Biosensors and Bioelectronics 61 (2014) 28–37

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Bioanalytical approaches for the detection of single nucleotide polymorphisms by Surface Plasmon Resonance biosensors Maria Laura Ermini a, Stefano Mariani a, Simona Scarano a, Maria Minunni a,b,n a b

Dipartimento di Chimica “Ugo Schiff”, Università di Firenze, via della Lastruccia 3-13, 50019 Sesto Fiorentino, FI, Italy Consorzio Sistemi a Grande Interfase, Department of Chemistry, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, FI, Italy

art ic l e i nf o

a b s t r a c t

Article history: Received 20 March 2014 Accepted 23 April 2014 Available online 5 May 2014

The mapping of specific single nucleotide polymorphisms (SNPs) in patients' genome is a main goal in theranostics, aiming to the development of therapies based on personalized medicine. In this review, Surface Plasmon Resonance (SPR) and Surface Plasmon Resonance imaging (SPRi) biosensors applied to the recognition of SNPs were reviewed, since these technologies are emerging in clinical diagnosis as powerful tools thanks to their analytical features, mainly the real-time and label-free monitoring based on array format for parallel analysis. Since the literature is heterogeneous, a critical classification and a systemic comparison of the analytical performances of published methods were here reviewed on the basis of the analytical strategy and the assay design. In particular, the use of helping agents (i.e. proteins, nanoparticles (NPs), intercalating agents) or artificial DNAs, often coupled to SPR to achieve allele discrimination and/or enhanced sensitivity, were here revised and classified. Finally, the real suitability of SPR biosensors to clinical diagnostics for SNPs detection was addressed by comparing their features and performances with those of other biosensors based on other techniques (e.g. electrochemical biosensors). & 2014 Elsevier B.V. All rights reserved.

Keywords: Optical biosensors Surface Plasmon Resonance (SPR) Surface Plasmon Resonance imaging (SPRi) Single nucleotide polymorphisms (SNPs) Nanoparticles (NPs) Clinical diagnostics

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Strategies in assay design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Exploiting proteins for SNPs detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Use of nanoparticles for SNPs detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Artificial DNAs for SNPs detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Intercalating agents for SNPs detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Methods for direct detection of SNPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Comparison with electrochemical biosensors for SNPs detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The rapid detection of nucleic acid sequences (DNA or RNA) represents a challenging research area of bioanalytical chemistry

n Corresponding author at: Dipartimento di Chimica “Ugo Schiff”, Università di Firenze, via della Lastruccia 3-13, 50019 Sesto Fiorentino, FI, Italy. E-mail address: maria.minunni@unifi.it (M. Minunni).

http://dx.doi.org/10.1016/j.bios.2014.04.052 0956-5663/& 2014 Elsevier B.V. All rights reserved.

28 29 29 30 33 33 34 35 36 37

applied to biosensors. Over the last few years, many works have been focused on this aim, with applications ranging from environmental monitoring to food and clinical analysis, developing platforms based on different transduction principles, i.e. piezoelectric, optical and electrochemical (D'Agata and Spoto, 2013; Lucarelli et al., 2004; Lucarelli et al., 2008; Mariani and Minunni, 2014; Šípová and Homola, 2013; Tombelli et al., 2005). Among the different fields, biosensors development for clinical diagnosis is undoubtedly one of the areas of most increasing interest due to the

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emerging challenge of personalized medicine. Indeed in personalized medicine, the so-called theranostics, the very final aim is the patient's characterization at molecular level, and this challenge can be achieved by low-cost and high throughput screenings, allowing molecular analyses. Among the various analytical devices, affinitybased biosensors currently play an important role in personalized medicine thanks to their performing features (Battersby et al., 2012; Turner, 2013). In this framework, the identification, the role and the frequency of certain polymorphisms is strategic to address tailored therapies. Human genome is characterized by DNA variability, indeed differences in nucleotide sequence of one or more bases are found in genes among members of the same population. If the frequency of one of these variations is lower than one percent, the variation is usually called mutation, if higher it can be classified as polymorphism. It is common to refer to polymorphism as a variation in the DNA sequence that generally does not cause debilitating diseases but could underlie differences in our susceptibility to a disease (Encyclopedia of Public Health, 2008). Polymorphisms may have no effect on the metabolism of the member but simply be involved in the development of certain phenotypic traits. Anyway, these definitions cannot be rigorously applied, since a rare disease in a specific population may become biochemically irrelevant in others. A polymorphism involving only one nucleotide in the gene sequence is called single nucleotide polymorphism (SNP) and its detection in a real sample is one of the most challenging and impressive results in clinical diagnostics, being SNPs involved in many diseases and in biological metabolisms. In many cases specific SNPs can be related to drug response and toxicity: if the involved gene codes for an enzyme or a transporter then the activity of the protein can be adjusted or inhibited by the variation. This can turn both into a reduced capacity of the involved biological process to absorb the drug, lowering its pharmacological effect on the patient or, counter, into a too rapid metabolization of the drug leading to a loss of its efficacy (Chorley et al., 2008). For drug response, the most common SNP is found in glutathione S-transferase P1 or xereoderma pigmentosum group D enzymes for the activity of oxaliplatin (Robert et al., 2005). Some other SNPs are related to responses to anticancer drugs activity, as for thiopurine methyltransferase and dihydropyrimidine dehydrogenase enzymes activity (Robert et al., 2005). It was also reported that different ethnic groups show differences in pain perception associated to OPRM1 A118G SNP (Rahim-Williams et al., 2012). Eventually, premature coronary artery disease and myocardial infarction are related by a SNP in the thrombospondin gene that causes marked effects on the structures and functions of the protein (Stenina et al., 2004). These selected examples just to give an idea of the role of SNPs in therapy responses. Therefore, the screening of patients' genome coupled to the mapping of specific SNPs is a target of relevance for theranostic approach aiming to the development of a personalized medicine also with tailored therapies. To achieve this goal, the patient should be characterized in its genetic fingerprint (Babić, 2012). Different sensing-based strategies for SNPs detection are reported in literature (Abi and Ferapontova, 2013; Bonanni et al., 2012; Ermini et al. 2013a, 2013b; Jiang et al., 2004, 2005; Loo et al., 2013; Suzuki et al., 2012; Xu et al., 2012; Wang et al., 2012; Wilson et al., 2005; Zhang et al., 2013) aiming to develop reliable detection methods for application to routine measurements in real matrices, such as biological specimens. The analytical problem in the detection of SNPs could be in principle reduced to the identification of the nucleic bases, i.e. A, T, G, and C, present in defined positions along the whole human genome. This review focuses on opticalbased biosensors applied to the recognition of SNPs and in particular on the recent literature on conventional Surface Plasmon Resonance (SPR) and Surface Plasmon Resonance imaging (SPRi). Other approaches based on biosensors are discussed here and

