6.2.1 Localized Surface Plasmon Resonance of Noble Metal Nanoparticles
Noble metal NPs have been used as decorative pigments since the time of the Romans, due to their brilliant colors. The physics behind this phenomenon is the excitation of surface plasmon resonance (SPR) by the incident electromagnetic field.9 When visible light shines on the particles, a certain portion of wavelengths is adsorbed to excite surface electrons oscillation, while other light is reflected that lends the material a certain color. Small spherical AuNPs (<50 nm in diam- eter), for example, adsorb green light, featuring a sharp adsorption peak (surface plasmon peak) at ∼520 nm in their adsorption spectrum (Figure 6.1A, curve a), and reflect red light (700 nm); therefore, the solution appears in wine red color.
Spherical silver NPs (AgNPs) have a bright yellow color due to the adsorption of
Wavelength (nm) (B)
(A)
Gradual aggregation
a
b c
d
Absorbance
300 400 500 600 700
400 500 600 700 800
Wavelength (nm) Gradual aggregation
a b c d
Absorbance
Figure 6.1 UV–vis adsorption spectra of (A) gold nanoparticles (13 nm) and (B) silver nanoparticles (27 nm) at different aggregation and dispersion status.
visible light of ∼400 nm (Figure 6.1B, curve a). For small NPs, surface electrons are oscillated by the incident light in a dipole mode. When individual particles are in close proximity or aggregate (e.g., the separation distance between par- ticles is comparable to or less than their radii), the oscillation of the plasmons from adjacent particles can become coupled.10 The strong enhancement of the localized electric field within the interparticle spacing broadens and red shifts the SPR spectra.10 For AuNPs, for example, progressively increased aggregation is characterized as a gradual drop of the plasmon peak at 520 nm and the appear- ance of a peak at ∼600 nm (Figure 6.1A, curves b–d) that are associated with solution color change from red to deep red, purple, blue, etc. depending on the degree of aggregation. The LSPR spectrum (solution color) may end up feature- less (colorless), reflecting extreme aggregation toward the bulk limit, where the plasmon wavelength moves into IR region and most visible lights are reflected.
For AgNPs (e.g., 27 nm), aggregation is characterized as the intensity decrease at 400 nm and the appearance of adsorption at ∼500 nm (Figure 6.1B, curves b–d) and the solution color change from bright yellow gradually to orange, depending on the degree of aggregation.
The sensitivity of a colorimetric assay is determined by the molar extinction coefficients of NPs’ plasmon bands that are determined by material composition and particle size.11,12 Larger NPs offer a higher sensitivity because they have a higher molar extinction coefficient for their surface plasmon bands.13–15 For AuNPs of 4–35 nm, for example, the molar extinction coefficients increase three orders of magnitude.15 A double logarithm plot of extinction coefficient against the particle size in diameter shows a good linear relationship that can be expressed in Equation 6.1, where ε is the extinction coefficient in M−1 cm−1, D is the core diameter of the AuNPs, and k = 3.32111, a = 10.80505.
lnε =k D aln + (6.1)
Based on an arbitrary estimation, 90% (or more) of the colorimetric assays in the literature use spherical AuNPs. This is apparently because gold is chemically stable and biocompatible. It is also easier to synthesis well-dispersed AuNPs of controlled size. Furthermore, bioconjugation chemistries have been well developed for AuNPs surface functionalization (an essential step in many assays). On the other hand, the use of silver particles (AgNPs) to construct colorimetric assays has received certain attentions more recently. The higher molar extinction coefficients of AgNPs rela- tive to AuNPs of same size provide a higher sensitivity for this material to be used as colorimetric probes.16–20 To circumvent the problem of chemical instability or chemical damping of AgNPs, core–shell structured particles (Ag@SiO2, Ag@Au) have been prepared and used in colorimetric assays.20,21 A thin layer of SiO2 or gold prevents the silver core from chemical damping and offers extended chemistries for biofunctionalization but does not alter silver’s plasmonic property. While most of the assays use either AuNPs or AgNPs separately, some assays have resourcefully
used a mixture of AuNPs and AgNPs. The two-component assays offer possibili- ties for multiplexing22 and enhance the assay reliability18,20 due to the presence of multiple spectrum signatures. In general, the extremely high extinction coefficients of metal NPs (e.g., 2.7 × 108 M−1 cm−1 at 520 nm for 13 nm AuNPs and 1.7 × 1010 M−1 cm−1 at 380 nm for 30 nm AgNPs13) make them ideal probes with high sensi- tive compared to fluorescent probes.14
6.2.2 Colloidal Stabilization
In aqueous solution, zero-charged bare NPs tend to aggregate under the van der Waals attractive forces. Colloidal stabilization is a matter of introducing repulsive forces between particles to prevent colloids from aggregating.23 There are three mechanisms for colloid stabilization, namely electrostatic stabilization (Figure 6.2A), steric sta- bilization (Figure 6.2B), and electrosteric stabilization (Figure 6.2C) that are usu- ally provided by charged small molecules, polymers, and electrolytes, respectively.
Under electrostatic protection, each particle carries a “like” electrical charge, a force of mutual electrostatic repulsion between adjacent particles is produced.
