Rev Anal Chem 2014; 33(3): 153–164

Sabine Szunerits*, Jolanda Spadavecchia and Rabah Boukherroub Surface resonance: signal amplification using colloidal for enhanced sensitivity

Abstract: Among all the detection methods developed for . The unique optical, electronic, and catalytic bioassays, optical-based approaches are the most popu- properties of gold nanoparticles put them into the fore- lar techniques. A widely used and accepted bioanalyti- front of interest for signal amplification as will be outlined cal technique for routine characterization of molecular here. recognition events at a is resonance (SPR) spectroscopy. Compared with other tech- Keywords: biosensing; gold nanostructures; localized niques, SPR offers a series of advantages including rapid surface plasmon resonance (LSPR); signal amplification; and real-time analysis, in situ detection, and the possibil- surface plasmon resonance (SPR). ity to determine kinetic as well as thermodynamic param- eters of interacting partners without the requirement of DOI 10.1515/revac-2014-0011 tagging one of the two partners. This method is nowadays Received April 16, 2014; accepted July 13, 2014 used in academic and industrial research laboratories for drug discovery processes, safety, and quality con- trol applications as well as for understanding bioaffinity reactions. Past studies have shown that SPR can detect Introduction approximately 100 ng/l of . Currently, much effort is still invested to further improve SPR with respect to the It was in 1983, that Liedberg, Nylander, and Lundström sensitivity in the detection of low molecular weight ana- proposed a new optical concept for the sensing of affin- lytes as well as in the detection limit for different analyte/ ity reactions based on the determination of tiny varia- ligand reactions. This review will focus on the use of gold tions of the refractive index in the surrounding medium nanoparticles as an amplification strategy in SPR sensing. of a thin gold film interface, when illuminated with Compared to other review articles highlighting the several under a certain angle (Liedberg et al. 1983). Made com- synthetic routes and properties of gold nanoparticles and mercially available in 1990 by Biacore® (GE Healthcare), research utilizing gold nanoparticles for various sensing SPR spectroscopy is now an accepted bioanalytical tech- strategies, this review will exclusively look at the impact nique for routine characterization of molecular recogni- of gold nanostructures for ultrasensitive SPR sensing. Sig- tion events at a solid interface. It has, in particular, proven nal enhancement by gold nanoparticles is caused by sev- to be a powerful approach for the determination of kinetic eral effects such as surface mass increase due to enhanced parameters (association and dissociation rate constants) surface area, larger refractive index changes by the par- as well as thermodynamic parameters, including affinity ticle mass, themselves, and electromagnetic field cou- constants of interacting partners without the requirement pling between the plasmonic properties of the of tagging one of the two partners. With such SPR-based (localized surface plasmon resonance) and propagating affinity sensors, it is now easy and quite fast to acquire the relevant data that serve as a basis to understand biologi- cal specificity and function. *Corresponding author: Sabine Szunerits, Institut de Recherche Interdisciplinaire (IRI, USR 3078 CNRS), Université Lille 1, Parc de The basis for the ability of SPR to determine binding la Haute Borne, 50 Avenue de Halley, BP 70478, 59658 Villeneuve affinities and kinetic parameters lies in the high sensitivity d’Ascq, France, e-mail: sabine.szuner[email protected] of surface plasmons excited at the metal- interface Jolanda Spadavecchia: Laboratoire de Réactivité de Surfaces, UMR to changes in the refractive index of the adjacent medium. CNRS 7197, Université Pierre and Marie Curie, Paris VI, Site d’Ivry, Le Surface plasmons, also often known in the literature as Raphaël, 94200 Ivry-sur-Seine, France Rabah Boukherroub: Institut de Recherche Interdisciplinaire (IRI, surface plasmon (SPPs) or surface plasmon USR 3078 CNRS), Université Lille 1, Parc de la Haute Borne, 50 (SPWs), are longitudinal charge-density distributions Avenue de Halley, BP 70478, 59658 Villeneuve d’Ascq, France generated at the metal/dielectric interface (typically gold 154 S. Szunerits et al.: Impact of gold nanoparticles for ultrasensitive SPR sensing or silver) when light propagates through the metal. Such AB SPPs are transversal magnetic waves that propagate parallel along the interface between the metal and a dielectric layer (e.g., air, , etc). According to the Drude model, the dis- n persion relation of an SPP, which essentially correlates the n+∆n Light intensity relationship between the vector along the interface and the angular frequency ω, can be described by: SPR or incident angle

