Research in nanomedicine has advanced many biomedical tools and applications of nanomaterials, primarily focused on new diagnostic platforms and strategies for therapy. While drug delivery was the first area to be more thoroughly examined and developed over the years, a more profound understanding of nanomaterial properties has allowed for the design of innovative diagnostics strategies. The unique properties of nanomaterials, coupled with the growing capability for tight control over manipulation and assembly, have furthered the concept of combining diagnosis and delivery into one device as an essential asset to allow precise treatment. Thus, nanomedicine may be perceived as a refinement of molecular medicine, integrating innovation in genomics and proteomics for more personalized medicine, allowing the precise characterization of molecular profiles of patients, from early diagnosis to precision therapeutics, improving outcomes while minimizing risks to the patients.1–2
These nanometric structures have found a wide range of applications within the biomedical field, with a particular focus on diagnostics, such as detection and identification of metabolites, proteins, and nucleic acids (DNA and RNA). Perhaps the most significant impact has been in molecular diagnostics, where the use of nanomaterials has prompted a change of paradigm in biodetection and analytical methodologies. Within this framework, diagnostic techniques based on nanoparticles offer an unprecedented gain in terms of sensitivity. Since most biomarkers are within a similar size range of these nanoscale structures, providing a 1:1 scale ratio response; this gain in sensitivity also allows to use of less sample, thus decreasing the need for robust apparatus for analytical procedures, which result in increased portability, bringing the conceptual lab to the point-of-need (patient bedside, clinician’s lab, etc.).3
Most platforms designed for protein detection customarily rely on molecular recognition using antigens and antibodies, whereas those designed towards nucleic acid-sensing rely on the complementarity of nucleotide sequences via hybridization protocols. Currently, biodetection of relevant nucleotide sequence biomarkers has progressively integrated nanoparticle-based systems, increasing sensitivity and lowering costs. Among these, noble metal nanoparticles (NPs), particularly gold, due to their optical and physic-chemical properties, have been used for the development of highly sensitive biosensing platforms.3
A key feature of AuNPs is their localized surface plasmon resonance (LSPR) that is responsible for their remarkable optical properties. The LSPR may be defined as the collective oscillation of electrons between empty orbitals in response to an incident electromagnetic wave, which generates a polarization in the nanoparticles, inducing the formation of dipolar moments. The LSPR is highly dependent on the size, shape, composition, distance between nanoparticles, and their interaction with the dielectric surrounding. This phenomenon is responsible for attributing different colors to colloidal suspensions of AuNPs, which can vary from different shades of red to blue and purple. Therefore, tight control of the AuNP size is paramount to tune the optical properties towards the desired biodetection wavelength. Also, to take full advantage of these optical properties, different routes for synthesis, means for dispersion in solution, and strategies for surface functionalization may be employed.3,4
The most common and straightforward method for synthesis of AuNPs is through the chemical reduction of gold salt, usually tetrachloroauric acid trihydrate (HAuCl4.3H2O), in the presence of reducing agents, such as sodium borohydride, ascorbic acid, or sodium citrate, which bind to the surface of the particles providing stability, reactivity and specific charge properties. Sodium citrate, for example, has been used since 1951 to produce monodisperse nanoparticles with sizes ranging from 1 to 150 nm.5 Although chemical reduction of metal ions is the most widespread method for synthesizing metal nanoparticles, some of these reducing agents are toxic, expensive, and their residues can be incorporated into the nanostructure, making characterization difficult and limiting in vivo applications. Furthermore, the control of process variables, such as pH and temperature, is critical for obtaining a homogeneous particle size dispersion.6 Due to its simplicity, while offering reasonable control over size dispersion and shape, the citrate reduction method proposed by Turkevich and later optimized by Frens makes it the most widespread method to produce AuNPs for biomedical applications.3,5,7,8 By simply varying the amount of reducing agent (e.g., sodium citrate) added to the mixture, it is possible to scale up production with reasonable size control (Figure 1). For example, when using a lower concentration of sodium citrate, the particle diameter produced is greater, and consequently, the number of agglomerates is greater. In fact, this process yields better results for smaller nanoparticles (10–30 nm in diameter), which are also more stable and, consequently, have less tendency to agglomerate and provide for better and more reproducible results down the line in biodetection. The stability of the colloidal AuNPs solutions depends on their interaction with the surrounding medium, either via electrostatic stabilization (ionic interactions, in which nanoparticles repel each other due to the presence of charged molecules on the surface) or steric stabilization mediated by covalent interactions with suitable moieties that prevent the approach of other AuNPs. Covalent interactions provide some advantages compared to ionic interactions, where modified AuNPs (e.g., bioconjugates) show a considerable increase in stability in diverse media.6,8
One way to minimize the tendency for the AuNPs to agglomerate is via the modification of the nanoparticle surface by the use of surfactants, polyelectrolytes, or ligands. Not only this improves colloidal stability, but it also provides for the possibility to derivatize AuNPs with a plethora of biomolecules suitable for biorecognition, such as antibodies and DNA/RNA oligomers. Still, the functionalization process quality is directly affected by factors such as substrate roughness, time of contact with the surface, concentration of the solvent, and temperature of the medium. In addition, the conjugated layers topography faithfully reproduces the surface of the AuNP and, consequently, its surface defects. One of the main types of defects found on gold surfaces is monoatomic vacancy, i.e., the surface presents irregularities, such as the reduction of gold atoms, directly affecting the formation, organization, and efficiency of functionalization.6,8,9
Figure 1.Characterization of AuNPs synthesized by the citrate reduction method. Several standard and straightforward techniques used for characterization of AuNPs, such as A) Transmission Electron Microscopy (TEM), showing the spherical shape with good size dispersion; B) UV-visible spectroscopy, where the LSPR peak is used for the assessment of size and stability; C) Dynamic Light Scattering (DLS), which measures the hydrodynamic radii of AuNPs, thus highlighting the size dispersion; and D) zeta potential, which provides information of the superficial charge of the AuNPs, thus an assessment of stability. Altogether, these data are easy to acquire and provide enough information for manufacturing characterization.
