Biosensors and Bioelectronics 54 (2014) 102–108

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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios

Hybridization chain reaction performed on a metal surface as a means of signal amplification in SPR and electrochemical biosensors Fabio M. Spiga a, Attila Bonyár b, Balázs Ring b, Manuele Onofri c, Alessandra Vinelli d, Hunor Sántha b, Carlotta Guiducci a, Giampaolo Zuccheri c,d,e,n a

Institute of Bioengineering at the Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland Department of Electronics Technology at the Budapest University of Technology and Economics, Budapest, Hungary c Interdepartmental Center for Industrial Research on Health Sciences & Technologies, University of Bologna, Via Irnerio 48, Bologna 40126, Italy d Department of Pharmacy and Biotechnologies at the University of Bologna, Via Irnerio 48, Bologna 40126, Italy e Istituto Nanoscienze of the National Research Council, S3 Laboratory, Italy b

art ic l e i nf o

a b s t r a c t

Article history: Received 24 July 2013 Received in revised form 1 October 2013 Accepted 22 October 2013 Available online 31 October 2013

A more specific and intense signal is desirable for most kinds of biosensors for biomedical or environmental applications, and it is especially so for label-free biosensors. In this paper, we show that hybridization chain reaction (HCR) can be exploited for the easily detectable accumulation of nucleic acids on metal surfaces as an event triggered by specific recognition between a probe and a target nucleic acid. We show that this process could be exploited to increase the sensitivity in the detection of nucleic acids derived from a pathogenic microorganism. This strategy can be straightforwardly implemented on SPR biosensors (commercial or custom-built) or on label-free electrochemical biosensors. Together with signal amplification, HCR can serve as a confirmation of the specificity of target recognition, as it involves the specific matching with a separate base sequence in the target nucleic acid. Furthermore, the kinetics of the target binding and the HCR can be easily distinguished from each other, providing an additional means of confirmation of the specific recognition. & 2013 Elsevier B.V. All rights reserved.

Keywords: DNA biosensors Hybridization chain reaction Surface plasmon resonance Label-free biosensors Electrochemical biosensors

1. Introduction The interest in the detection of ever smaller amounts of DNA or RNA and of a high fidelity in sequence recognition has recently increased, due to the need of better performance in the detection of pathogenic organisms (Wang et al., 1997) in diagnostics and environmental analysis, in cancer diagnosis (Topkaya et al., 2012), and in the identification of genetic diseases (Marrazza et al., 2000). Several laboratory-based analytical techniques, such as real-time PCR, have proven to be very sensitive in DNA detection (Zhu et al., 2012), however they are often complex, expensive and not apt for field deployment. Biosensors have attracted increasing interest due to their easy deployment in point-of-need automated analysis systems (Choi et al., 2011b). Surface plasmon resonance (SPR) is an optical phenomenon that occurs in the dielectric medium close to a thin metallic surface. It is exploited for the characterization of molecular binding to surface-immobilized ligands, and therefore also as a detection technique in biosensing. SPR allows for the realtime and label-free detection of interactions amongst nucleic n Corresponding author at: Interdepartmental Center for Industrial Research on Health Sciences & Technologies, University of Bologna, Via Irnerio 48, Bologna 40126, Italy. Tel.: þ39 512094388. E-mail address: [email protected] (G. Zuccheri).

0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.10.036

acids, with low sample consumption. The standard SPR-based analytical technique measures the average binding kinetics on one or a few sensor-surface area(s) with respect to a reference area. A development of the standard SPR technique is represented by SPR imaging (SPRi), in which parallel measurements are performed by imaging the change of reflectivity (dependent on the SPR phenomenon) of many areas on a sensor surface (Ouellet et al., 2010). In especially designed and custom developed instrumentations, the SPR setup can enable the detection of DNA targets down to the femtomolar range or lower (Luan et al., 2010). A reduction of the instrumental limit of detection can be achieved using enhancement strategies such as the use of metal nanoparticles (He et al., 2000; Yao et al., 2006) or through complex enzyme-dependent reactions (Goodrich et al., 2004). In these cases, the recognition-dependent binding triggers reactions that lead to the increase (or the decrease) of the surface-bound mass, to a consequent change in the interface refractive index or the electromagnetic coupling with the sensor surface. While these methods greatly increase the sensitivity of DNA detection, they commonly imply a quite different experimental setup with respect to the standard use of a surface-tethered oligonucleotide probe: probes of a different kind (RNA), or secondary probe oligonucleotides with complex functionality. For these reasons, efforts are justified to develop label-free enhancement strategies that could