Light line SPP C Analyte flow ω

kSP Change in SPR signal 1/2 ω εε k = md (1) SP c εε+ Time md Figure 1 (A) Excitation of surface plasmons by the attenuated total where kSP is the component of the wave vector parallel to reflection (ATR) method in the so-called Kretschmann configuration; the interface, c is the speed of light in a vacuum, and εm and (B) illustration of angular modulation as the refractive index of the sensed dielectric changes from n (solid line) to n+Δn (dashed line); εd are the dielectric constants of the metal and dielectric, respectively. To allow optical excitation of surface plas- (C) SPR sensorgram angle change can be monitored as a function of time. mons, as kSP is outside the light cone, the SPP wave cannot be directly excited from the far field by a plane wave, and the wave vector of the incident light must be increased. Physically, this can be achieved by passing the light wave Many approaches have been explored to improve through an optically denser medium in the attenuated the LOD of propagating SPR such as the use of fluores- total reflection (ATR) configuration (Figure 1A). The suffi- cence (Yu et al. 2004), modification of the sensor chip ciently thin metal film allows some of the incident light to with metallic particles (Lyon et al. 1998), or performing be transmitted through the metal-air boundary, where the a labeled assay using large particles (He et al. 2000). surface plasmon is excited. Prism-based angular modula- Among these, the use of gold nanoparticles (Au NPs) has tion with a conventional SPR structure consisting of a high attracted significant attention. Unlike propagating plas- refractive index prism and a thin metallic layer (typi- mons supported on a bulk metal surface, nanoparticles’ cally around 50 nm of gold) has become the most com- plasmons are quantized electron oscillations confined to monly used configuration in SPR sensing technology and nanoscale volumes, which provide a means for manipu- is commercialized by several companies. lating light- interactions while circumventing the The excitation of surface plasmons is characterized by diffraction limit. Although Mie’s theory of light scatter- a dip in the angular spectrum of the reflected light at the ing and absorption by gold nanospheres was reported angle of resonance (Figure 1B). The promising potential of more than a century ago (Mie 1908), it is only recently SPR sensors lies in the high sensitivity of the position and that improved computational approaches and theoretical shape of the SPR dip to a change in the refractive index of developments have allowed the mapping of plasmon reso- the dielectric layer or formation of a nanoscale-thick film nances in more complex nanoparticles and assemblies. In (Wijaya et al. 2011, Szunerits et al. 2012, 2013). particular, a new theoretical approach known as plasmon While this technique has proven to be a robust tool hybridization theory (Prodan et al. 2003) draws on the and is now used in research as well in industrial laborato- parallels between the behavior of plasmons in assemblies ries, much effort is still invested to improve SPR, especially of metallic nanoparticles and that of electrons in quantum with respect to sensitivity in the detection of low molecu- molecular orbitals. This theory predicts that the plasmons lar weight analytes. Low molecular analytes ( < 1000 Da) on neighboring metallic nanostructures interact, mix, and are more difficult to quantify by SPR than larger ones hybridize just like the electronic wave functions of simple (Mann et al. 1998). atomic and molecular orbitals. A critical prerequisite for S. Szunerits et al.: Impact of gold nanoparticles for ultrasensitive SPR sensing 155 the use of these materials is the production of high-quality in the collective oscillation of electrons, termed localized metallic nanoparticles with tunable size and controllable surface plasmon (LSP) (Figure 2A) (Szunerits and Bouk- shape to tailor their distinct plasmonic signatures. This herroub 2012, Akjouj et al. 2013). The LSP has two major can be achieved through wet chemical synthesis tech- effects. The electrical fields near the ’s surface are niques, in which careful optimization of synthesis condi- greatly enhanced, and the particle’s optical extinction tions allows rational control over the nanoparticles’ size displays a maximum at the plasmon resonance frequency, and morphology (Pèrez-Juste et al. 2005, Xia et al. 2009). occurring in the visible wavelength for noble metal parti- Before illustrating the examples of Au NP-enhanced SPR cles (Figure 2B). Gold nanoparticles are the most widely sensing, the synthetic aspects of gold nanostructures and used ones as they are chemically stable and resistant to the way they enhance the SPR signal will be discussed in surface oxidation. more details. Numerous preparation methods for Au NPs have been reported, including both top-down and bottom-up approaches (Daniel and Astruc 2009, Mayer and Hafner 2011, Saha et al. 2012). They allow to form gold nanostruc- Interest of gold nanoparticles for tures of different sizes, shapes, and composition. The signal enhancement effect of the metallic nanoparticles’ shape on LSPR can be assessed using a boundary integral method. Shape varia- The bright of metal nanoparticles have attracted tions change not only the eigenvalues but also their cou- considerable interest right since historical times, where pling weights to the electromagnetic field. Thus, rather they have been used as decorative pigments in stained small changes in the shape may induce large variations glass windows. Their tuneable optical properties and their of the coupling weights. It has been found that shape addressability via spectroscopic techniques (UV/Vis, dark changes that bring volume variations > 12% induce struc- field microscopy, etc.) have made metal nanoparticles of tural changes in the extinction spectrum of metallic nano- particular interest for SPR signal enhancement. The excep- particles. Also, the largest variations in eigenvalues and tional optical properties of gold and silver nanostructures their coupling weights are encountered by shape changes result from the participation of the particle’s free electrons along the smallest cross-sections of nanoparticles.

