher probe density and B) the electrode was rinsed with distilled water to remove any remain- accumulation of more Ru(NH3)63+ on the electrode surface due ing storage buffer, then, 100 L of buffer solution (50 mM to the presence of more probe/target adducts. Regarding the phosphate buffer containing 150 mM NaCl (pH=7)) was drop lack of signal change for the sensor fabricated using 50 nM casted onto the electrodes. Voltammetric measurements were probe, it depends on the fact that we are in the high ligand-de- then recorded until a constant peak signal was obtained (this pletion regime (probe concentration approximately of 5 nM vs “pretreatment” ensures that the signal decrease observed during a Kd of 25 nM).25 Another important aspect to keep in mind the measurement is related to the target binding rather than the when using low deposition concentration in a signal ON format removal of weak adsorbed probes). Successively, the single on a paper-based device is that it requires harsher washing step stranded DNA target was added and quantified by performing than those generally used on plastic/ceramic SPE or glassy car- voltammetric method in the range between -0.1 and -0.5 V as bon electrode. This harsher washing can indeed affect the num- optimized and reported elsewhere.14,19 Experimentally, when ber of capture probes present on the working electrode, showing the target was present, a decrease of the recorded peak was ob- a lower signal that expected (probes/AuNPs can be washed served due to the formation of a rigid duplex structure which away during the washing step thus the available negative reduces the interaction, i.e. electron transfer, between MB and charges are lower thus the measurement will produce a lower electrode. signal), Figure S2 in Supporting Information File. Given the higher signal change, the 200-nM DNA probe modified strip RESULTS AND DISCUSSION has been adopted to detect breast cancer target in the concentra- tion range between 0.1 and 3000 nM (Figure 2). Moreover, the use of single-use platforms allows for evaluating the repeatabil- DNA detection with signal ON approach. In analytical chem- ity of the platform. istry, the so-called “signal ON” approaches are those that pro- vide an increase of detectable signal when the target analyte is present. In electroanalysis, classic are enzymatic biosensors where one of the enzymatic by-product gives the signal for quantification, for example the operation mechanism used in a glucometer.22 In the field of nucleic acids detection, in particu- lar for liquid biopsy, a novel class of electrochemical-based genosensors for detection of nucleic acids (both DNA and RNA) has been reported. Ru(NH3)63+ is accumulated at the working electrode through electrostatic attraction to the nega- tively charged DNA, and targets are detected adding ferricya- nide (Fe(CN)63−).23 In our case, the addition of ferricyanide was omitted as it did not improve the sensitivity (Supporting Infor- mation, Figure S1): under the experimental conditions, both the 3 electrode). As for the previous study, the same solution of probe densities has been taken into account for strip modification, i.e. 50, 100 and 200 nM, and the results are displayed in figure 3. Figure 3. Evaluation of the DNA probe density on the Signal ON system, using a) 50, b)100, and c) 200 nM of recognizing probe in absence (black line) and in presence (red line) of 30 nM target. Working solution is 50 mM phosphate buffer con- Figure 2. Binding curve (fitted to the hyperbolic Langmuir iso- taining 150 mM NaCl (pH=7). therm - R2 value of 0.990) obtained with the Signal ON ap- proach by using screen-printed electrodes onto filter paper chal- Unlike the behavior observed for the signal ON platform, here, lenged with target concentration ranging between 0.1-3000 nM the maximum signal change, -40%, was obtained for the sensors target. Working solution is 50 mM phosphate buffer containing prepared using the 50 nM probe solution. While 100 nM and 150 mM NaCl (pH=7). 200 nM produced respectively ca. -25% and -6%. The complete opposite behavior compared to signal ON sensors depends on The detection of mutations related to breast cancer in standard the sensor architecture itself. In fact in signal OFF sensors we solution has been consistent with a detection limit of 5 nM start from the maximum signal already and we want to see a (computed using the 3 sigma method), a calculated affinity of signal decrease for the minimum amount of target possible. 26  5 nM (calculated fitting the data to the hyperbolic Lang- Thus having a higher packing density means that we need more muir isotherm), and a linear range between 10 and 100 nM (cal- target molecules to observe a signal decrease (the more probes culated as the transition from 10% to 90% site occupancy).26 on the surface, the more probes need to change conformation The repeatability of the system was described by a relative before seeing a signal change). Although the largest variation standard deviation of 15% (n=8) calculated on the binding current was obtained with the lowest probe density, 50 nM, the curve. The eight calibration points of each binding curve are the signal acquired contained a high level of noise. Thus, further result of three experiments that have been performed with sin- measurements were conducted using densities of 100 nM as the gle-use strips: in total, twenty-four strips have been used for best compromise for current variation and signal quality. Simi- each binding curve, evidencing a satisfactory reliability associ- lar