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be used as a simple add-on to standard nucleic-acids sensing procedures, making use of only DNA through triggered nanoassembly. This would ensue a simplification of the protocols and guarantee the compatibility between probe material and enhancement reagents and the same long shelf-life (not always shared by proteins or complex functionalized nanoparticles). The versatility of DNA as a nanoscale building material, due to its ability to self-assemble into complex structures, has spurred several interesting strategies to improve sensitivity and multiplexing in biosensor-based techniques. An interesting concept is the ability of specifically designed DNA sequences to self-assemble in response to a specific stimulus, such as the presence of a specific target sequence. Dirks and Pierce embodied this concept in the hybridization chain reaction (HCR) (Dirks and Pierce, 2004). Here, two different singlestranded DNA oligonucleotides locked in a hairpin conformation and present in equimolar amounts are triggered to assemble into a long double-stranded structure after a third oligonucleotide is added to the solution. This interesting approach does not involve the use of enzymes, and does not require an accurate temperature control, making it very attractive for use in label-free biosensors. Niu and colleagues demonstrated the possibility to combine HCR with enzyme signal-amplification in order to improve fluorescence detection on magnetic beads. They can detect as low as 8.1  10–16 M target DNA (Niu et al., 2010), albeit through a relatively complex number of labeling and transduction steps. Huang and co-workers, instead, labeled DNA hairpins with two pyrene moieties (Huang et al., 2011). HCR with these hairpins determines the formation of pyrene excimers which exhibit easily detectable fluorescence emission. In the work of Willner and colleagues, DNA recognition triggers HCR to form peroxidase-like hemin-G4 complexes (Shimron et al., 2012). Furthermore, HCR has also been used in an immunosensor assay by Choi and colleagues (Choi et al., 2011a), while Dirks and Pierce suggested an aptamer-HCR system, in which the chain reaction is triggered by the molecular recognition event (Dirks and Pierce, 2004). In this paper, we describe the exploitation of HCR performed on a surface as a strategy to amplify the DNA hybridization signal for the detection in label-free biosensors. Interestingly, HCR also serves to confirm the specificity of the biosensor signal, therefore reducing the false positives also when the biosensor signal could be detected without amplification. Experiments performed on a commercial SPR device and on a custom made SPRi show that our strategy is viable. Beside optical methods, label-free detection of DNA can also be achieved in electrochemical biosensors. Electrochemical impedance spectroscopy (EIS), for example, has been widely used to detect changes in the electrical properties of the electrode interface after the hybridization event (Wang and Kawde, 2001; Wang et al., 1998). Faradaic impedance biosensors measure changes in the charge transfer resistance upon DNA hybridization. In order to enhance the sensitivity, redox species such as [Fe(CN)6]4  /3  can be used as signal reporter. Liu et al. reported the detection of a 1 nM solution for a 15-mer oligonucleotide target, using PNA as probe and ferricyanide as signal reporter (Liu et al., 2005). NonFaradaic EIS can be exploited to detect DNA hybridization on the electrode surface (Ma et al., 2005). As proposed by Berggren and coworkers (Berggren et al., 1999, 2001; Berggren and Johansson, 1997; Bontidean et al., 1998), and reported by our former work (Guiducci et al., 2004), when an electric potential step is applied to the electrode it causes a current response that can be fitted to a simple RC model and thus informs on the electrical capacitance of the interface. This approach has been used in immunosensors (Hedstrom et al., 2005), and DNA sensors (Berggren et al., 1999), allowing detection of very low target-DNA concentrations (reportedly sub attomolar). Following the method of Berggren et al., we herein also present preliminary data showing that the HCR add-on can similarly enhance the signal coming from label-free capacitive biosensors.