A Electric field

Metal sphere


B 1.5 0.4 Gol d nanoparticles 5 nm 10 nnm 0.3 20 nm

1 u.) (a.

on 0.2 ncti ti Ex Extinction (a.u.) 0.5


0 202 nnmm 400 500 600 700 800 0 400 500 600 700800 Wavelength (nm) Wavelength (nm)

Figure 2 (A) Formation of LSPR bands, (B) UV-Vis absorption spectra of gold nanoparticles of different size (left), and gold nanorods and nanostars (right) (Spadavecchia et al. 2013). 156 S. Szunerits et al.: Impact of gold nanoparticles for ultrasensitive SPR sensing

Fabrication of different gold nanostructures using strong thiol-gold interactions to protect Au NPs with thiol ligands. The Au NPs are generated in organic While the synthesis of can be tracked to such as toluene with controlled diameter in the , it was Turkevich who developed one range of 1.5–5 nm. The thiol-protected Au NPs feature of the most popular approaches for the synthesis of gold superior stability because of the strong thiol-gold inter- nanostructures. It is based on the use of citrate reduction action (Figure 3A). Such particles can be dried and then of HAuCl4 in water (Figure 3A). In this method, citrate re-dispersed in organic solvents without any aggregation acid acts as both reducing and stabilizing agent (capping or decomposition. They can be further modified by ligand agent) and provides Au NPs with a diameter of 20 nm. A exchange reactions and by any chemical coupling strate- further significant breakthrough was achieved by Brust gies depending on the application sought after (Figure 3B). and Shiffrin, who reported a two- synthetic strategy Indeed, the choice of the stabilizing ligand is important.

- + A HAuCl4 + 3 Na3C6H5O7 2Au + 3 Na3C5H6O5 + 3NaCl+ 5Cl + 5H + 3CO2 - O O - HO O - Lewis bond O OH O -O O O OO -O (loosely bond) O - O O O Au NP - O OH O O O O OH Citrate O O -O -O Citrate capped Au NPs

1. nOct4NBr R HAuCl4 S n R 2. HS n Thiol capped Au NPs 3. NaBH4

B Product - R O O H - -COOH HO O N OH -NH2 -O O O EDC/NHS OO O -O R O HS n R O O OH O O S n O -NH Ligand exchange 2 C -COOH OH Thiol capped NH2 Citrate capped O EDC/NHS O Au NPs Au NPs -O - R=COOH, NH , etc O 2 O O R R-SH S N N O O R O N NH R-NH-NH2