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compared to SPR results, such as electrochemical detection using nanoparticles (Lucarelli et al., 2008) and PNA (Kerman et al., 2008) or locked nucleic acid (LNA) as signaling probes (Berti et al., 2011), chronocoulometry (Kelley et al., 1999), alternating-current voltammetry (ACV) (Xiao et al., 2009), differential pulse voltammetry (DPV) (Nakayama et al., 2002), cyclic voltammetry (CV) (Wan et al., 2009) and impedance spectroscopy (Akagi et al., 2006). An overview on these approaches can be also found in some interesting recent reviews (Hvastkovs and Buttry, 2010; Paleček and Bartošík, 2012; Sassolas et al., 2008). Concerning optical transduction, a related work by Raman spectroscopy (Moody and McCarty, 2009) and fluorescence spectroscopy has also been reported (Schulze et al., 2012). The principle of SPR detection is widely described by excellent reviews and books (D'Agata and Spoto, 2013; Homola, 2006; Scarano et al., 2010; Šípová and Homola, 2013). SPR phenomenon is widely applied as transduction principle in DNA biosensing to different fields of analysis. Moreover, the recent possibility to have commercial devices based on imaging technology has pushed SPRi a step forward in emerging fields, since parallel analysis of many binding events, i.e. target sequences, can be monitored simultaneously, among others anti-doping analysis (Scarano et al., 2011; Llop et al., 2011) and personalized medicine (Helmerhorst et al., 2012). Recent advancements of SPR technology relevant for SNPs detection such as employment of gold NPs (Bedford et al., 2012; Spoto and Minunni, 2012; Zanoli et al., 2012) or artificial DNA (D'Agata and Spoto, 2012) was recently reviewed. Bedford et al. (2012) reported the recent trend of apply gold NPs in SPR biosensors incorporating them within the sensing surface or using them labeled with a detecting biomolecule. Spoto and Minunni, 2012 described increases in sensitivity with related DLs decreases for a wide range of analytes by the mass enhancement effect of the NPs in the EW (evanescent wave) as well as by the optical coupling between surface plasmons (SPs) and electric fields of localized surface plasmons (LSPs) of NPs when they are close to the metallic surface. The employment of functionalized gold NPs was also reviewed by Zanoli et al. (2012) for ultrasensitive oligonucleotide detection with different transducers (including SPR), confirming the usefulness of the strategy for bypassing the enzymatic amplification of DNA before biosensor analysis. D'Agata and Spoto (2012) reported the employment of artificial nucleoside probes in combination with SPR transducer to enhance the nucleic acid sensing sensitivity and selectivity, thus expanding the diagnostic potentialities. In this review the panorama of SPR-based methods for SNPs detection will be reviewed and critically compared by grouping existing approaches on the base of the different strategies proposed. SNPs detection is reported mainly coupling SPR technology with helping agents, involving the use of proteins, NPs, as well as intercalating agents added to the hybrids; on the base of their use different detection strategies can be designed. Since the literature is quite heterogeneous, a rational classification and a critical comparison of the analytical performances of the different systems are presented here. Finally, a brief comparison between the analytical performances of SPR-based biosensing and other sensing techniques (e.g. electrochemical biosensors) is reported, discussing the real-applicability of these systems to clinical diagnostic and, in particular, to polymorphisms detection.

2. Strategies in assay design 2.1. Exploiting proteins for SNPs detection Biomolecules, and in particular proteins, have been exploited in SPR assays focused on SNPs detection, essentially playing the role

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of mass enhancers and/or discriminating agents. Among examples of discriminating agents, MutS is undoubtedly the most exploited protein (Babic et al., 1996; Gotoh et al., 1997; Nakano et al., 2011; Su et al., 2005; Wilson et al., 2005) thanks to its specificity in recognizing only mismatching sequences which, in living cells, can be corrected and repaired through this mechanism. From a biological point of view, MutS is in fact involved in the methyl-directed MutHLS mismatch repair pathway (Lahue et al., 1989), a strand-specific pathway that finds and repairs base-pair mismatches or DNA regions containing short deletions or insertions (Au et al., 1992). MutS not only binds heteroduplexes DNA, identifying the presence of a mismatch, but it does it with varying affinities towards the eight possible single-base mismatches (Su et al., 1988). One of the first attempts in this sense was a SPR-based study aiming to the determination of MutS affinities for various types of mismatches present on a 30 mers sequence (Babic et al., 1996). Different mismatch combinations were tested in order to evaluate the behavior of a specific couple of bases, with MutS finally added to heteroduplex. Comparing SPR signals from different mismatch-MutS complexes, MutS binding was the strongest to a G–G mismatch, followed by G–T 4A–A 4C–T, A–C. Binding to A–G, T–T and C–C mismatches was marginally higher than that observed between MutS and homoduplex DNA. Further, other SPR studies using MutS for SNP detection appeared in literature (Babic et al., 1996; Gotoh et al., 1997; Li et al., 2006; Nakano et al., 2011; Su et al., 2005; Wilson et al., 2005). In these studies the selectivity of the protein was evaluated testing its ability in discriminating different SNPs. The protein-based recognition system resulted in able to distinguish DNA sequences differing only for one base and this was assessed by the simultaneous monitoring on different channels of DNA interactions in presence of SNPs and the relative full-matching sequences used as reference. The selectivity was clearly confirmed since only negligible signals were recorded from interactions between MutS and DNA homoduplexes. Generally, the selectivity of the recognition can be further tuned varying some experimental variables such as the buffer composition and the assay design, e.g. by introducing helping agents. For instance, Gotoh et al. (1997) demonstrated how MutS pre-treated with ATP enhances the specificity of MutS toward MutS-mismatch complexes rather than fully matching hybrids on the biosensor. In combination with MutS, the singlestrand binding protein (SSB) was also applied to prevent possible unspecific binding of MutS to the immobilized ssDNA. In fact SSB selectively binds to single-stranded DNA, preventing annealing and avoiding digestion by nucleases (Wold, 1997). The same MutS/SSB synergic activity was reported by Wilson et al. (2005, p. 53) gene mutation detection by conventional SPR, using a portable instrumentation, coming to similar conclusion drawn by Gotoh et al. (1997), i.e. the application of SSB prior to MutS should not affect its ability to bind to mismatches, but would prevent non-specific binding of MutS to DNA probes and chain ends. Moreover, the SSB itself would be informative about the SNP presence since a clear difference in SSB binding on matching or mismatching DNA duplexes was found, with significant higher signals in presence of mismatch. Interesting considerations were underlined by Wilson et al. (2005) about the surface mismatch density in relation to SPR signal obtained by using MutS. This topic was addresses by Nakano et al. (2011) together with the importance of a careful choice of the sequence length to be used in the SNP assay. In this study they demonstrated that MutS binding was significantly influenced by amount and length of the interacting DNA sequence on the chip. They prepared a substrate density-controlled DNA chip exposing the immobilized DNA probes to a mix of complementary target of two different lengths. After, the free extremities of the longer targets were hybridized with single nucleotide mismatching sequence, to