Generally, for the surface functionalization of AuNP, the so-called alkanethiols are used for chemical stability and easy formation of self-organizing monolayers. Several stabilizing agents and polymeric compounds may also be used, such as sodium citrate, cetrimonium bromide (CTAB), polyethylene glycol (PEG), silica, among others.6,8 The choice of stabilizing agent is critical for diagnostics applications; this interface is often used as a linker for the biorecognition element (e.g., DNA, RNA, aptamer, peptides, antibodies) that should not disturb the intrinsic nanoscale property being used for signal transduction upon recognition of target analyte. This stabilization of AuNPs can also be steered through multifunctional polymers with the capability to bind the gold surface and provide for the simultaneous biding to the recognition element (e.g., methyl, amino, carboxyl, carbonyl, hydroxyl, and even sulfhydryl groups). Perhaps the most used linkers are those relying on (at least) one thiol-reactive group, which binds strongly and spontaneously to gold due to the strong interaction between gold atoms and sulfur. In fact, this is the preferred strategy for directly binding DNA, RNA, and aptamer oligomers to the AuNPs’ surface, as shown in a diversity of molecular diagnostics concepts.10,11
Antibodies are glycoproteins belonging to the class of immunoglobulins commonly associated with the defense of organisms against foreign antigens. Antibodies can bind to various surfaces and have been used as biorecognition molecules for biodetection, particularly associated with AuNPs. Antibodies have a high degree of affinity and specificity, with the ability to detect, recognize and bind to the target antigen.12 The principle of the association between an epitope of an antigen and its antibody involves the complementarity between them and a reversible bond via electrostatic interactions, hydrogen bonds, hydrophobic and van der Waals interactions. Since direct functionalization of antibodies to the AuNPs’ surface is not as straightforward as for DNA/RNA, the use of heterofunctional polymers such as PEG (HS-PEG-NH2) provides a route to the nanoparticle functionalization, where there is a covalent bond through the –SH group, while the NH2 group is available to bind the antibody via free COO- groups. Functionalization is typically achieved via prior activation of one of the many COOH groups of the antibody through the use of coupling agents, such as 1-ethyl-(3-dimethylaminopropyl)-carbodiimide (EDC).10
Several colorimetric approaches based on AuNPs have been proposed for the detection of biomolecules (i.e., nucleic acid and proteins) with high levels of sensitivity and specificity.3,13 Most of these methods rely on the colorimetric change of an AuNPs solution upon aggregation, a process that may be mediated either by changes to the media dielectric or interaction with the designated target. In the former, binding and adsorption of the target modulated the effect of changes to the medium dielectric on the AuNPs; the latter relies on the capability of targets to mediate interparticle interactions, either by promoting cross-linking of AuNPs or keeping them apart via steric hindrance. The resulting aggregation leads to a shift of the LSPR band, which may be perceived by the naked eye or measured through standard spectrophotometry. This is the case of several hybridization-based protocols, where ssDNA probes are functionalized onto the AuNPs and used to identify DNA/RNA targets in a sequence-dependent manner (Figure 2).10
Figure 2.Gold nanoparticles (AuNPs) for molecular diagnostics. AuNPs may be used in a plethora of diagnostics schemes based on antibody-antigen recognition or DNA hybridization, such as colorimetric assays based on inter-particle distance; fluorescence modulation as a function of the distance between the fluorophore and the AuNP surface; Surface Enhanced Raman Spectroscopy (SERS); and the application of AuNPs as tags in lateral flow platforms (LFAs) for point-of-need.