Protein capped R N N Au NPs 3 N N EDC/NHS R

Figure 3 (A) Formation of citrate-capped gold nanostructures (Turkevich method) and thiol-protected gold nanostructures; (B) chemi- cal functionalization of gold nanostructures through ligand exchange reactions and by covalent coupling strategies using different end ­functionalized gold nanostructures. S. Szunerits et al.: Impact of gold nanoparticles for ultrasensitive SPR sensing 157

The capping agent can interfere in the particle growth, silver than for gold resulting in higher efficiency, but also prevents agglomeration thereby rendering stable less plasmon damping, and narrower plasmon line width colloidal . A widely used capping agent in the (Johnson and Christey 1972, Mayer and Hafner 2011). synthesis of anisotropic gold nanostructures such as gold nanorods is cetyl-trimethylammonium bromide (CTAB) (Jana et al. 2001, Murphy 2002, Spadavecchia­ et al. 2012). Enhancement of SPR signals - 0 The conproportionation reaction of AuCl4 and Au into - AuCl2 is believed to be favored by the presence of CTAB The general principle behind LSPR-based sensors is the (Nikoobakht and El-Sayed 2003), attributed to the high shift in wavelength and/or the change in absorption inten- binding constant of Au+ to CTAB. It was suggested that sity of the LSPR band upon analyte detection. Colorimet- oxygen plays the role of oxidizing agent, while Br- acts as ric detection-based assays are one of the earliest assays a complexing agent, in a mild oxidation process using O2 showing the interest of gold nanostructures. Mirkin et al. to decrease the aspect ratio of AuNRs (Tsung et al. 2006). (1996) and Rosi and Mirkin (2005) reported on an assay The use of such as polyvinylpirrolidone (PVP) using oligonucleotide-functionalized Au NPs that exhibit as stabilizing agent has allowed the formation of highly strong red shifts upon aggregation in the presence of branched gold nanostructures such as gold nanostars complementary nucleotides. The strong change is a (Kawaguchi et al. 2008, Kumar et al. 2008). result of particle-particle plasmonic coupling, producing Sharper plasmonic bands are obtained with silver pronounced red shift in the LSPR frequency. The clearly nanoparticles, but they suffer from being easily oxidiz- distinguishable color shift facilitates a very simple sensor able under ordinary laboratory conditions (Haes and Van readout that often can be performed with the naked eye. Duyne 2002, Haes et al. 2004, 2006). The enhanced plas- In the case of Au NP-mediated SPR signal amplification, monic performance originates from the intrinsic optical with the aim to increase the shift in ΘSPR and (ΔR/R) of the properties of the silver particles: (i) the real part of the die- propagating SPR interface, different concepts can be used lectric function of silver varies more with wavelength than (Figure 4A). The first is based on the direct deposition of that of gold over the visible light region, (ii) the imaginary gold nanoparticles onto the SPR surface, the second on part of the dielectric function is, in addition, smaller for the deposition of Au NPs on a spacer layer coating the gold


Glass Glass Glass Au NPs deposited Au NPs deposited Modified Au NPs in directly onto on spacer layer interact with SPR interface modified SPR interface

BCGold NPs amplified Gold NPs amplified immunoassay sandwich immunoassay 1.0

R 0.5

25 A 25 B 0 20 20 42 44 46 15 15 10 θ (°) 10 5 5 54 55 56 57 54 55 56 57 Angle (°) Angle (°)

Figure 4 (A) Gold nanoparticles mediated SPR signal amplification strategies; (B) reflectivity of a gold film measured as a function of the incident angle in air for gold (circles), for a 1,6-hexanedithiol modified Au film (triangles) and for a layer of Au NPs deposited onto Au ­film-HDT (squares). The solid lines area calculated fits to the exp. data [reprinted with permission from Hutter et al. (2001); copyright © 2001, American Chemical Society], (C) Au NP-amplified immunoassays and the corresponding SPR signals. 158 S. Szunerits et al.: Impact of gold nanoparticles for ultrasensitive SPR sensing

