Naked Eye Immunosensing of Food Biotoxins Using Gold Nanoparticle-Antibody Bioconjugates

Colorimetric immunoassays using gold nanoparticles (AuNP) form a special class of assays where AuNP act as a transducer to monitor binding events between an antigen and an antibody. Indeed, AuNP display unique optical properties that can been exploited in various ways to develop biosensors. One of the most striking properties of colloidal AuNP (and more generally of noble metal nanomaterials) is their extremely high extinction coefficient in the visible range of the spectrum owing to the localized surface plasmon resonance (LSPR) phenomenon. This feature makes AuNP detectable down to very low concentrations by absorption spectroscopy or even by the naked eye. Herein we took advantage of the high detectability of AuNP to design a solid-phase, sandwich-type, colorimetric immunosensor aiming at the detection of staphylococcal enterotoxin A (SEA). A test zone comprised of a polyclonal anti-SEA antibody was created at the surface of amino-functionalized glass slides via high affinity binding to covalently immobilized Protein A. The same antibody was conjugated to 13 nm diameter AuNP to afford the nanoimmunoprobe. After the glass slides were successively exposed to SEA and AuNP-antibody bioconjugate, a distinct red spot appeared at the detection zone from as low as 1 ng SEA in buffer. Quantification of SEA in the 10–500 ng/mL range was established using a benchtop UV–visible spectrometer by integration of the LSPR band centered at 530 nm. Eventually, this biosensor was applied to the detection of SEA in milk with a limit of detection of 1.5 ng/mL

Investigation of an Allergen Adsorption on Amine- and Acid-Terminated Thiol Layers: Influence on Their Affinity to Specific Antibodies

This work describes the controlled immobilization of a recognized allergen, beta-lactoglobulin, onto gold transducers with the aim of optimizing the elaboration of a biosensor directed against allergen-produced antibodies. This protein was immobilized on both amine- and acid-terminated thiol self-assembled monolayers, and the influence on its affinity to a specific IgG was investigated. For amine-terminated layers, the β-lactoglobulin was immobilized via its surface acid functions implying an activation step with 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride/ester of N-hydroxysuccinimide (EDC-NHS). Conversely, the grafting on acid-terminated layer takes advantage of the accessible amine groups that react with the activated acidalkylthiols. The resulting layers of β-lactoglobulin were then submitted to various concentrations of rabbit serum containing β-lactoglobulin specific rabbit immunoglobulin (rIgG), and the antigen/antibody affinity was evaluated using modulated polarization-infrared absorption spectroscopy (PM-IRRAS) and Fourier transform surface plasmon resonance (FT-SPR). Even though for similar concentration, the amount of adsorbed β-lactoglobulin was identical on both surfaces, atomic force microscopy (AFM) images showed a better dispersion for amine-terminated layers. Moreover, the affinity to specific IgG, estimated under static conditions by PM-IRRAS and under dynamic conditions by SPR, was different. Grafting β-lactoglobulin via its acid groups gave an affinity constant 3 times higher than its immobilization via its amine groups despite the fact that the amount of accessible recognition sites appeared to be similar for both systems. This work underlines the importance of the involved chemical groups upon protein immobilization on their biological activity and will be essential for the construction of nondirect biosensors for detecting specific immunoglobulin E (IgE) of allergens

Hollow Gold Nanoshells for Sensitive 2D Plasmonic Sensors

The interaction of incident light with noble metal nanoparticles engenders a fascinating phenomenon known as localized surface plasmon resonance (LSPR). This results in the presence of single or multiple intense absorption bands in the visible to near-infrared spectral range whose position is affected by the refractive index of the surrounding medium. In this comprehensive study, we thoroughly investigated the experimental parameters governing the size, aspect ratio, and optical properties of hollow gold nanoshells (hAuNSs) synthesized through the galvanic exchange of cobalt-based nanospheres. Subsequently, we rigorously determined both the empirical and the theoretical refractive index sensitivity (RIS) and figure of merit (FoM) of these engineered nanostructures. Notably, hAuNS with an external diameter of 98 nm and a shell thickness of 13 nm demonstrated a noteworthy RIS of 360 nm/RIU and an FoM of 2.0 in solution. In contrast, solid gold nanospheres (sAuNSs) of a similar diameter exhibited a significantly lower RIS of 136 nm/RIU. Following the transfer of both of these nanostructures onto glass slides for the development of LSPR sensors, it was intriguing to note that the RIS and FoM remained largely unaffected. These findings underscore the potential of these plasmonic nanoparticles as promising candidates for the design of sensitive solid-phase LSPR sensing devices