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2. Materials and methods All reagents are from Sigma-Aldrich unless otherwise noted, and have been used as such without further purification. Oligonucleotides were obtained from Eurofins MWG Operon (Ebersberg, Germany) and Sigma-Genosys. 2.1. HCR in solution Hairpin samples were heated to 95 1C for 2 min and then allowed to cool to 20 1C for 1 h before use. Native polyacrylamide gel electrophoresis (10% in TBE 1X) was run at 8 V/cm for 1 h, stained with SYBR Gold (Invitrogen), and viewed with a Gel Doc 1000 (BioRad). For the HCR reactions, stock solutions of target (T), hairpin 1 (H1) and hairpin 2 (H2) were diluted in running buffer (0.5 M NaCl, 50 mM Na2HPO4, pH 6.8) to 15 μM, 30 μM and 30 μM respectively. Then 9 μl of each solution was combined to allow HCR for 1 h at room temperature. 2.2. Surface functionalization The thiol modified oligonucleotide stocks were incubated with a 300 μM solution of Tris(2-carboxyethyl) phosphine hydrochloride (TCEP) to ensure reduction of the disulfides, if present. Efficacy of reduction is checked on a PAGE. Gold surfaces (SPR chips or freshly exposed TSG (Hegner et al., 1993)) were functionalized with an overnight incubation of 3 μM thiol-modified oligonucleotides in PBS buffer. Subsequently, the whole chip surface was passivated with 1 mM 6-Mercapto-1-hexanol (MCH) for 30 min at room temperature in order to reduce nonspecific binding of DNA on the gold surface. 2.3. SPR SPR experiments were performed on Biacore X100, a commercial SPR device from Biacore (GE Healthcare). Once the functionalized chip (Biacore, GE Healthcare) was docked into the Biacore X100, the hybridization protocol was performed by cycles of subsequent injections starting by an injection of running buffer (0.5 M NaCl, 50 mM Na2HPO4, pH 6.8) for 1080 s at 5 μl/min to obtain a baseline, and then several injections of oligonucleotides (1 μM of the target sequence and hairpins solutions both 0.5 μM in running buffer), for 1080 s at 5 μl/min, followed by an injection of running buffer for 300 s at 5 μl/min. After each cycle the surface was regenerated by DNA denaturation with an injection of 7 M urea, followed by a stabilization period. 2.4. SPRi The HCR control and multi-detection measurements were performed with a custom-built SPR imaging instrument which utilizes Kretschmann optical configuration with a 680 nm superluminescent light source and a 1 MP CCD camera with 251 range of incident angle (see Scheme 1B). In this configuration, the position of the light source and the camera is fixed and only the prism holder platform could be rotated to scan and find the inflexion point of the SPR peak in order to maximize sensitivity. There are no moving parts during the measurements. The experiments were done in a custom PDMS flow cell designed and fabricated specifically for our SPRi instrument. The flow cell has four parallel channels and their assignment during the HCR control and multidetection measurements are discussed in the following sections. Gold surfaces were functionalized with an overnight incubation of 3 μM thiolated oligonucleotides. Then the surface was docked in the flow cell and 1 mM MCH was injected in each channel for 20 min to passivate the surface. The running buffer was injected for 10 min at 5 μl/min after every hybridization and HCR steps (20 min at 5 μl/min) to obtain a baseline.

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H1

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Scheme 1. (A) Depiction of the surface-bound HCR. The binding of the target nucleic acid (T) to the specific surface-bound oligonucleotide probe (P) enables the further binding of multiple instances of the two hairpins (H1, then H2, and so forth) to the surface. Segments with same shadings are complementary. Numbered phases are (1) clean functionalized surface; (2) target hybridization; (3) Hairpin 1 nucleation; (4) Hairpin 1 opening and full hybridization to target portion; (5) Hairpin 2 binding and opening; (6) binding of another Hairpin 1 to the now available Hairpin 2 site. The reaction follows like this until saturation or chainbreakage events. (B) A scheme of the custom-built SPRi instrumentation. See also Bonyár et al. (2011).