SPR interface, while the third one uses analyte-labeled Au and Hall 1983). The LSPR-SPR interaction is, indeed, a hot NPs to interact with the ligand on the SPR surface. topic in near-field and far-field optics. One of the first con- The first reports on the role of Au NPs on SPR signal were tributions to this question is the work by He, Keating, and by Lyon et al. (1999) and Hutter et al. (2001). By comparing coworkers. They investigated SiO2-coated gold films as a experimental SPR data for gold substrates with and without substrate for Au NP-amplified SPR (Figure 5A). Coating the particles attached (25–30 nm in diameter) to the substrate SPR surface with an additional dielectric layer can be bene- metal via a sandwiched monolayer of 1,6-hexanedithiol, it ficial in several regards. While the surface devel- has been shown that the width and the efficiency of SPR are oped on gold has been of great value (Ulman 1996, Love effected (Figure 4B). The interest of using antigen-modified et al. 2005), the limitations of working on gold are becom- Au NPs for colloidal gold enhanced SPR sandwich immuno- ing more noticeable with increasingly complex fabrication sensing was demonstrated by Lyon et al. (1999) (Figure 4C). requirements for biometric systems and arrays. Alternative In this approach, anti-human immunoglobulin G (h-IgG) routes and improvements were, thus, sought after. was immobilized onto a carboxylate-modified SPR inter- dioxide-based materials such as glass (silicate) are standard face via traditional carbodiimide coupling chemistry to materials for biosensing being inexpensive and benefiting free amine moieties on the protein. Two architectures for from a rich variety of well-developed attachment schemes particle-enhanced SPR immunosensing were investigated. based on silane-coupling chemistry. The classical strategy The first involved direct binding of antigen-Au NP conju- for adding functionality to such interfaces is based on the gate to the antibody-derivatized surface, while the second reaction of the SiO2 layer with functional silanes (Figure 5B) comprises the same antibody-derivatized surface followed (Manesse et al. 2008). In this case, reaction of the SiO2 layer by binding of a free antigen and then a second antibody- with 3-aminopropyltrimethoxysilane (APTES) results in the Au NP conjugate (Figure 4C). Tremendous signal amplifica- introduction of amine terminal groups, onto which citrate- tion with a picomolar detection of human immunoglobulin capped Au NPs can be integrated by drop coating. A pro- G has been obtained using this approach. The factors for nounced SPR angle shift is observed at each coating step. such an amplification are multiple. Interaction of the ana- The change of the SPR angle shift upon Au NP binding was lyte-modified Au NPs in solution with ligands on the SPR investigated as a function of the SiO2 layer thickness. The interface results in a significantly higher increase in the observed angle shift increases upon increasing Au NP-gold refractive index at the metal surface than in the absence surface separation, reaching a maximum value at 32 nm of the Au NPs. The fundamental ideas behind this signal before decreasing. An immunochemical assay was carried amplification concept are that the artificial increased mass out using these interfaces to demonstrate the feasibility of of the analyte due to the linked Au NPs results in higher distance-amplified SPR for protein detection. Although the refractive index changes on the SPR surface, leading to SiO2-coated SPR surface gave a broader curve compared to larger SPR shift (Lyon et al. 1998, He et al. 2000, Hutter et al. the uncoated surface, it resulted in a threefold-enhanced 2001, Matsui et al. 2005, Li et al. 2006). This is furthermore SPR angle shift. This comes on top of the 1000-fold increase amplified by the increased amount of analyte loaded onto in sensitivity reported for the Au NP-amplified SPR. the Au particles. The other reason for signal enhancement However, a fundamental question in the design of the is linked to electromagnetic field coupling between SPR LSPR-SPR setup is the contribution from the coupling effect and LSPR. This coupling factor is, indeed, expected to play on the analytical signal (rather the mass increase effect), the dominant role of the signal enhancement. This electro- whether it can play a dominant role in the signal enhance- magnetic coupling between metal nanoparticles and the ment and can be optimized for such an analysis format. thin metal films used in SPR substrates has been found to The Au NP density in the vicinity of the gold surface might be a complex distance-dependent phenomenon (Holland have an important influence as it changes the interparticle