subsequently interact with MutS. Thus, they found that in order to obtain accurate kinetic measurements for the SNPs detection, the density on the surface need to be tuned to guarantee a minimal distance between DNA substrates, which should be greater than the MutS size avoiding steric crowding. Thanks to these studies, aimed to optimize assays conditions for MutS application to SNPs detection, the overall system selectivity was improved so that no binding with double stranded DNA occurred. MutS-based strategies in SPR sensing resulted fast and selective, hence SPR biosensors were further used as reference method for the significant results achieved in SNPs detection with MutS, in parallel with other label-free approaches such as quartz crystal microbalance (QCM). In particular Su et al. (2005) employed SPR and QCM-based sensing to evaluate MutS binding to single Thymine–Guanine (T–G) mismatched DNA. The combined SPR and QCM data provided information on the structural properties of the DNA and MutS/DNA complexes, confirming the general validity of the use of MutS for SNPs detection. Corn at coworkers (Li et al., 2006) reported a successful application of proteins by SPRi for SNPs detection, taking advantage of the DNA ligase as discriminating agent. Its specificity in elongating double-stranded DNA having sticky ends (cohesive ends) allowed a two-steps strategy leading to a yes/no response to the presence of a mismatching end, i.e. the SNP (see Fig. 3 in the next paragraph). The assay involved also the use of nanoparticles and therefore it is further detailed in the following paragraph, however the key role in SNP detection was played by ligase that guarantees the specificity of the discrimination. Regarding the use of proteins for mass enhancer-based strategies applied to the detection of SNPs, a short synthetic analog of TP53, sequence frequently mutated in germ line cancer, was detected by Šípová et al. (2011) (Table 1) by exploiting the streptavidin/biotin binding (in a 1:4 molar ratio) in creating streptavidin–oligonucleotide complexes (SON) carrying a DNA probe (biotinylated, probe B) designed to be fully complementary to the wild type TP53 sequence displaying polymorphisms. A primary probe (thiolated, probe A) is immobilized on the biochip to bind part of the TP53 sequence, leaving exposed a protruding end (11 mers) free to bind with higher (in case of full match) or reduced (in case of mismatches) affinity the SON complex via probe A recognition (Figs. 1 and 2). Results in presence of SON complexes were compared with those obtained by the direct SNP detection in their absence, showing a 14 fold signal amplification with 40 pM of detection limit. Moreover, by playing on buffer composition, temperature control and assay design, authors gained stringency in specificity and SNP discrimination. 2.2. Use of nanoparticles for SNPs detection Together or alternatively to the use of proteins, also nanoparticles (NPs) have been employed in the design of biosensors devoted to SNPs detection. In SPR-based biosensing, NPs are mainly employed to obtain strong optical coupling of electromagnetic radiations to electric fields of localized surface plasmons (LSPs) (Shalabney and Abdulhalim, 2011). LSPs induce a local field-enhancement in form of hot-spots with properties depending on composition, size and shape of the nanostructure and also on surrounding local media (Haes and Van Duyne, 2004). Currently, a wide range of nanosized materials with different physicochemical properties has been used to achieve optical coupling (Bedford et al., 2012; Chen et al., 2012; Kwon et al., 2012; Mariani et al., 2013; Torun et al., 2012). NPs have been already employed in detection strategies for biomolecules such as DNA, RNA, and proteins to improve sensitivity in SPRi-based sensing. Most of these strategies deal with the use of them in the design of sandwich-like assays, using

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Table 1 SPR based assay for single nucleotide polymorphism discrimination. Author

Year

Recognition method

Šípová et al. (2011)

2011 Streptavidin–oligonucleotide complexes

Babic et al. (1996)

1996 MutS protein

Gotoh et al. (1997)

1997 MutS protein incubated with ATP

Wilson et al. (2005)

2005 MutS

Nakano et al. (2011)

2011 MutS

Su et al. (2005) 2005 MutS

Li et al. (2006)

2006 Taq DNA ligase and NPs

Sato et al. (2006)

2006 Gold NPs

D'Agata et al. (2008)

2008 PNA probes and gold NPs for signal enhancement

D'Agata et al. (2011)

2011 PNA probes and gold NPs for signal enhancement

Lao et al. (2009)

2009 PNA probes

Corradini et al., 2004 PNA (chiral boxes) 2004

Ananthanawat et al. (2010)

Nakatani et al. (2001)

2010 pyrrolidinyl PNA bearing a d-prolyl-2aminocyclopentanecarboxylic acid backbone, acpcPNA 2001 ligands of naphthyridine family

Y. Wang et al. (2013)

2013 ligands of naphthyridine family

Milkani et al. (2010)

2010 Label-free

Song et al. (2002)

2002 Label-free

Detection principle

Instrumental setup

Four-channel SPR biosensor developed at the Institute of Photonics and Electronics, (Prague) MutS with the greatest affinity for BIAcore™ (Pharmacia the G–G heteroduplex with Biosensor AB, different affinities: G–T, A–C, A–A, Sweden) T–T, G–G, A–G, C–T, C–C. Streptavidin–oligonucleotide complexes attached to the target and then injected in the sensor in a single step.

Unspecific interaction between protein and fully matching hybrid on the sensor lowered to zero employing MutS with ATP. MutS coupled to single strand binding protein to reduce the unspecificity. Probe density tuned in order to have an interacting substrate suitable for the size protein.