2.5. Capacitive measurements The measurements of the electrical capacitance of the biosensor electrodes were performed applying the method described by Berggren and colleagues (Berggren and Johansson, 1997; Bontidean et al., 1998). All the measurements were performed in a three electrodes cell, connected to a μAutolab III electrochemical workstation (Metrohm Autolab B.V., Utrecht, The Netherlands) and using the following solution as measuring buffer: 10 mM NaCl, 197 μM KCl, 291 μM Na2HPO4, 131 μM KH2PO4, pH 7.4. These experiments were performed using ultraflat gold surfaces commonly referred to as ‘template stripped gold’ (Hegner et al., 1993). A potential step of 50 mV was applied to the working electrode and the resulting current decay was sampled at 20 kHz. The measurement was performed before the injection of the target, until a stable capacitance was reached. After the hybridization occurred, the measurement was repeated, and the capacitance variation upon the hybridization event was calculated.

3. Results and discussions 3.1. Oligonucleotides sequence design for HCR-assisted detection and verification with experiments in solution The gene or sequence that serves as target for the nucleic acids detection needs to be defined, for instance through bioinformatics analysis or database mining. A proper set of oligonucleotide probe

and hairpins must consequently be designed for, respectively, binding and detecting the target sequence from the analyte solution and then performing HCR amplification and confirmation of the signal. For the experiments herein reported, the DNA sequences of the necessary probes, targets and hairpins were designed starting from sequences of target pathogenic microorganisms (e.g. Cryptosporidium parvum) or viruses. The sequence of a selected gene known to be specific for a strain, for example the COWP gene in C. parvum, GenBank AF248743 (Yu et al., 2009), was first screened automatically for potential candidate regions (for good potential binding and low interfering secondary structure and dimerization of the target). Then, a restricted number of candidate sequence portions were individually checked in a manual fashion to extract the candidate sequence with the lowest predicted structural interference to HCR. The preliminary screening was performed with a custom-made script (written in MATLAB). The script uses RNAcofold ver. 1.80 (Bernhart et al., 2006; Hofacker et al., 1994) to calculate the thermodynamic stability and the dimerization energy of portions of the sequence of a length suitable to bind an oligonucleotide probe and, adjacently, the recognition portion of the hairpin (40 nt). This search is thus limited to short-range interactions, suitable for modeling the structure of oligonucleotides, partially denatured DNA sections, and fragmented or PCR-amplified sections of the gene to be detected. Subsequently, the binding energy of a probe oligonucleotide chosen to be 16 nucleotides (nt) long, was calculated for a perfect match with the end portion of the respective 40 nt target sequence. For each position along the sequence, a penalty parameter was assigned. The penalty parameter is directly proportional to the free energy of the secondary structure of the target, to its possible self-binding stability and to the amount of secondary structure upon binding the probe (as this could hinder the subsequent binding of hairpins), while it is inversely proportional to the probe-target binding energy (see Fig. SD1, in Supporting information). Several combinations of the above energy contributions in a single penalty parameter have been attempted (see Fig. SD1 for one of these) giving equivalent results. Probe, target and hairpin candidates, derived from regions of minimal penalty along the target gene sequence, were then manually re-checked using the Vienna package and NUPACK (Zadeh et al., 2010) testing for probe-target and probe-hairpin binding. HCR hairpins were then designed using Nanev software (Goodman, 2005), by imposing the selected target sequence as the recognition portion of probe and hairpins, and then letting the software call the rest of the sequence in order to optimize the correct assembly. Sequences from the pathogens C. parvum (the COWP gene for the Cryptosporidium oocyst wall protein (Yu et al., 2009)), Giardia lamblia (the β-giardin gene (Guy et al., 2004)) and Hepatitis E virus, (HEV, the DNA conversion of a portion of the polyprotein gene of the RNA virus (Orrù et al., 2004)) were chosen as an example test-set with possible practical applications. Sets of probe and HCR hairpin oligonucleotides towards these waterborne pathogens were designed and are listed in Table SD1. In order to verify the ability of three sets of initiators and hairpins to trigger HCR in solution, HCR reactions were assembled and electrophoresis PAGE assays were performed essentially as in Dirks and Pierce (2004). HCR reaction was limited to 1 h, as to test its usefulness in conditions comparable to point-of-need assays (better performance is expected for longer reaction times, but practical usefulness would be limited). We show in Fig. SD2 that only the presence of the initiator (the target DNA) and the two hairpins leads to the formation of high molecular weight products, the result of HCR. This demonstrates that our oligonucleotide sets are efficient in generating the HCR in response to the detection of a target nucleic acid, such as for the three waterborne pathogens listed above.