to the signal ON platform, the analytical performance of the ated with the entire manufacturing process. After evaluation of signal OFF platform was evaluated in buffer solution with var- the performance of the signal ON platform in standard solution, ying concentrations of the target (Figure 4) a real matrix scenario was simulated using whole and 50%, 25% and 12.5%-diluted blood as model. Due to the complex compo- sition of blood, containing proteins, lipids, salts, etc., the foul- ing of the modified electrode, due to stacking and interactions of the components and the surface, becomes problematic (it ste- rically prevents the accumulation of Ru(NH3)63+ on the elec- trode surface and negatively charged contaminants can actively sequestrate it) and the washing step proved to be inadequate for the complete removal of the matrix from the working electrode. We obtained no electrochemical signal. While this problem could be solved using plastic or ceramic SPE, which can be mildly washed and the complete removal of contaminant achieved, in paper-based platform is more problematic. The net of cellulose fibers entraps cells and proteins making the removal of fouling agents extremely difficult, whilst a vigorous washing step can cause damages to the hybrid nanocomposite, i.e. AuNPs/DNA, onto the working electrode surface. Figure 4. Binding curve (fitted to the hyperbolic Langmuir iso- therm - R2 value of 0.993) obtained with the Signal OFF ap- DNA detection with signal OFF approach. The second plat- proach by using screen-printed electrodes onto filter paper chal- form tested was made with the same recognition probe as afore- lenged with target concentration ranging between 0.1-3000 nM mentioned; however, in this case, MB was covalently attached target. Working solution is 50 mM phosphate buffer containing at the free end of the probe. Although it could appear as a small 150 mM NaCl (pH=7). Inset: 0.1-3000 nM target detected in difference, this characteristic makes this platform reagentless 12.5-% diluted blood. (the redox mediator is already included within the paper-based strip) and utilizes a different sensing mechanism (the formation of a rigid probe/target duplex reduces the electron transfer at the In this case, as shown in Figure 4, the limit of detection was calculated to be 6 nM (using the 3 sigma method), the binding 4 constant was 40  6 nM (for the fitting to the hyperbolic Lang- strategy requires extra steps, namely additional washing steps muir isotherm), and the linear range was between 20 and 300 where specific attention is required, as there is a high risk of nM (calculated as the transition from 10% to 90% site occu- removal of the pre-embedded recognition elements, thus affect- pancy). The repeatability of the system was described by a rel- ing the repeatability of the measurements. We must say though ative standard deviation of 12% (n=8). One of the main differ- that additional washing steps may result useful to discriminate ences of the signal OFF platform, in comparison to the signal between target with one or more mismatches, whose weaker ON, is the non-incorporation of the redox mediator onto the bindings to the probe could be more easily detected. Moreover, working surface during the fabrication steps. This detail is ad- the post washing step also limits the platform for analysis of vantageous to this type of detection as it negates the require- whole blood. In fact, because of the time required for sufficient ment of post washing steps for the removal of excess mediators, hybridization between probe and target, build up or fouling as seen with signal ON platforms, and allows for testing with from the other components of the blood matrix, such as blood complex matrices. As aforementioned, complex matrices, such cells and proteins, can occur and the post washing step becomes as blood, present their own challenges, namely fouling of the ineffective, resulting in false positive measurements. However, electrode surface in this case. As such, a comparison of various once again, it should be noted that the use of other substrates, dilutions of whole blood, 50%, 25% and 12.5%, and its effects i.e. glass, plastic or ceramic, could minimize this issue during on the signal OFF platform was conducted. The optimal dilution the washing steps. Another possible solution is the implemen- was revealed to be 12.5% and, upon interaction with the syn- tation of blood filters on top of the working electrode, minimiz- thetic H1047R (A3140G) DNA strands between 0.1-3000 nM, ing the presence of cells on the electrode surface. On the other the binding constant was observed to be 23  4 nM, which was hand, the measurement with the Signal OFF platform proves to of the same order of magnitude of the measurements obtained be easier in terms of operation steps; however, it should be con- in standard solution. In addition, the calculated detection limit sidered that the cost of a tagged probe is 4-5 fold higher than was also similar within the experimental errors. It should be that of the un-tagged probes used in the former system. In addi- noted that the possibility of working in the cathodic range of tion, from an analytical point of view, the signal OFF could suf- applied potentials, (MB exchanges electrons from -0.5 to -0.1 fer from the presence of background signals which may limit its V) reduces the possible interference by easily oxidizing com- application for trace detection. Instead, regarding the signal ON pounds which are physiologically occurring,27 e.g. ascorbic platform, the