BIAcore™ (Pharmacia Biosensor AB, Sweden)

TI-SPR-1 Spreeta™ (Texas Instruments, Texas, USA) Model SPR670 system, (Nippon Laser & Electronic Laboratory, Nagoya, JP) Information about MutS-DNA AutoLab ESPR (Eco complex. Chemie, The Netherlands) Enzyme catalyzingcovalent bonds SPR imager (GWC formation between ssDNA for fully Technologies, Madison, USA) matching target, then discrimination with DNA functionalized NPs. 2D-SPR04A (NTT At high salt concentrations Advanced Technology, ( 40.5 M NaCl), fully matched duplexes with blunt ends induced Atsugi, Japan) aggregation on NPs treated surface. In presence of a SNP, no aggregation was recorded. Sandwich strategy on PNA probes SPR imager (GWC and SNPs revealing with NPs. Technologies, Madison, USA) SPR imager (GWC On genomic DNA isolated from Technologies, patients, sandwich strategy on Madison, USA) PNA probes and SNPs revealing with NPs. AutoLab ESPR (Eco Selective destabilization of the hybrids containing mismatches in Chemie, The Netherlands) presence of a denaturing agent. Biacore 1000, Modified PNA with chiral chains with greatly improved mismatch BIAcore™ (Pharmacia recognition ability respect to PNA. Biosensor AB, Sweden) The acpcPNA showed the highest AutoLab ESPR (Eco Chemie, The mismatch discrimination Netherlands) efficiency than DNA or PNA. Biacore 2000, Ligands of naphthyridine family specifically bound to the guanine– BIAcore™ (Pharmacia Biosensor AB, guanine mismatch Sweden) BIAcore 3000 system Naphthyridine-ligand (BIAcore, Uppsala, immobilized on the sensor for recognize eteroduplex in solution Sweden) Comparison between fully match TSPR1K23 (Nomadics, Inc. Stillwater, USA) and single mismatch in different position on target sequence. High-resolution SPR incorporated FI-SPR developed at with a bi-cell photodiode detector the Department of Chemistry and combined with a flow injection Biochemistry, device California State University (Los Angeles, USA)

Running buffer

Detection limit (or lower concentration tested)

Tris Mg buffer

40 pM

10 mM PBS, 150 mMNaCl, 0.1 mM 570 mM EDTA, and 0.005% P20 surfactant (single-stranded and homoduplex binding) 10 mM PBS, 150 mM NaCI, 0.1 mM EDTA, and 0.005% P20 surfactant (binding base mismatches and alkylated bases) 0.9 M NaCl, 90 mM sodium citrate, 1 mM pH 7.2

300 mM NaCl, 20 mM Na2HPO4, 1 mM 0.1 mM EDTA, pH 7.4 and 0.05% TWEENs 20 5 mM MgCl2, 20 mM Tris-HCl (pH 1 mM 7.5), 1 mM DTT and 0.1 mM Na2EDTA HEPES buffer

0.3 M NaCl/10 mM phosphate buffer pH 7.4

1 pM

10 mM PBS, pH 7.0, with 0.1 M NaCl, 0.01 wt% Tween 20

16–32 nM

10 mM PBS, pH 7.4, with 137 mMNaCl, 2.7 mMKCl

1 fM

10 mM PBS, pH 7.4, with 137 mMNaCl, 2.7 mMKCl

2.6 aM

10 mM PBS, pH 7.4, with 137 mMNaCl, 2.7 mMKCl

500 nM

10 mM PBS, 100 mMNaCl, 0.1 mM 5 mM EDTA, pH 7.0

10 mM PBS, pH 7.4, with 137 mMNaCl, 2.7 mMKCl

200 Nm

8 mM HEPES, pH¼ 7.4 with 120 mMNaCl

125 nM

10 nM 10 mM HEPES, pH 7.4; 150 mMNaCl; 3 mM EDTA; 0.005% Surfactant P20 3 M NaCl, 0.3 M sodium citrate, pH 20 pM 7.0 TE (10 mMTris–HCl and 1 mM EDTA) and 0.1 M NaCl

54 fM

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Table 1 (continued ) Author

Year

Recognition method

Bassil et al. (2003) Ermini et al. (2013a)

2003 Label free 2013 Label free

Detection principle

Instrumental setup

Running buffer

Detection limit (or lower concentration tested)

Multi-array asset

SPR imaging

PBS (25 mM phosphate buffer with 0.137 M NaCl, pH¼7.4) 300 mM NaCl, 20 mM Na2HPO4, 0.1 mM EDTA, pH 7.4

1.4 pM

Rational probe design and optimized pre-analytical step (ultrasonic fragmentation and thermal denaturation)

Fig. 1. Detection of all eight possible mismatches reported by Gotoh et al. (1997) Complementary and eight possible mismatched dsDNA were immobilized on a sensor chip surface and then exposed to SSB. ATP-treated MutS was applied to the sensor chip surface and binding was monitored in resonance units. MutS did not bind to complementary dsDNA; however, MutS did bind all eight possible mismatched pairs. Reprinted with permission from Elsevier.

Fig. 2. Assay based on streptavidin–oligonucleotide (SON) complexes: capture of SON–target complex by the DNA probes immobilized on the sensor surface.

nanostructures also as mass enhancers (Spoto and Minunni, 2012). Despite the great improvement brought by NPs to SPR sensing, their use still presents some drawbacks ascribable to their synthesis, possible aggregation phenomena and unspecific interactions at the SPR surface. Besides, conditions oriented to a selective measurement must be carefully studied and optimized, starting from surface functionalization and coming to suitable buffer composition. An assay strategy for SNPs detection involving gold NPs was successfully provided by D'Agata et al. (2008, 2011) to improve SPRi signal obtained from the target hybridization by a sandwich format, both on synthetic DNA, i.e. PNA (D'Agata et al., 2008), and genomic DNA isolated from patients (D'Agata et al., 2011) to reveal the presence of mismatches in DNA samples down to 2.6 aM. In the last study, a whole and non-amplified genomic DNA was injected on the PNA capturing probe immobilized on the biochip giving a primary SPR signal. The subsequent injection of gold NPs carrying a nucleotide sequence able to bind the captured target near the SNP successfully allowed its discrimination. However,

SPRi Lab France)