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Time (s) Fig. 1. HCR-generated signal is visible in real-time with a commercial SPR system. SPR curves depicting the response after the consecutive injections of (1) running buffer, (2) Target_Giardia (1 μM) and (4) Hairpin1_Giardia (1 μM, dashed line) or of (1) running buffer, (2) Target_Giardia (1 μM) and (3) Hairpin1_Giardia/Hairpin2_Giardia mix (0.5 μM each, solid line). The sensorgrams represent the response of the working cell (functionalized with the probe Probe_Giardia) minus the response recorded in the reference cell (functionalized with a control oligonucleotide). The SPR protocol automatically includes short running buffer phases (also marked with 1) before any solution change.

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Aiming at implementing HCR as a surface-bound signal amplification strategy for biosensors, we evaluated the efficiency in performing HCR on a sensor surface according to the mechanism depicted in Scheme 1A. Commercially available SPR gold chips (Biacore, GE Healthcare) were functionalized with thiol-modified probe oligonucleotides, complementary to a portion of the initiator sequence (specifically Probe_Giardia, see Table SD1 in the Supporting information), and then passivated with MCH. A commercial SPR instrument (Biacore SPR X100, GE Healthcare) was used to simultaneously perform and detect the hybridization of the target DNA and the formation of the HCR products on the surface. Measurements are made with respect to an identical flow cell where the gold surface is functionalized with a non-specific probe oligonucleotide, and then passivated with MCH as for the measurement cell. This enables the determination of the signal amplification factor (Fig. 1). The injection of the target oligonucleotides (Target_Giardia, 1 μM) in the SPR microfluidic detection cell produces an evident signal due to the DNA binding on the surface, which quickly reaches a plateau possibly due to surface saturation. The subsequent injection of Hairpin1_Giardia (1 μM), performed after Target_Giardia injection, produces a binding signal similar in amplitude to the target, albeit with a slower kinetics (vide infra). On the other hand, the injection of an equimolar mix of Hairpin1_Giardia and Hairpin2_Giardia (both 0.5 μM) after the binding of Target_Giardia to surface-anchored Probe_Giardia, clearly shows a peak of a larger magnitude. This indicates the formation of high-molecular weight HCR products on the surface. The HCR signal is 4 times bigger than the signal from the Target_Giardia alone or the signal of Hairpin1_Giardia alone (over Target_Giardia). The results obtained are highly reproducible. SPR chips were regenerated with 7 M urea treatment between experiments. In order to better test the possibility of using HCR to amplify the label-free detection of the DNA target, a further experiment was performed on the Biacore SPR X100. Target_Giardia was injected at different concentrations over Probe_Giardia, followed by a second injection of Hairpin1_Giardia/Hairpin2_Giardia mix (both 1 μM) for the HCR. As shown in Fig. 2, for low Target_Giardia concentrations (0.1 nM and 1 nM), the signal after target binding remains practically indistinguishable from the background signal. However, the subsequent HCR reaction determines a visible signal

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Fig. 2. HCR is able to reveal Target_Giardia binding on the surface at concentrations below the direct-detection limit. Response value after the injection of different concentrations of the target Target_Giardia (lighter bars) and the following HCR (darker bars). The inset shows the higher signals measured with 100 nM target, compared with lower signals of 10 and 1 nM. Error bars represent the standard deviation of multiple experiments.