AB R R Si OH OH OH Si-OC2H5 OOO SiO C H O OC H 2 SiO2 2 5 2 5 Piranha SiO2 SPR SPR SPR R=NH2, CN, SH, etc Glass-based SiO2 spacer 0-50 nm SPR interface

Figure 5 (A) Distance-dependent LSPR-SPR surface assembly. (B) Surface modification strategy of SiO2-coated SPR interfaces. S. Szunerits et al.: Impact of gold nanoparticles for ultrasensitive SPR sensing 159 space and, consequently, the position of the LSPR wave- of established procedures for the preparation of these length as well as the surface mass and, thus, the dielectric conjugates. Such -enhanced SPR detection properties of the layer next to the gold layer. This important has been used for the detection of proteins (Lyon et al. point was recently investigated by Hong and Hall using a 1998, Pieper-Furst et al. 2005, Cao and Sim 2007, Huang spectral rather than an angle resolved SPR read out (Hong et al. 2008, Kawaguchi et al. 2008, Mitchell and Lowe and Hall 2012). They showed that the LSPR-SPR coupling 2009, Wang et al. 2009) and DNA probes (He et al. 2000, effect diminishes when the spacer layer is thicker than Bailey et al. 2003, Fang et al. 2006) and most recently ≈10 nm. The coupling effect is distance dependent and for the study of -lectin interactions (Huang dominates the signal enhancement particularly when the et al. 2013). Detection limits ranging from picomo- distance is small ( < 10 nm). This conclusion was confirmed lar to attomolar levels, corresponding to a 1000 to 107 through the study of DNA hybridization in the presence improvement in sensitivity when compared to standard and absence of Au NP-labeled DNA. SPR have been reported utilizing in most cases - wich-like assay formats. Larger particles are expected to result in larger refractive index changes, following specific interaction onto the SPR chip. The increased Some examples of gold-nanoparticle- difficulty in bio-functionalizing larger particles, while amplified SPR studies maintaining good colloidal stability in saline solutions, has made the use of larger and differently shaped gold The majority of Au NP-enhanced SPR studies reported nanostructures a big challenge. Recent work by Lee have been focused on the use of DNA-functionalized Au and co-workers investigated the enhancement factor of NPs of 10–15 nm in diameter due to the wide availability SPR detection of thrombin by using differently shaped

Amide bond A a O OH NHSS Au NPs (i) EDC/NHSS EDC

(iii) Antibody MUA R’-NH S 3 c S b O O O Th H3C CH3 O OO O

OR O SH HS O Prolinker B S

B a :638 646 nm b :644 653 nm λ :531 535 nm 1.2 λmax λmax 1.2 c max 1.2 1.0 1.0

0.8 0.8 0.8 0.6 0.6 Extinction Extinction Extinction 0.4 0.4 0.4 0.2 0.2

0 0 0 400 500 600 700 800 400 500 600 700 800 400 500 600 700 800 Wavelength (nm) Wavelength (nm) Wavelength (nm)

Figure 6 (A) Schematics showing the anti-thromin linking to the various Au nanostructures and the modification of the SPR interface with prolinker B or a of prolinker B and thiol-terminated PEG, followed by linking of 5-amine terminated thrombin-aptamer; (B) UV-Vis spectra of the different Au nanostructures before and after antithrombin modification [reprinted with permission from Kwon et al. (2012); copyright © 2012, American Chemical Society]. 160 S. Szunerits et al.: Impact of gold nanoparticles for ultrasensitive SPR sensing