þ

(Horiba,

2.8 fM

Fig. 3. Schematic representation of the SNP genotyping method based on a combination of surface ligation chemistry and nanoparticle enhanced SPRI. Two array elements with different DNA probes are shown. The array probes (PA and PG) differ only by the last nucleotide at their 30 ends. When target DNA (T), ligation probe DNA (L) and Taq DNA ligase are simultaneously introduced to the array, surface duplexes form at both array elements. However, ligation only occurs if the duplex is perfectly complementary. After denaturation with 8 M urea, the perfectly matched PG is extended with the L sequence while PA returns to its original state. The presence of L is then detected by the hybridization adsorption of gold nanoparticles modified with oligonucleotides (LC) complementary to L. Li et al. (2006). “Reprinted with permission from (Li, Y., Wark, A.W., Lee, H.J., Corn, R.M., 2006.Anal.Chem. 78, 3158–3164.). Copyright (2006) American Chemical Society.”

after a single measurement, gold NPs aggregated on the surface prevented the further use of the sensor, and this drawback may require additional investigation and improvement. A very original approach, based on Taq DNA ligase activity for SNP genotyping in combination with NPs, was reported by Li et al. (2006). In particular, enzyme surface ligation chemistry was used coupled to NPs-enhanced SPR detection. Taq DNA ligase catalyzes covalent bonds formation along a single ssDNA only when the single chains of the duplex are perfectly complementary. In this work, two fragments of targets DNA, mapping in congruous probe region and Taq DNA ligase were simultaneously introduced on immobilized probes on the same surface as displayed in Fig. 3. At this stage, only the fully complementary hybrids, in presence of the enzyme, were subjected to the enzyme ligation. Differently from other strategies involving proteins for SNPs detection, here Taq DNA ligase did not recognize directly the genotype, but its activity was strictly related to the presence of a SNP on the target sequence. Furthermore the recognition ability of the enzyme is highly selective versus the mismatches sequence, bringing to a high selectivity of the whole strategy. In presence of a matching target, i.e. no SNP, the result was the elongation of the probe; conversely, in presence of SNPs, no elongation occurred. The elongated portion was finally revealed by hybridization with the complementary DNA probes bound to gold NPs, generating a SPR signal only in case of fully complementary DNA, while no signal could be recorded in presence of SNPs. Using different probes, it was possible to identify

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selectively the SNPs under study down to 1 pM, by combining detection with NPs as signal enhancers in a “switch on/off” detection strategy. SPR signal due to hybridization was observed on array elements: it increased where the probe sequence was perfectly matched to the target. Non-specific adsorption was not observed either on the other probe array elements or on the blanks. Behind this approach, a very original work was also reported by Sato et al. (2006), where the hybridization between gold NPs modified with a DNA probe and the target sequence (fully matching or mismatching) took place in solution, closely in contact with the gold chip surface. At high salt concentrations (4 0.5 M NaCl), in presence of fully matched duplexes, NPs aggregated, while no aggregation was observed in presence of SNP, indicating that NPs were crucial in sequence discrimination. The method showed selectivity against terminal mismatches, inferring that single-base mismatches at distal ends of DNA sequences investigated stabilized the NPs dispersion. 2.3. Artificial DNAs for SNPs detection DNA detection can be achieved using Watson-Crick base pairing with oligonucleotides or their analogs, the so-called artificial DNAs: Peptide Nucleic Acids (PNAs), Locked Nucleic Acids (LNAs), morpholino oligonucleotides (MOs), and other modified probes. Artificial DNA has shown improved properties of affinity and selectivity in target recognition compared to simple DNA probes. Sequence selectivity is a very hard issue, mainly in the case of single mismatch detection, since the stability constant and the melting temperature of the mismatched duplex could be very close to those of the fully matched one. Differences in a single base pushed the recognition properties of the probes to the maximum efficiency (Bertucci et al., 2012) and a key role is played by the probe to be immobilized on the chip surface. PNAs are nucleic acid chains that mimic DNA but with the sugar-phosphate backbone replaced by a polyamide chain composed of N-aminoethylglycine. PNAs were introduced by Nielsen et al. (1991) and frequently coupled to SPR detection (D'Agata and Spoto, 2012) to enhance the biosensor sensitivity and selectivity and for the first time they were reported combined to SPR for SNPs detection by Feriotto et al. (2001) in 2001. PNA-DNA hybridization is less sensitive in aqueous solution of salts, which are necessary to attenuate electrostatic repulsion in a DNA duplex. However the relative independence of their performances to the environment makes bio-analytical methods robust, especially with analytes in complex matrices such as biological fluids. PNA is also characterized by good stability both to nucleases and peptidases, since its “unnatural” skeleton prevents recognition by natural enzymes, making them persistent in biological fluids. A major drawback is that enzymatic reactions, which are often used in combination with DNA probes, are not possible using PNA substitutes. Therefore, detection schemes involving e.g. DNA-ligase or DNA polymerases cannot be performed with PNA. However PNA can be easily functionalized with biotin and then coupled to an enzymatic assay. Lao et al. (2009) demonstrated that hybridization conditions for SNP discrimination using SPR-based sensing needed to be separately optimized for PNA and DNA probes, as a consequence of their distinct properties. As stringency control strategy for single base mismatch discrimination, a denaturant, i.e. formamide, was added to the hybridization buffer as suppressor agent. In this way, hybrids containing mismatches can selectively be destabilized, resulting in a successful discrimination of the single base mutation. In these conditions, the stability of PNA/DNA hybrids was greatly affected by the presence of a single base mismatch, thus as a consequence, PNA probe allowed more efficient mismatch discrimination than the corresponding DNA probe. For this reason PNA can be very useful for biosensor development aiming to

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SNPs detection (Ananthanawat et al., 2010; Corradini et al., 2004; D'Agata et al., 2010 Feriotto et al., 2001;Lao et al., 2009). In addition to the examples reported above, a very interesting application is about chiral boxes (Corradini et al., 2004) consisting in PNA chains containing an insert of three chiral monomers based on D-lysine. This structure was able to recognize complementary DNA oligonucleotides with sequence selectivity higher than the corresponding achiral PNA, and in particular showed greatly improved mismatch recognition ability, proving that chiral PNAs could be highly selective probes in DNA recognition of SNPs. Another application of modified PNA for SPR-based SNPs detection was recently reported by Ananthanawat et al. (2010), which used as probe a 13 mers pyrrolidinyl PNA sequence bearing a d-prolyl2-aminocyclopentanecarboxylic acid backbone, called acpcPNA. Its performances, in terms of specificity to detect the mismatch in the target DNA (1 mM), were evaluated and compared to the same ability of DNA and conventional peptide nucleic acid (aegPNA). acpcPNA showed the highest mismatch discrimination efficiency between fully matched DNA and single mismatching targets, indicating that introducing suitable modifications to PNA chain could be of advantage, emphasizing its peculiar characteristics in term of SNPs mismatch discrimination. Enantiomers have also been successfully proposed for SNPs detection, as in the case of MOs having standard nucleic acids bound to morpholino rings, which are linked through phosphorodiamidate groups. Their particular structure makes them resistant to nucleases and thus very stable in water solutions. MOs were reported for SNPs detection by using a Surface Acoustic waves system (Bertucci et al., 2012) but never, so far, with SPR-based sensing systems.