increase for a Target_Giardia concentration of 1 nM. Here, the dispersion of the SPR signal also increases, albeit not linearly with the signal: the global effect is an improvement of the limit of detection in this concentration range. Higher SPR signals were obtained with target concentrations of 10 nM and 100 nM where target signals are detectable also prior to HCR. Signal flattening is reached at concentrations higher than 100 nM due to surface saturation enabled by the very efficient SPR microfluidics used for target hybridization. This data demonstrate that performing HCR does decrease the detection limit for the bound target. When using higher-performance SPR equipment and microfluidics (such as those shown in (Luan et al., 2010)), it is reasonable to expect that the limit of detection could drop much below the nanomolar regime. It is also to be noted here that, due to the small operational volumes of the SPR, the nanomolar limit of detection coincides with the detection of only 10–100 femtomoles of target DNA. Since HCR is performed after target binding, and exploits the same means of detection (SPR signal), it can be considered as a simply implementable add-on that increases the detection signal, extends the dynamic range of the detected concentrations, or simply gives a positive signal correlated with the target recognition thus confirming that the detection is not a false positive. As the HCR hairpins bind a different sequence region with respect to the biosensor probe, their binding is an independent event that further increases the detection specificity. HCR displays a slower kinetics than the simple binding of an oligonucleotide (such as a secondary label, which could as well serve as confirmation of target recognition). The slow kinetics of mass accumulation on the SPR probe is very likely due to the HCR mechanism, which requires the unfolding of one hairpin in concert with its binding to the target. Likewise, hairpin unfolding is also required to subsequently bind other hairpin molecules. As surface polymerization takes place progressively during injection of hairpins, this kinetics is a result of both the slow binding, but also of the progressive polymerization that continues during the injection. At some limiting point, conditions such as steric hindrance, possible hydrodynamic shearing, or spontaneous depolymerization will lead to a steady SPR signal over time. The HCR slower kinetics could itself be considered a distinctive feature in the label-free biosensor and serve as a confirmation that the target signal comes from the specific recognition. A relatively quick steady-state is reached during HCR in the SPR microfluidics, and thus the signal amplification is evident albeit not as high as it might be desirable. With relatively high concentrations of target DNA exposed to the surface (100 nM–1 mM), HCR visibly amplifies the binding signal of about 4–7 times with

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respect to the target DNA alone. Saturation of the SPR signal should not be due to growth of the HCR chains beyond the depth of the SPR evanescent field, as chains are not expected to grow straight but to be in a random coil state. The limited SPR amplification factor might be due to chains being in a random coil and taking up the volume with a small amount of mass and thus changing the refractive index only limitedly. Additionally, a reduction in sensitivity could be expected when the growth occurs further from the surface. HCR on the surface was also tested with a custom-built SPR imaging instrument (see instrument design in Scheme 1B and raw data in Fig. SD4 in Supporting information) (Bonyár et al., 2011). Such custom made SPRi was used to measure the signal variation after the injection of the target sequence Target_Giardia and the HCR hairpins, analogously to what was done in the commercial SPR instrumentation (Fig. 3). The injection of the target DNA determines a clear signal increase, while the subsequent injection of Hairpin1_Giardia/Hairpin2_Giardia mix yields a significantly higher signal, consistent with the formation of HCR products on the sensor surface. Several control experiments were then performed. First, HCR was carried out stepwise on the surface: after the injection of the target sequence Target_Giardia, an injection of Hairpin1_Giardia was performed (Target þH1, in Fig. 3). After this step, a signal increase is visible, although lower than what observed after Target_Giardia injection in the first step. A further signal increase of similar magnitude is produced after the subsequent injection of Hairpin2_Giardia (TargetþH1 þ H2). This data validate the molecular mechanism underlying the HCR on the sensor surface. The specificity of the HCR for the target sequence was assessed by performing different experiments. First, a non-complementary target (Target 2, see Table SD1) was injected: no signal change was recorded (Fig. 3). The following injection of Hairpin1_Giardia/ Hairpin2_Giardia mix (NC targetþHCR) determines only a slight non-specific signal, suggesting that, as expected, no reaction occurs on the surface in the absence of the specific target sequence. In a second control experiment, Hairpin2_Giardia was injected on the specific target-probe complex, recording no signal change. This was expected as Hairpin2_Giardia should only hybridize to the bound Hairpin1_Giardia (Fig. 3). Subsequent injection