Au particles (Figure 6A) (Kwon et al. 2012). The parti- undergo a significant red shift (100 nm) when placed cles were modified by antithrombin antibodies using close to a gold metal film (He et al. 2004). Plasmonic EDC/NHS coupling chemistry of 11-mercaptoundeca- coupling is, thus, likely to occur to various extents for all noic acid-modified Au nanostructures. The SPR surface particles. The poor sensing performance of the nanoc- was modified with prolinker B or a mixture of prolinker age bioconjugate can be attributed to a lower material B and thiol-terminated PEG, followed by exposure to density compared to rod and spherical particles even 5-amine-modified thrombin aptamer. Figure 6B shows though further work is still needed to understand the the UV-Vis spectra of the different Au nanostructures spectral changes. The Au spheres showed detection with LSPR bands at 646 nm (gold nanocages), 653 nm limit of 1 aM for thrombin compared to 10 aM for the (gold nanorods), and 535 nm (quasi-spherical parti- nanorods. cles). In terms of sensing performance, the response of Corn et al. investigated the possibility of ultrasensi- quasi-spherical particles was more than double when tive sensing of microRNAs by nanoparticle-amplified SPR compared to the others. The difference is believed to imaging (Figure 7A) (Fang et al. 2006). MicroRNAs are originate from the changes each adsorbed NP induces small RNA (19–23 mers) that can regulate the on the real and imaginary components of the refractive expression of genes in plants and animals by binding to index of the at the SPR chip/solution interface. the 3′ untranslated region of messenger RNA. The mul- The presence of a high density of Au NPs will affect the tiplexed detection of mRNAs with DNA microarrays is a real component value, while near-field plasmonic cou- particular appealing method for miRNA profiling in bio- pling will primarily affect the imaginary component. logical samples. The direct hybridization of miRNA onto Plasmonic coupling will occur when there is sufficient complementary DNA microassays is problematic owing to overlap between the wavelength used to excite the the short length of the miRNA sequence. Polyadenylation plasmon (760 nm in this case) in the gold film and the of the 3′-end of target miRNAs in solution using poly-A local SPR profile of an individual particle. One has to polymerase is widely used. Using florescence imaging, keep in mind that the spectrum of individual Au NPs will the detection of miRNAs down to a of 10 pM

-T-T n A -A-A -A-A n n 3’-OH T-T-(T) A-A-(A) A-A-(A) -T(T)-T-T n -T-T T- 5’ n T-T-(T) 3’ poly-A SPR SPR SPR miRNA SPR

B a 200 fM miR-122b


b 50 fM miR-16 + 50 fM miR-23b


c 10 fM miR-23b

miR-122b 0.2% miR-16 miR-23b 0 1.0 2.0 3.0 Distance (mm)

Figure 7 (A) Schematics showing the detection of microRNAs using a nanoparticle-amplified SPR strategy; (B) SPRi images and the cor- responding line profiles obtained for the detection of 200 fM miR-122b, 50 fM miR-16, and 50 fM miR-23b [reprinted with permission from Fang et al. (2006); copyright © 2006, American Chemical Society]. S. Szunerits et al.: Impact of gold nanoparticles for ultrasensitive SPR sensing 161 is possible. A considerable improvement was achieved by Very recently, Au NP-based SPR was employed for amplification of the SPR hybridization signal with Au NPs the sensitive detection of carbohydrate-lectin interactions modified with poly-T DNA with detection limit of 10 fM (Figure 9) (Huang et al. 2013). Indeed, with half or more of (Figure 7B) (Fang et al. 2006). all proteins containing some carbohydrate motives, the use A rather different approach for enhanced SPR-based of a bioanalytical tool allowing glycosignatures to be scruti- DNA sensing was reported by Yang et al. (2007) (Figure 8). nized rapidly is without doubt one of the principal driving The Au NP size was enlarged through a catalytic growth. forces behind the rapid development of SPR-based glyco- To inhibit metal deposition on the Au film in the process nomics. The interaction between and specific of catalytic growth of Au NPs, a 25-nm-thick SiO2 layer was proteins mediate many important physiological processes, deposited on the SPR chip. Under these experimental con- including bacterial and viral infections, inflammation, and ditions, the detection limit of direct hybridization meas- cancer metastasis. Concanavalin A (Con A), a plant lectin, urement, Au NPs amplified SPR, and catalytic growth of can specifically bind to different membrane receptors con- Au NP-enhanced SPR were 1.2 nM, 17.3 pM, and 4.8 pM, taining mannose and glucose residues. Con A was chosen as respectively (Yang et al. 2007). a protein model here. The assay is based on , a linear Nanoparticle-amplified SPR interfaces were recently polysaccharide composed of 1,6-α-d-glucopyranoside, employed for the detection of mercury (Chang et al. coated Au NPs, which can interact specifically with ConA- 2011). Mercury ions have shown to coordinate to DNA modified SPR interfaces. The Con A-functionalized SPR duplexes that feature thymine-thymine base pair mis- interfaces were achieved by first thiolation with negatively matches. Hairpin DNA probes containing a 21mer T-rich Hg2+ charged 3-mercapto-1-propanesulfate, followed by immer- binding sequence loop and a 24 mer sequence at each end sion into poly(diallyldimethylammonium) chloride (PDDA) of the strand were linked to SPR interfaces. In the pres- and adsorption of graphene oxide (GO). Phenoxy-derivat- ence of Hg2+, the hairpin loops of the DNA strand form a ized dextran (DexP) was assembled onto GO through π-π duplex-like structure through the formation of a T-Hg2+-T stacking interactions followed by incubation with Tween complex. As a result, a terminal DNA binding domain is 20 and sodium dodecylsulfate (SDS) to prevent nonspecific available. This terminal DNA binding domain was further adsorption. The resulting sensing interface could specifi- hybridized with Au-NP-conjugated DNA strands. A linear cally capture Con A, which could further react with dextran- correlation was observed over a mercury concentration modified Au NPs through the specific interaction between range of 5–5000 nM with high selectivity. Con A and dextran. Sensitive detection of Con A in the range of 1–20 μg/ml with a detection limit of 0.39 μg/ml was reported. Compared to the direct assay format, the sandwich SPR sensors led to an improvement of 28.7-fold in the sensi- tivity (Huang et al. 2013).