2.4. Intercalating agents for SNPs detection The most frequent approach for SNPs detection by SPR biosensors is based on the immobilization of a probe sequence on the sensor surface and the subsequent discrimination of the mutation applying different strategies after target hybridization. In this context, the work of Nakatani et al. (2001) results innovative because the discriminating molecule was immobilized on the surface. They designed and synthesized ligands of naphthyridine family that specifically bind to the G–G mismatch, one of the possible SNPs (Fig. 4). They demonstrated that the G–G mismatch was significantly stabilized when two naphthyridine intercalators simultaneously bound to both G bases, within the base stacks of the DNA duplex. The panel of mismatched bases was further enlarged (Kobori and Nakatani, 2008) using three different intercalating molecules (naphthyridine dimers) immobilized on the biochip, and capable to stabilize specific mismatched base pairs by selective binding. By this approach it was possible to detect DNA duplexes containing a single-base mismatch in 10 nM DNA samples. The explanation of this finding was that the site with bulged nucleotide base, without complementary base to form a hydrogenbonding pair, was susceptible to the ligand intercalation, which might produce a highly organized and stable complex with the bulged DNA; moreover, for the various mismatching sites, different affinities were expected for the mismatch-binding ligands, since the number of hydrogen-bonding groups complementary to the bulged base was different in the distinct cases. Recently Y. Wang et al. (2013) applied 2-(2-aminoacetyl)amino5,6,7-trimethyl-1,8-naphthyridine in an SPR assay for the specific recognition of G-bulged dsDNA. The ligand in this case was immobilized on the SPR sensor surface via amino coupling on a carboxymethylated dextran surface. The ligand recognized the bulged DNA giving a response approximately 109 fold higher than that from the fully matched dsDNA. The sensor specificity for the guanine mismatch was also studied and confirmed since T, C, or

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Fig. 4. (A) Structures of naphthyridine dimers 1 and 2 (green), and hydrogenbonding pattern to guanine (red). (B) An illustration of duplex containing a G–G mismatch. V, W, X, and Y can be any nucleotide bases. (C) Hypothetical structure of the G–G mismatch regarded as two consecutive guanine bulges. Both G bases have no complementary base to form a hydrogen-bonding pair. (D) A proposed binding model for ligand 1 to the G-G mismatch. Naphthyridine moieties of ligand 1 (shown with green boxes) intercalate into the GV and GY steps and produce hydrogen bonds to two guanines. (E) Molecular models of the simulated complex of ligand 1 (colored green) and DNA containing the G–G mismatch (colored red) viewed from the major groove side (left) and the minor groove side (right) (Nakatani et al., 2001). Reprinted with permission from Nature Publishing Group.

A bulge produced only weak responses. The linear range was limited from 20 nM to 1.0 μM with a detection limit of 10 nM. 2.5. Methods for direct detection of SNPs SNPs detection is achievable also without the use of additional biomolecules or nanoparticles or intercalating agents, thus these strategies are here classified as direct. Milkani et al. (2010) reported a direct assay on synthetic 21 mers oligonucleotide aimed to study the influence of the mismatch position along the double helix on the probe/target interaction. They compared the SPRi signal for three targets differing only in the position of the non-complementary base along the fragment. In their experimental asset, the comparison was performed among 30 end, 50 end, and middle mismatches on the target DNA oligonucleotide. The effects of buffer concentration,

flow rate, and temperature were also studied with regard to the detection of single nucleotide mismatches using conventional SPR. The observed response for the 50 mismatch (distal end) was similar to the fully matching strand one, while single mismatches at the proximal end of the probe resulted in a less efficient hybridization of target to probe compared to the distal end, which is closer to the solution. Song et al. (2002) used a high-resolution SPR incorporated with a bi-cell photodiode detector combined with a flow injection device. The influence of a mismatch on the signal was evaluated on a target of 47 mers by using a 30 mers probe, obtaining a detection limit of 54 fM without any label, higher than other conventional SPR systems. Moreover, the selectivity resulted to be very good, achieving single SNP detection. Kwon et al. (2012) reported a study on binding of fully matching and single base mismatching DNA towards a probe immobilized on a sol–gel silica film modified with a dendron, a branched molecule. The gel was deposited onto a gold surface and further modified with N-(triethoxysilylpropyl)-o-polyethyleneoxide urethane on which the dendron was attached. After deprotection, the resulting amines were chemically linked to amine-modified probes. Surface plasmon field-enhanced fluorescence spectroscopy was employed to investigate the kinetic rate constants and the affinity constants between the target DNA, fluorescently labeled, and the probe on the dendron-modified surface. In this case a label was used on both matched and mismatched target sequences since it was essential for the detection, even if not for the strict mismatch discrimination mechanism. The same group previously demonstrated that this technique succeeded in determination of intrinsic rate constants comparing fully matching and single base mismatching binding on a streptavidin modified surface (Liebermann et al., 2000). In the paper they found that the presence of a mismatch was directly detectable and that rate constants on this surface were higher by one order of magnitude than those inferred from a biotin–streptavidin modified surface, except for dissociation constant of the mismatched case. The label free detection was also accomplished by Bassil et al. (2003) on cystic fibrosis transmembrane regulator (CFTR) gene, using a SPR imaging coupled with a multi-array asset. Different mismatches were studied using the same surface where different specific probes were immobilized using a spotting robot on a polyethylenimine-avidin functionalized gold surface. Four mixtures of DNA targets mimicking the three possible different genotypes (homozygous wild type, homozygous mutant or heterozygous) were tested, resulting able in discriminating each of these cases. Three years later the group studied the discrimination of frequent point mutations occurred in the CFTR gene by the same approach (Mannelli et al., 2006) and by a multi-dimensional approach (Lecaruyer et al., 2006). More recently, the direct detection of SNPs by SPR imaging was reported by Ermini et al. (2013a). DNA probes were designed by an in silico approach (Ermini et al., 2011) allowing the rational selection of the DNA probes to be used in the assay design. In particular, the detection strategy was based on the use of a highly specific ‘capturing probe’ mapping a specific region of the target sequence (62 mer from the SNP to be detected) and followed by a secondary hybridization on the DNA region containing the SNP. The SNP investigated here as case study was the well-known rs1045642 occurring in the human gene ABCB1, a key gene in pharmacogenomics, whose SNP is related to the incidence of many diseases. The probes to be used in the discrimination of the SNP were studied and optimized in length and for the position of the polymorphic site along the DNA sequence by a computationalbased approach. This in silico design of probes aimed to reduce intra-strand bonding and cross reactions with other sequences present in the genome, thus improving the system selectivity in complex matrices. Finally, SNP detection was achieved by a