For spherical AuNPs and AgNPs, electrostatic stabilization is usually achieved using a coating of citrate ions that is formed during particle formation using the classic citrate reduction reactions.24 The surface charges, together with the counter ions in solution, form a repulsive electrical double layer that stabilizes the particles against van der Waals attractive forces.25 Since the thickness of the elec- trical double layer is determined by the bulk ionic strength of liquid medium, the electrostatic stabilization is highly sensitive to salt concentration. This explains why citrate ion-coated spherical NPs are stable in water but undergo aggregation when salt (NaCl, KCl, etc.) are added. In the case of steric stabilization, polymers adsorbing onto the particle surface or in solution provide a barrier that prevents the particles from crowding. The strength of this stabilization effect is not sensi- tive to salt concentration but determined by the molecular size and the capping
(A) –
– –
– –
– – –
– –
– – – – –
– –
–
– –
– –
– – – – –
– – –
–
–
(B) (C)
Figure 6.2 Nanoparticle stabilization mechanisms: (A) electrostatic stabiliza- tion, (B) steric stabilization, and (C) electrosteric stabilization.
density. When a polymer stabilizer is rich in charge, a double protection (i.e., electrostatic and steric) can be expected. This effect, termed electrosteric effect, is perhaps the most effective stabilization strategy. Biomolecules and their bind- ers, for example, drugs, peptides, nucleic acids, and proteins, are usually rich in charge and have polymeric properties. They can be effective stabilizers or coagu- lants of metal NPs.
6.2.3 Control of Nanoparticles Aggregation and Dispersion in Colorimetric Assays
It is the unique LSPR properties and the elegant colors associated with the aggre- gation and dispersion status that makes metal NPs ideal colorimetric reporters for biological analysis. In the design of colorimetric assays, the key is to control NPs aggregation and dispersion using biomolecules and biological processes.
There are two fundamental mechanisms to aggregate metal NPs in colorimetric assays: (1) through interparticle bond formation and (2) through removal of col- loidal stabilization effects.7 The interparticle bond formation-based assays rely largely on the use of bioreceptor-functionalized NPs. Network NPs aggregates can be formed through interactions with an analyte that carries multiple binding sites for the receptors on NPs surface or by direct interactions between receptor modified NPs when the receptors on each set of NPs are complementary with each other. In either case, specific binding forces (H-bonding, electrostatic inter- action, metal–ligand coordination, etc.) associated with biological recognition events (DNA hybridization, ligand–DNA binding, DNA cleavage, etc.) over- come the interparticle repulsive forces. Referring to the formation of network aggregates through interparticle bonds, this type of assay is termed “cross-link- ing aggregation” assay. On the contrary, in the case where assays are designed based on the removal of colloidal stabilization effects, NPs aggregates are formed without forming interparticle connects. This type of assay, termed “non-cross- linking aggregation,” usually uses unmodified NPs (i.e., no functionalization of NPs with bioreceptors), in which van der Waals attractive forces dominate the aggregation.
In this review, the assay schemes are categorized based on not only their aggre- gation mechanisms (i.e., cross-linking and non-cross-linking) but also the type of NPs used. Those involving bioreceptor-functionalized NPs are defined as Type I and those using unmodified NPs as Type II. These classification methods have been commonly used7,26,27 and would be widely acceptable, despite the presence of some other classification methods (i.e., “labeled assay” and “label-free assay” for the Type I and Type II, respectively).28,29 It is worth mentioning that not all the Type I assays (using bioreceptor-functionalized NPs) involve interparticle bonds formation. They can be based on control of colloidal stabilization.11,28,30,31 In the following application
sections, the terms Type I, Type II, cross-linking aggregation and non-cross-linking aggregation will be frequently quoted.
6.2.4 Quantification of Nanoparticle Aggregation and Dispersion
Since metal NPs’ aggregation can generate a huge SPR band shift up to hundreds of nanometers, the color change can be easily visualize by naked eye; therefore, no sophisticated instrument is needed for qualitative analysis (color photographs in Figure 6.1). To quantify the aggregation or dispersion, LSPR spectra of NPs under different status are recorded using a UV–vis spectrophotometer. As shown in Figure 6.1, the progressive particle aggregation is recorded as a gradual spectrum shift, that is, a gradual increase of intensity at the longer wavelengths, representa- tive of aggregated particles, and decrease of intensity at the original wavelength for dispersed particles. The ratio of absorbance at a longer wavelength and original wavelength (e.g., A600/A520 for AuNPs and A500/A400 for AgNPs) at a given time point is a quantitative measure of the aggregation and dispersion status. Since par- ticle aggregation is a continuous process, the ratio of absorbance can be plotted as a function of time to show how fast the aggregation process is (i.e., aggrega- tion kinetics). Figure 6.3A shows the typical aggregation kinetics (using AuNPs as an example). Sometimes, the variation of the integrated absorbance between two selected wavelengths is used for quantitative analysis.32–34
In some of the Type I cross-linking aggregation-based assays (for details, see Section 6.3), disassembly or dissociation of NPs aggregates, accompanied by a blue-to-red color change for AuNPs and orange-to-yellow change for AgNPs, is the measure of a particular biological process (e.g., DNA melting,8,16,20 bind- ing of DNA binders,35,36 and DNA cleavage37). In these cases, the assays are quantified by the plots of intensity of dispersed particles (A520 or A520/A600 for
More aggressive aggregation Less aggressive aggregation
(A)
A600nm/A520nm
t/min
Faster dissociation
Slower dissociation (B)
A520nm
t/min (or Temp/°C, etc.)
Figure 6.3 (A) Kinetics of AuNPs aggregation. (B) Dissociation of AuNPs aggre- gates over time or under parameters that can trigger the dissociation process (e.g., temperature).
AuNPs; A400 or A400/A500 for AgNPs) over time and/or any parameters that can trigger the disassembly, for example, temperature. Figure 6.3B shows typical the dissociation curves of AuNPs aggregates with different transition speed.