The Prevailing Role of Hotspots in Plasmon-Enhanced Sum-Frequency Generation Spectroscopy

The plasmonic amplification of non-linear vibrational sum frequency spectroscopy (SFG) at the surfaces of gold nanoparticles is systematically investigated by tuning the incident visible wavelength. The SFG spectra of dodecanethiol-coated gold nanoparticles chemically deposited on silicon are recorded for twenty visible wavelengths. The vibrational intensities of thiol methyl stretches extracted from the experimental measurements vary with the visible color of the SFG process and show amplification by coupling to plasmonics. Since the enhancement is maximal in the orange-red region rather than in the green, as expected from the dipolar model for surface plasmon resonances, it is attributed mostly to hotspots created in particle multimers, in spite of their low surface densities

Biomineralization in Barnacle Base Plate in Association with Adhesive Cement Protein

Barnacles strongly attach to various underwater substrates by depositing and curing a proteinaceous cement that forms a permanent adhesive layer. The protein MrCP20 present within the calcareous base plate of the acorn barnacle Megabalanus rosa (M. rosa) was investigated for its role in regulating biomineralization and growth of the barnacle base plate, as well as the influence of the mineral on the protein structure and corresponding functional role. Calcium carbonate (CaCO3) growth on gold surfaces modified by 11-mercaptoundecanoic acid (MUA/Au) with or without the protein was followed using quartz crystal microbalance with dissipation monitoring (QCM-D), and the grown crystal polymorph was identified by Raman spectroscopy. It is found that MrCP20 either in solution or on the surface affects the kinetics of nucleation and growth of crystals and stabilizes the metastable vaterite polymorph of CaCO3. A comparative study of mass uptake calculated by applying the Sauerbrey equation to the QCM-D data and quantitative X-ray photoelectron spectroscopy determined that the final surface density of the crystals as well as the crystallization kinetics are influenced by MrCP20. In addition, polarization modulation infrared reflection–absorption spectroscopy of MrCP20 established that, during crystal growth, the content of β-sheet structures in MrCP20 increases, in line with the formation of amyloid-like fibrils. The results provide insights into the molecular mechanisms by which MrCP20 regulates the biomineralization of the barnacle base plate, while favoring fibril formation, which is advantageous for other functional roles such as adhesion and cohesion

Enzyme Immobilization on Silane-Modified Surface through Short Linkers: Fate of Interfacial Phases and Impact on Catalytic Activity

We investigated the mechanism of enzyme immobilization on silanized surfaces through coupling agents (cross-linkers) in order to understand the role of these molecules on interfacial processes and their effect on catalytic activity. To this end, we used a model multimeric enzyme (G6PDH) and several cross-linking molecules with different chemical properties, including the nature of the end-group (-NCO, -NCS, -CHO), the connecting chain (aliphatic vs aromatic), and geometrical constraints (meta vs para-disubstituted aromatics). There did not seem to be radical differences in the mechanism of enzyme adsorption according to the linker used as judged from QCM-D, except that in the case of DIC (1,4-phenylene diisocyanate) the adsorption occurred more rapidly. In contrast, the nature of the cross-linker exerted a strong influence on the amount of enzyme immobilized as estimated from XPS, and more unexpectedly on the stability of the underlying silane layer. DIC, PDC (1,4-phenylene diisothiocyanate), or GA (glutaraldehyde) allowed successful enzyme immobilization. When the geometry of the linker was changed from 1,4-phenylene diisothiocyanate to 1,3-phenylene diisothiocyanate (MDC), the silane layer was subjected to degradation, upon enzyme adsorption, and the amount of immobilized molecules was significantly lowered. TE (terephtalaldehyde) and direct enzyme deposition without cross-linker were similar to MDC. The organization of immobilized enzymes also depended on the immobilization procedure, as different degrees of aggregation were observed by AFM. A correlation between the size of the aggregates and the catalytic properties of the enzyme was established, suggesting that aggregation may enhance the thermostability of the multimeric enzyme, probably through a compaction of the 3D structure

Gold Nanorod Coating with Silica Shells Having Controlled Thickness and Oriented Porosity: Tailoring the Shells for Biosensing