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sandwich like assay in whole human genomic DNA, quantitatively enriched by using a non-conventional amplification method, different from PCR reaction, i.e. Whole Genome Amplification (WGA), (Ermini et al., 2013a). The sensor was reusable, allowing repeated measurements on the same chip. This approach is currently under optimization to detect rs1045642 SNP directly in human genomic DNA extracted from blood (i.e. Lymphocytes) by avoiding any pre-concentration step (Wu et al., 2012). Ultrasensitive genomic detection by SPRi has been already previously reported in an optimized assay (Ermini et al., 2013b), opening perspectives in parallel sensitive DNA-analysis by reusable sensors. Finally, considerations on advisable SPR instrumental assets for SNPs detection were underlined by D'Agata and Spoto (2013) in their very recent review. Surely, the possibility of having instrumentation at controlled temperature is an interesting point, since the presence of mismatches in DNA sequences influences the hybridization thermodynamics; thus information on hybridization kinetics is relevant also to detection of SNPs. This feature was clearly demonstrated in 2008 by Livache group, which reported data relative to oligonucleotide (15-bp) probe–target melting behavior under controlled temperature. After probe–target hybridization, label-free melting curves were obtained by acquiring SPRi data while heating the system at a rate of 2 1C/min. Denaturation curves were useful to detect the genotype of the target sequence by distinguishing between homozygous and heterozygous cases (Fiche et al., 2008). Behind this, new approaches for fluidic control would be strategic, to reduce reagents consumption, sample volumes, analysis time and material loss, eventually integrated to achieve in one shot sample-pretreatment and SNPs detection.

3. Comparison with electrochemical biosensors for SNPs detection Over years, different transduction approaches for SNPs detection have been reported. An overlook to current available strategies is here presented from an analytical point of view, to better place SPR biosensing within the panorama of molecular diagnostics. Among transduction principles applied to SNPs detection, electrochemical offers molecular strategies similar to those attempted with SPR platforms. For example, concerning proteins able to recognize SNPs, the performances of MutS were also confirmed by other transduction principles, basically electrochemical. Masarík et al. (2007) reported a voltammetric method where a DNA duplex (complementary or SNP containing) was bounded to streptavidin-coated magnetic beads. After incubation in solution, MutS was dissociated from the beads and electrochemically determined down to 56 aM, through oxidation of aminoacids residues (tyrosine and tryptophan) at a CPE (Carbon Paste Electrode). Signals related to different mismatches were distinguishable. However, the whole procedure resulted laborious due to the many steps required, in comparison to affinitybased ones. Looking at other electrochemical techniques, an attempt in the femtomolar range of sensitivity using proteins was described using magnetic beads (MBs) covered with streptavidin for amplification on a home-made electrically controllable magnetic gold electrode (ECM-GE), obtaining a detection limit of 0.37 fM (Zhang et al., 2013). In this chronoamperometric technique, streptavidinmodified MBs were bound to biotinylated capture probes. If biotinylated DNA targets hybridized with capture probes on the surface of the MBs then a streptavidin–horseradish peroxidase (HRP) could bind to free biotin of DNA target. Finally the structure was adsorbed onto the surface of the electrode (ECM-GE) thanks

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to MBs and chronoamperometric measures in H2O2-3,30 ,5,50 -tetramethylbenzidine (TMB) sulfate media were performed. The electrochemical current by oxidation of hydrogen peroxide, mediated by TMB, was proportional to HRP concentration and then to the target concentration in MBs hybridized structure. The mechanism took place spontaneously at room temperature and by adjusting the length and sequence composition of the toehold the kinetic rate was controlled. Further, different detection principles were recently associated to nanoparticles and nanomaterials used as promising tools to improve systems sensitivities in SNPs detection (Akhavan et al., 2012; Zamborini et al., 2012; Lu et al., 2013; X. Wang et al., 2013). SNPs detection reported on dually labeled probes (with thiol at its 50 end and biotin at its 30 end) on gold nanoparticles (AuNP)modified screen-printed carbon electrode (SPCE) using a cascadeevents leading to colourimetric detection of silver was recently reported (Liu et al., 2012). The DNA detection limit was 1.5  10  17 M and the selectivity was very high versus singlebase mismatched DNA. By the use of electrochemical approaches, very interesting results, in terms of excellent sensitivity can be also reached as demonstrated by using differential pulse voltammetry (DPV) with reduced graphene nanowalls (RGNWs) in developing an ultra-highresolution electrochemical biosensor for SNPs (Akhavan et al., 2012). The four bases of SNP (G, A, T, and C) could be detected by monitoring the oxidation signals of the individual nucleotide base. Single nucleotide polymorphisms detection was achieved down to 20 zM of DNA, resulting ultra-sensitive electrochemical biosensors. This work is representative of the most recent advances in SNPs detection by coupling the use of nanomaterials to different transduction principles, here electrochemical one. The detection limit reported in this example is excellent, although not so far from reported results obtained using nanoparticles coupled to SPRi, allowing attomolar detection of SNPs (Lao et al., 2009). Carbon nanotubes were also applied coupled to oligonucleotides analogs, i.e. locked nucleic acid (LNA) probes, andenzyme reaction driven by alkaline phosphatase, on PCR sample for SNP detection by electrochemical transduction. DL in nanomolar range, using sandwich-like assay format (Berti et al., 2011) was reported. However, this approach resulted to be less sensitive compared to what reported with NPs-DNA analogs (i.e. PNAs) by SPRi (D'Agata et al., 2008, 2011; Li et al., 2006; Sato et al., 2006) where a detection limit lower of three orders of magnitude was found (picomolar vs nanomolar). Enzymatic activity was also used in ligase-based approaches where the enzyme reaction occurred only in presence of fully matching sequences, allowing thus discrimination of SNP presence, as performed by SPR (1 pM as detection limit) (Li et al., 2006) and by electrochemical sensing in a sandwich-like assay in array format (Wan et al., 2009). The detection limit of the electrochemical sensing resulted 10 nM, comparable to a common simple SPR measurement without any signal improvement, although signal amplification, driven by peroxidase–avidin conjugate, was used in the former assay. Moreover, the whole procedure resulted quite complex by itself, involving two kinds of enzymes with precise temperature control. Finally intercalating agents are found also for SNPs detection by electrochemical transduction thanks to their charge transport through DNA films. In particular daunomycin (DM, a redox-active antitumor agent) binds DNA duplexes in a site-specifically manner and a difference in the electron transfer efficiency was observed comparing matching and mismatching targets. The coupling of the redox reactions of intercalated species with electrocatalytic processes in solution drove the sensitivity of this assay to subnanomolar range (Wong and Gooding, 2006). Daunomycin was also used as indicator in chronopotentiometric stripping analysis (PSA)