The AuNPs’ plasmonic specific and sharp electrical shifts may also be used to detect a biomarker based on light scattering. These approaches rely on the intense scatter of larger and anisotropic AuNPs, whose spectral changes are associated with the binding or association to molecules.14
AuNPs are also useful for electrochemical detection, namely, to bind enzymes to electrodes and mediate electrochemical reactions as redox catalysts.15 In addition, AuNPs are well-known quenchers of fluorescence dyes that are positioned close to their surfaces, a property that has been widely used for molecular detection schemes. In these platforms, binding to the specific target triggers a conformational change of the recognition moiety, which moves the fluorophore further apart from the AuNP or closer to the surface, thus increasing or decreasing the fluorescence emission.16
Raman spectroscopy has also profited from the use of AuNPs, particularly the anisotropic and irregular-shaped structures. Surface Enhanced Raman Spectroscopy (SERS) considerably enhances the metal surface association of the intrinsically weak Raman signal intensity of biomarkers. For example, non-spherical AuNPs, where the edges of the nanoparticles act as a hotspot for up to 1012 to 1014 enhancement of the SERS signal, offer a new dimension to multiplexing detection of biomarkers.17
Lateral flow devices (LFA) are portable devices capable of transferring biological fluids, such as blood or serum, through their capillary action, without requiring an external power supply. LFAs are fast and straightforward to handle, low cost, with acceptable specificity without the need for refrigeration. Several diagnostics have primarily incorporated AuNPs as labels/tags for probes, adding sensitivity to the system. The most well-known LFA immunoassay is the pregnancy test; however, any molecule may be a target, and there are already rapid tests for SARS-CoV-2, HIV, HCV, among others.3,18,19 The principle mechanism of LFAs is the capillary migration of the biomarkers within the sample towards the region where a first recognition element is conjugated to the AuNPs, typically a capture ssDNA oligomer probe or an antigen/antibody. Then, this complex migrates to the detection region through a hydrophobic nitrocellulose membrane or cellulose acetate and is immobilized by a second recognition/capture element. In the control section of the device, the second capture/recognition element immobilizes the initial complex but not the complex of interest, yielding the result. The striking vibrant red color of the AuNPs makes them ideal tags for these systems, rendering them suitable for evaluation by the naked eye.3
The growing number of conceptual devices and platforms for in vitro diagnostics using nanomaterials and AuNPs has sparked a need to create standardized methodologies and regulatory protocols for the fabrication and characterization of nanomaterials.20 However, the worldwide regulatory agencies’ capability to regulate these products is limited by the sheer nature and scale of the nanomaterials. One such regulation effort has been undertaken at the Nanotechnology Characterization Laboratory (NCL), a formal scientific interaction of three US Federal agencies: National Cancer Institute (NCI), Food and Drug Administration (FDA), and National Institute of Standards and Technology (NIST), created to support the growing need for characterization and standardization. The proposed guidelines and protocols span over physic-chemical characterization (i.e., size, morphology, shape, surface charge, etc.), in vitro characterization (i.e., sterility, drug release, targeting, toxicity, etc.), and in vivo characterization (i.e., efficacy, exposure, etc.).
The International Organization for Standardization (ISO) has already implemented some guidelines and certifications, such as ISO/TS 12901: 2012 for the management of risks applied to nanomaterials engineering; ISO/TR 11360: 2010 for classification of nanomaterials, ISO/TS 12025: 2012 for quantification of NOAAs generated by aerosols, and ISO/TS 16195: 2013 with guidelines for tests on materials incorporating nanoscale parts. All the guidelines and certifications must be aligned with the more general directive governing the production and commercialization of in vitro diagnostics (IVD) (Directive 98/79/ EC of the European Parliament and the Council of 27 October 1998 on in vitro diagnostic medical devices). Despite these efforts for standardization, the estimated time to market molecular diagnostics systems incorporating nanoparticles and nanomaterials is still considerably long.
Despite significant advances in the use of AuNPs for molecular diagnostics, much remains to be investigated, including improving the robustness and reproducibility of the proposed systems. Perhaps the biggest challenge is the production scale-up of the most innovative concepts and their translation to use in clinics. Several concerns about the control and characterization of each manufactured batch of nanoparticles and subsequent functionalization need careful evaluation under the existing regulatory guidelines and require suitable methods for the required quality control. For all these innovative systems based on AuNPs to reach the in vitro diagnostics market, they must abide by the emergent standards and regulations associated with the existing ISO certificates and guides to impact and change the way we perform diagnostics.
The authors wish to thank FCT/MCTES for funding to UCIBIO (UIDB/04378/2020). We also thank Catarina Roma-Rodrigues for graphical support.