of Hairpin1_Giardia (Target_Giardiaþ H2 þH1 in Fig. 3) increases the signal analogously to the injection of Hairpin1_Giardia on the pristine target-probe complex. Our custom SPRi system allows for multiplexed detection (see Scheme 1B and (Bonyár et al., 2011)). The SPRi sensor surface can be turned into an array of up to 16 different cells. Using the same experimental setup as in the experiments above, it was showed that HCR could be effectively employed in a multi-receptor array for selective signal amplification. For this purpose, an array of four areas was prepared on the SPRi sensor surface. The four areas were defined by the four channels of a PDMS microfluidic cell placed over the sensor surface so that in-situ probe immobilization could be performed overnight. The four surface areas were functionalized respectively with Probe 2, Probe_Giardia, Probe 3 (see Table SD1), and without any DNA (running buffer only). Following this, the chip was rotated of 901 with respect to the microfluidic cell, thus generating a 4 by 4 array where each channel feeds a portion of all the four surface areas. The whole sensor surface was then passivated with MCH. In order to verify the specificity of the signal, Target 2, Target_Giardia, Target 3 and running buffer were injected in the cell, one per channel. As anticipated, the hybridization signal is visible only in the area where the matching combination of Probe/Target is present (Fig. 4). In order to use HCR on these sensing areas, all the channels were injected with the Hairpin1_Giardia/Hairpin2_Giardia mix (both 1 μM). Only the area where the specific Target_Giardia oligonucleotide is bound binds the hairpins and hence leads to a HCR signal increase. In agreement with the data on the commercial SPR system, the HCR reaction on the SPRi amplifies the specific target signal about 5 times. Taken together, these data demonstrate that HCR can specifically amplify the DNA binding signal, and that it is promptly suitable for both commercial SPR and multiplexed SPRi detection systems. Positive HCR-related signal change not only serves as a signal enhancement, but it also enhances the target detection specificity by requiring the presence of a further target sequence portion, therefore reducing the chance of false positive detections.

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Fig. 3. HCR increases the detected signal also in a custom-made SPRi system. Several SPRi experiments were performed with injections of complementary target, Hairpin1_Giardia (here marked ‘H1’ which should bind the target and H2), Hairpin2_Giardia (here marked ‘H2’, which should bind H1 but not the target) or Hairpin1_Giardia/Hairpin2_Giardia mix (here marked HCR). ‘Target_Giardia þ H1 þH2’ for example means H1 was added and then H2 was added as a later step. Control experiments were performed with non-complementary Target 2 (here marked as ‘non comp. target’). Target concentration in each experiment was 1 μM.

Fig. 4. SPRi data showing the specificity of HCR amplification. A three-component array (Probe_Giardia, Probe 2 and Probe 3) was created on a gold surface. After the injection of the target sequences (Target_Giardia, Target 2 and Target 3) the resulting signals were collected (light gray bars). Then HCR was performed in each channels (dark gray bars represent the cumulative signal after HCR).

3.3. Capacitive biosensor In order to prove the generality of the HCR strategy for amplifying the label-free detection of nucleic acids, we assessed

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its use in a simple and miniaturizable electrochemical label-free biosensor. Additionally, to further prove the generality of the approach, in this preliminary set of experiments, we tested for the detection of a different DNA sequence, one from the protozoan C. parvum, another socially relevant waterborne pathogen. The surface functionalization and the hybridization procedures were analogous to the ones used for SPR for the sequences for Giardia. The biosensor was made out of a three-electrode cell. The working electrode (a disc of ultraflat gold) was functionalized with an overnight incubation with 3 μM thiolated oligonucleotide Probe_Parvum (see Table SD1) and then passivated with 1 mM MCH. Subsequently, the biosensor cell was incubated with the target DNA (Target_Parvum) for 1 h in order to allow its hybridization on the probe DNA. Different target DNA concentrations were used in separate experiments. DNA binding on the electrode surface was then detected using the potentiostatic-step method described by Berggren and coworkers (Berggren and Johansson, 1997; Bontidean et al., 1998) that we also implemented before (Guiducci et al., 2004). The measurements were made in a low ionic strength buffer (very diluted phosphate buffer with 10 mM NaCl) in order to slow down the current decay following the potential step. This allows an easier sampling of the discharge current. In our experiments, a sampling frequency of 20 kHz was used (higher sampling frequencies permit measurements in higher ionic-strength solutions). The subsequent data analysis is used to calculate the electrical capacitance by fitting the initial portion of the current decay with an RC model (in these experiments, linear regressions with r ¼ 0.99 were obtained from fitting the first 500 ms of the decay). The capacitance at the electrode-solution interface was monitored before and after the hybridization events, therefore allowing the measurement of the capacitance variations due to the hybridization of target DNA and the HCR. Calibration curves were obtained working with series of different target concentrations (Fig. 5). The results obtained with the capacitive biosensor show that the hybridization of Target_Parvum on the electrode surface produces a signal that is proportional to the concentration of the DNA (Fig. 5) even though the dispersion of the data obtained in these initial measurements is quite large. When the HCR was performed on the electrode surface, the resulting capacitive signals increased, while still being proportional to the Target concentration. Although the standard deviation of the HCR signal is bigger than the one of the Target, the measurements on the