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Fig. 5. Mechanism of the regenerable E-SNP sensor, where in detection is based on conformational rearrangement of the triple-stem DNA probe upon binding a perfectly matched target. In the absence of perfectly matched target (left), the triple-stem probe forms a discontinuous, rigid, 21-base duplex, inhibiting electron transfer between the MB redox label and electrode. Upon target binding (right), the triple-stem structure is disrupted, liberating a flexible, single-stranded segment and enabling efficient collisions between the MB and electrode. Xiao et al. 2009.“Reprinted with permission from {Xiao, Y., Lou, X., Uzawa, T., Plakos, K.J.I., Plaxco, K.W., Soh, H.T., 2009. J. Am. Chem. Soc. 131, 15311–15316}. Copyright {2009} American Chemical Society.”

(Marrazza et al., 2000) to detect SNPs of apoE in PCR-amplified DNA extracted from human blood. Single-stranded DNAs were adsorbed at monitored potential on graphite screen-printed electrodes (SPEs). Methylene blue (MB) has been also exploited, as reported by Xiao et al. (2009), where SNP detection was achieved by electrochemical biosensing, in a single-step at room temperature using a probe folding into a triple-stem structure and labeled with MB (Fig. 5). A perfectly matched target causes probe rearrangement with release of the intercalating agent bound to single-stranded segment and allowing interaction of the redox reaction of MB with the free electrode with consequent faradic current increase. The reported detection limit was 5 nM, similar to those of obtained using molecular beacons and other hybridization probes. This assay was really simple and fast but, considering the reported level of sensitivity, precluded the detection of SNPs directly in genomic DNA of biological samples since the concentration of genomic DNA, obtained from standard phenol/chloroform-based extractions, is typically about 30 ng/μL. Finally, although direct comparison between SPR and non-SPR strategies for SNPs detection is demanding, due to significant differences in the detection strategies, some considerations of the relative ability to detect base substitution can be done. Compared to the most of electrochemical biosensors cited above, SPR allows multiple analyses in real time, tens of measurements on a single biochip, improving analytical productivity, which could be theoretically previewed by approaching SNPs electrochemical detection with high-density electrodes platforms, but not yet appeared as real clinical application. Further commercially available SPR instrumentations allow more controlled experimental conditions, such as flow and temperature, ensuring greater reproducibility for ready to use clinical diagnostics application, so real application in the very next future can be foreseen as also underlined by Helmerhorst et al. (2012). In summary, interesting and challenging work is running relatively to SNPs detection, using different sensing-based approaches. Conventional SPR and emerging SPRi applications result competitive and very promising when compared with other transduction principles in terms of sensitivity, assay design and parallel analysis. In all systems the use of auxiliary detection systems are welcome and successfully employed, i.e. proteins, oligonucleotides analogs, intercalating reagents and metallic nanostructures. Computerassisted strategies for optimizing probes design in assay formats,

reported for SNPs detection by SPRi, resulted to be of key importance for achieving ultra-sensitive and selective detection and can be of general validity, independently from the transduction principle used for SNP detection. Strategies based on coupling nanotechnology with various detection techniques look also very promising for the improvements of detection limits of the assays, thus brilliant and creative studies at this regards are expected and strongly encouraged. Ultra-sensitive SNPs detection, directly in genomic DNA, is at the forefront of bioanalytical chemistry but efforts for reliable analysis in real matrices are still necessary. Most of works deals with synthetic oligonucleotides but application to clinical diagnostics, pharmacogenomics, and theranostics should rely on sensing-platforms providing accurate results in real samples.

4. Conclusion On the whole, many of the work presented, SPR and not, showed sensitivity in the nanomolar range. These methods, for being applied to real samples analysis, need an amplification of the DNA to analyze. As instance, genetic material can be found in saliva down to femtomolar concentration (Zhang et al., 2013), thus an ultra-sensitive, selective and stable method for the specific detection is required. The application of nanoparticles or nanomaterials to biosensors permits to achieve high levels of sensitivity, by both SPR (D'Agata et al., 2008, 2011; Li et al., 2006; Sato et al., 2006) and electrochemical-based biosensors (Liu et al., 2012), even reaching detection limits at zeptomolar level, as previously discussed. Electrochemical methods can be susceptible to false positives arising from nonspecific binding of redox reporters and require exogenous reagents and post-hybridization washing steps. A control of the selectivity of the strategy is necessary and from this point of view, a multi-array system can be of great help. It allows the control surface and the coexistence of different receptors can confirm the selectivity of the system. Obviously the presence of different probes can also shorten the analysis, allowing the parallel analysis of a number of sequences in single samples. Non-SPRi systems were recently applied for SNPs detection, exploited as above reported, resulting less challenging than optical-based ones. Furthermore, SPRi allows a real-time image analysis in array format, and these are unique features in the perspective of having new bio-analytical methods for the high

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