alternative use of neutral probes, e.g. PNA, as re- acid, uric acid, and acetaminophen. ported in literature,28 can represent an advantage this method, Signal ON vs. Signal OFF. Within the field of (bio)sensors, by lowering the background current (because the positively and more specifically in the field of nucleic acid detection, this charged redox probe is not electrostatically attracted in presence technical note is a demonstration of an experimental tutorial to- of a neutral probe), but it would make the system even more wards optimal selection of an electroanalytical platform. Of expensive, so it should be carefully weighted. Moreover, the se- course, factors such as facilities, specialization of personnel and lectivity of both approaches has been tested in presence of 1 specific analytical needs, will influence the selection of the mismatch, 3 mismatches and fully-random probes (Supporting most suitable platform. A comparative overview of the differ- Information, Figure S3 and Figure S4): as reported, no relevant ences between these techniques are summarized in table 1. differences have been highlighted. Table 1. Comparison between Signal ON and Signal OFF onto CONCLUSIONS paper-based strips for nucleic acid detection. The rising demand for user-friendly analytical devices high- lights the versatility and potential of paper-based tools as a piv- Signal ON Signal OFF otal forerunner for the development of (bio)sensors. Emerging 3+ Principle Ru(NH3)6 electro- Probe is MB- technologies, such as liquid biopsy, can be coupled with elec- statically attracted to tagged. The pres- troanalytical platforms and corresponding technologies with DNA. More is the ence of [Target] easy fabrication routes. In this technical note, the two most [Target], signal in- reduces the MB common strategies for detection of nucleic acids are compared creases. contact with the in a paper-based platform. Signal ON and Signal OFF systems electrode, and the have been implemented towards the determination of a single signal decreases. strand DNA relative to a missense mutation in breast cancer. Procedure 1.Probe/Target bind- 1.Probe/Target Although the two methods, within the experimental setup, have ing, 2. Addition redox binding, 2. Meas- showed similar analytical performance, e.g. detection limit in probe, 3. Washing, 4. urement. the low nM level and calculated binding constant in the nM Measurement. range, some differences have been observed. For each platform, Performance Low nM Low nM some key factors are to be considered prior to the fabrication and use of the paper-based strip. For the signal OFF platform, Real matrices Requires washing No washing as the redox mediator is already integrated into the platform, needed this negates the requirements of any additional reagents. How- Cost Cheap Expensive ever, the cost of the signal OFF platform is typically 4-5 fold higher than the signal ON platform, due to the modification of the probe with a tag. From the other side, the main limitations Although the performance of the two detection systems, devel- of utilizing the “cheaper” signal ON system is the necessity of oped onto paper-based electrochemical strips, display similar external chemicals, the need of extra washing steps and the pos- detection limits in the range of low nanomolar, there are still sibility of electrode fouling from complex matrices. Both ap- various considerations that need to be addressed. The signal ON proaches are promising in terms of costs and miniaturization; 5 however, for in-situ application performed by non-specialized 11) López-Marzo, A. M.; Merkoçi, A. Lab Chip 2016, 16, 3150- users, the Signal OFF represents the optimal compromise of 3176. manufacturing and ease of use. 12) Arduini, F.; Cinti, S.; Scognamiglio, V.; Moscone, D. Compr. Anal. Chem. 2017, 77, 385. AUTHOR INFORMATION 13) Yang, Y.; Noviana, E.; Nguyen, M. P.; Geiss, B. J.; Dandy, D. S.; Henry, C. S. Paper-based microfluidic devices: emerging Corresponding Author themes and applications. Anal. Chem. 2016, 89, 71-91. *E-mail: stefano.cinti@unina.it *E-mail: arben.merkoci@icn2.cat 14) Patterson, A.; Caprio, F.; Vallée-Bélisle, A.; Moscone, D.; Plaxco, K. W.; Palleschi, G.; Ricci, F. Anal. Chem. 2010, 82, 9109-9115. ACKNOWLEDGMENT 15) Ricci, F.; Plaxco, K. W. Microchim. Acta 2008, 163, 149-155. S.C. acknowledges Marie Skłodowska-Curie Actions Individual 16) Li, H.; Somerson, J.; Xia, F.; Plaxco, K. W. Anal. Chem. Fellowship, this project has received funding from the European 2018, 90, 10641-10645. Union’s Horizon 2020 research and innovation programme under 17) Das, J.; Ivanov, I.; Sargent, E. H.; Kelley, S. O. J. Am. Chem. the Marie Skłodowska-Curie grant agreement No 794007. C.P. Soc. 2016, 138, 11009-11016. acknowledges Marie Skłodowska-Curie Actions Individual Fel- lowship, this project has received funding from the European Un- 18) Levine, D. A.; Bogomolniy, F.; Yee, C. J.; Lash, A.; Barakat, ion’s Horizon 2020 research and innovation programme under the R. R.; Borgen, P. I.; Boyd, J. Clin. Cancer Res. 2005, 11, 2875- Marie Skłodowska-Curie grant agreement No 795635. The ICN2 is 2878. funded by the CERCA Programme / Generalitat de Catalunya. The ICN2 is supported by the Severo Ochoa program of the Spanish 19) Cinti, S.; Proietti, E.; Casotto, F.; Moscone, D.; Arduini, F. Ministry of Economy, Industry and Competitiveness (MINECO, Anal. 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Sci. 2005, 102, 16368- 16373. 10) Labib, M.; Sargent, E. H.; Kelley, S. O. Chem. Rev. 2016, 116, 9001-9090 Table of Contents 6 7