NADH/HAuCl4 SiO2 SiO SiO SiO DNA 2 2 2 SPR SPR Au NP-DNA SPR SPR Perspectives and conclusion

Figure 8 Enhanced SPR sensing with catalytic growth of Au NPs The advancement in the fabrication and manipulation of (see Yang et al. 2007). gold nanostructures as bio-recognition labels and their use

H3C CH H3C CH3 H3C.N.CH3 N 3 N O- + + + O u u u O- S O O O O O S O O S O O S O O S O n + S N 1. PDDA CH S S S HS H3C 3 1. DexP SPR SPR 2. Graphen oxide (GO) SPR SPR SPR 2. Tween 20, SDS Con A Con A modified SPR interface

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Sabine Szunerits has been a Professor of Chemistry at the Univer- Rabah Boukherroub is a research director at the CNRS, Lille, sity Lille 1, France, since 2009 and was nominated as a member France. His research interests are in the area of chemistry, surface of the “Institut Universitaire de France” (IUF) in 2011. Her current chemistry, functional materials, and photophysics of research interests are in the area of material science with an nanostructures with an emphasis on and lab-on-chip emphasis on the development of novel analytical platforms and applications, and development of new tools for studying molecu- interfaces for the study of affinity binding events and in the modi- lar dynamics in vivo. He is a co-author of more than 290 research fication of nanostructures (diamond particles, magnetic particles, publications and wrote several book chapters on subjects related to nanographene) for biomedical applications. She has co-authored nanotechnology, materials chemistry, and biosensors. He has eight more than 170 research publications, wrote several book chapters, patents or patents pending. and has six patents.

Jolanda Spadavecchia obtained her degree in Pharmaceutical Chemistry and Technology from the University of Bari, , in 2000 following postgraduate studies at the University of Lecce on the synthesis of phthalocyanines from natural products. From 2001, she was a PhD student of Material Engineering in the Engineer- ing Faculty of the University of Lecce (Italy). She worked at the Laboratoire Charles Fabry of the Optic Institute, in Orsay, France, for 3 years (2006–2009). Her past research activity has mainly been devoted to surface plasmons resonance imaging systems and biomolecular surface interaction characterizations, for dynamical biochip applications. Since 2010, she has been a researcher for CNRS at the Laboratory of Reactivity and Surface (Université Pierre Marie Curie Paris VI, France). Her research interests concern the synthesis of organic compounds, gold nanoparticles, sensor appli- cations and characterization, DNA-based sensors, and the synthesis of TiO2 nanocrystals and nanohybrid systems (Au/TiO2; ZnO/organic molecules) for biomedical applications. She has co-authored about 48 publications and 50 conferences.