The coating of gold nanorods with a silica shell (AuNR@SiO2) is an effective way to extend their use in a wide variety of biomedical applications including biosensing, drug delivery and photothermal therapy. A silica shell offers numerous advantages as it provides more stability, frees the surface from toxic cetyltrimethylammonium bromide (CTAB), and preserves the rod shape under photothermal conditions. This shell needs to be very thin for applications such as plasmonic biosensing, while a thicker and porous shell is suited for drug encapsulation and further controlled release. We introduce herein a strategy to perform silica coating based on dissociation of tetraethylorthosilicate (TEOS) hydrolysis and condensation reactions. This dissociation is achieved by a pH modulation of the reaction medium, and, depending on selected pH conditions, AuNR@SiO2 with a thick silica shell having an organized mesoporosity aligned either parallel (AuNR@//m-SiO2) or perpendicular (AuNR@⊥m-SiO2) to the AuNR surface was generated. Moreover, when mercaptopropyltrimethoxysilane (MPTMS) was used as a surface primer prior to TEOS condensation, ultrathin and homogeneous silica shells (AuNR@t-SiO2) of controllable thickness in the range 2–6 nm were produced. While formation, at high TEOS concentration, of core-free silica nanoparticles is evidenced by TEM analysis before the purification procedure, their total elimination during the purification step was achieved by addition of a suitable amount of CTAB to ensure the colloidal stability of the core-free and core–shell nanoparticles. Complete elimination of CTAB from AuNR@SiO2 was demonstrated by XPS, Raman, and ζ-potential measurements. Finally, the efficiency of AuNR@t-SiO2 in label-free plasmonic biosensing of a model target was demonstrated and their refractive index sensitivity factor was improved by 30% compared to CTAB-capped AuNRs

Revealing the Interplay between Adsorbed Molecular Layers and Gold Nanoparticles by Linear and Nonlinear Optical Properties

Gold nanoparticles (AuNPs) chemically grafted on substrates are widely used as sensors due to their plasmonic properties. The efficiency and robustness of such sensors strongly depend on the molecular sublayer structure, which influences the distribution of AuNPs, and therefore the plasmonic properties of the layer. Few spectroscopic tools are able to sense the grafting layer both before and after particle deposition. Here, we use sum-frequency generation (SFG) spectroscopy to deeply investigate both the grafting layer and the immobilized AuNPs. We combine SFG with reflectance UV–visible spectroscopy and scanning electron microscopy (SEM) for 14 nm diameter AuNPs, dispersed on modified silicon surfaces with either amine or mixed amine/thiol terminated layers. SFG spectra show the specific vibrational fingerprint of each supporting layer through the amplitudes of methylene and methyl vibration modes and prove the presence of unreacted ethoxy groups from (3-aminopropyl) triethoxysilane. We establish a linear evolution of the absorbance amplitudes with AuNP surface coverage, a relationship valid up to the aggregation limit of 10¹¹ AuNPs·cm^–2. In the same way, SFG amplitudes follow a quadratic dependence with the UV–vis absorbance amplitudes, showing the close correlation between nonlinear and linear optical properties. In addition, the optical properties of the AuNP layers are stable for several months (plasmon position and damping) despite their storage in ambient air and long exposure to visible laser light

Gold Nanoparticles Assembly on Silicon and Gold Surfaces: Mechanism, Stability, and Efficiency in Diclofenac Biosensing

We investigated the assembly of gold nanoparticles (AuNPs) on gold and silicon sensors with two final objectives: (i) understanding the factors governing the interaction and (ii) building up a nanostructured piezoelectric immunosensor for diclofenac, a small-sized pharmaceutical pollutant. Different surface chemistries were devised to achieve AuNPs assembly on planar substrates. These surface chemistries included amines to immobilize AuNPs via electrostatic interaction or a mixture of amines and thiols to covalently attach the AuNPs. We also generated PEG-amine-terminated surfaces to benefit from the well-known non-biofouling properties of PEG-coated surfaces. The functional substrates and the resulting gold nanoparticle layers were characterized in detail by surface IR, contact angle measurements, and scanning electron microscopy (SEM). The mechanism of adsorption is discussed herein considering the nature of the terminal groups and their charge at the pH of AuNPs adsorption. The coverage and the dispersion of AuNPs were strongly dependent on the anchoring points on the surfaces; the optimal were reached when the attachment layer offered multiple interaction points, in particular, for NH₂/SH- and PEG/NH₂-terminated surfaces, where the percentage of isolated particles was up to 78%. In addition, PEG-coated surfaces led to a stable AuNPs layer resistant to ultrasounds and to further functionalization of the immobilized nanoparticles. These surfaces were used to engineer quartz crystal microbalance (QCM) biosensors for diclofenac detection. The AuNPs nanostructured substrates significantly enhanced the biosensor sensitivity as compared to planar substrates (up to 6 times higher). This enhancement presages a higher sensitivity in the competitive detection of diclofenac on these systems. More importantly, despite the biorecognition and the drastic regeneration conditions, SEM images show that gold nanoparticles layers are stable and reliable, which paves the way for their use as nanostructured platforms for multiple applications