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capacitive biosensor confirm that the HCR allows the detection of a smaller concentration of target than the measurements of the target alone. Even in this non-optimized case, the limit of detection after amplification lies between 0.1 and 0.5 mM, while it is close to 1 mM for the target alone. Our data on the capacitive biosensor indicate that the HCR approach can be implemented on a label-free electrochemical biosensor, and that it can therefore be exploited to improve the limit of detection of potentially simple and miniaturizable sensors. 3.4. Conclusions In this work we investigated the application of the hybridization chain reaction (HCR) as a label-free strategy to amplify DNA hybridization signal on label-free biosensors. HCR brings about a generally large signal increase with respect to the target binding to the probe. Furthermore, HCR can also serve as a confirmation of the specific target hybridization, as it provides a signal enhancement only where the target has bound, by using a different sequence portion than the one used for probe binding. Indeed, only the target DNA bound on the surface-anchored probe can trigger the HCR reaction. The HCR follows a generally different kinetics than the binding of a secondary oligonucleotide to the probe-target complex and thus it can be easily differentiated. We showed that the DNA oligonucleotide sets designed from the genomes of human pathogens can trigger HCR in solution and on the surface of biosensors. The SPR results confirmed the proposed molecular mechanism for the HCR. Moreover, the HCR performed very well in real-time multiplexed DNA detection on SPR array systems, therefore opening new possible uses for it. HCR has proved capable of amplifying the hybridization signal, even though moderate amplification factors were observed (further optimization is possible). The maximum amplification obtained in the reported experiments was approximately 5-fold. As the standard deviation does not grow accordingly, a decrease of the limit of detection by about one order of magnitude is observed in SPR experiments. The results obtained with the capacitive sensor further indicate the possibility of applying HCR to parallelized, automated and point-of-care label-free biosensors. While at the current state the final limit-of-detection certainly relies on the detection technique, HCR can be used to gain an increase in sensitivity and a confirmation of specificity. For SPR or label-free capacitance detection, HCR (in our hands) cannot eliminate the use of pre-amplification of the target nucleic acid, such as PCR or cultural enrichment of pathogens, when very low limits of detection are needed. Still, HCR can prove useful in practical applications where it can cut down detection times by limiting cultural enrichment of slow-growing microorganisms. Further experiments on the use of HCR to discriminate the presence of single base mismatches in DNA targets are in progress.

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Target concentration / µM Fig. 5. HCR can improve the signal detection in a capacitive DNA sensor. Calibration curves of a capacitive DNA sensor obtained using different target concentrations (different concentrations of Target_Parvum on Probe_Parvum, solid line) and after 1-h sequence-specific HCR amplification (dashed line). Error bars represent the standard deviation of multiple experiments. The data points have been offset graphically on the x axis to avoid overlapping.

The Authors would like to acknowledge support from the 6th European Framework Program Project 'DINAMICS' (IP 026804-2) and from the Swiss NanoTera.ch initiative (project 128852 “ISyPeM”). G. Z. would like to acknowledge support from POR-FESR 2007-2013 (European Fund for Regional Development) Regione Emilia-Romagna.

Appendix. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2013.10.036.

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