An emergent topic in the development of new bioanalytical procedures, structures, and systems is nanotechnology, particularly for the generation of useful nanostructures for diagnostic applications; this is the so-called field of “nanobiotechnology”. Novel and improved electronic devices and biosensor platforms have emerged as a consequence of the inherent small size, enlarged surface area, and unusual optical, magnetic, catalytic, and mechanical properties of nanomaterials, unlike those of bulk materials [24]. Depending on their specific nature, for biosensing, nanomaterials may act as labels (including signal amplification), as biomolecule immobilization supports, or even as probes for specific biotarget anchoring. Certain nanomaterials can also be used for pre-concentration of biological targets. Among these applications, labeling has been the most commonly employed. Label-based detection methods are usually more time-consuming and labor intensive than label-free methods due to the labeling steps. Labels have also limited shelf lives and are subjected to leakage from sensing surfaces. However, label-based methods usually provide superior performance, especially in terms of sensitivity, than label-free ones. Moreover, standardized protocols with labeling procedures are already available. Fluorescence labeling has been, by far, the most common approach in this regard, although suffering from pH sensitivity and photobleaching over time [53]. Such handicaps and the advent of nanoengineering have propelled the search and development of new and improved labels.

Nanoparticles (NPs) have been the most widely employed type of nanomaterials for biosensing, especially metallic NPs. Metallic NPs are inorganic NPs that exhibit improved physicochemical characteristics compared to fluorescent labels, including higher sensitivity. In general, they are suitable for construction of high-density bioanalytical devices, taking advantage of their high signal-to-noise ratio (S/N). They are easily synthesizable and functionalized (by simple mixing at room temperature) and have a controlled, self-assembled surface structure [24]. Gold nanoparticles (GNPs), in particular, are already used frequently in molecular diagnosis; some of their advantages include low toxicity and versatility for many specific biorecognition applications and schemes. One common way to enhance the GNP signal even further, and thus the sensitivity of detection, is the inclusion of a final step of silver staining (“silver enhancement”), yielding detection schemes able to preclude the use of a prior PCR amplification step. The high sensitivity exhibited by many NP-based detection layouts, especially in the form of microarrays, has enabled to avoid a prior step of nucleic-acid amplification [69].

Quantum dots (QDs) constitute another class of metallic NPs, able for fluorescence tagging. They are much brighter and more photostable than conventional organic fluorophores. Plus, their color can be directly correlated with size, while exhibiting very broad excitation wavelength windows, very narrow emission wavelength windows, and large Stokes shifts, allowing excitation at wavelengths far removed from their emission peaks [70]. Since QDs of different emission peaks (according to their different sizes) can be excited using a single wavelength excitation source, detection of multiple targets in complex biological systems is a hallmark of these NPs [71]. Another alternative to the overlapping of closely spaced fluorescence emission peaks and consequent limitation of the maximum number of fluorescent dyes that can be discriminated when simultaneously testing multiple pathogens in a single PCR tube is the use of masscode tagging, with a panel of distinct labels with different molecular weights. After an initial step of multiplexed (RT-)PCR using primers labeled with the masscode tags, unincorporated primers are removed, and the photo-cleavable tags of amplifying primers are then released by UV irradiation. Subsequent mass spectrometry analysis assigns each identified tag to its specific pathogen [1]. In principle, the multiplexing ability will only be limited by the highest primer concentration contained by a PCR mix. The method was applied to the identification of respiratory pathogens [72] and hemorrhagic viruses [73]. It offers a rapid, specific, sensitive, and cost-competitive alternative to conventional PCR and RT-PCR for disease diagnosis through POC devices. Nevertheless, some difficulties persist in miniaturizing mass spectrometer devices.

Among metallic NPs are also magnetic NPs (MNPs). Equivalent designations frequently found in the literature include “magnetic nanobeads”, “nanomagnets”, “nanomagnetic beads”, “nanomagnetic spheres”, and “nanospheres”. They have been vastly employed in many biosensor layouts for diagnosis. Despite not matching the nanosize of molecular recognition probes and targets, their microscaled counterparts, magnetic microparticles, are frequently preferred as magnetic labels for biosensing in view of the easiness for detecting the lesser abundant microbeads by routine optical microscopy or by magnetic detection and by the easiness of the purification process, thus allowing more efficient removal of nonspecifically bound labels, with enhancement of the assay performance [74]. However, the higher S/V of nanobeads provides much more binding sites for bioprobe and biotarget anchoring and hence a higher S/N [5]. Very often, magnetic particles are used for target pre-concentration from large initial sample volumes and purification, in parallel with the detection step itself being carried out through another particle that works as the label (e.g., fluorophore or GNP). In this case, there is an initial capture of the target by the probe-functionalized magnetic particle, followed by releasing of the target (“debinding”) for final detection. Through magnetically controlled removal of nonspecifically bound beads (magnetic washing), improved sensitivity can be achieved upon elimination of the time-consuming washing step of nonspecifically bound molecules [75]. This process can, for example, improve significantly the detection specificity of genomic RNA, since RNA enrichment due to magnetic confinement also precludes the effect of common interfering substances and common RNA inhibitors [76]. In addition, the use of magnetic beads permits testing optically opaque samples [29], which is the case of many crude biological samples. Magnetic particles can be manipulated off-chip by a permanent magnet, making easier the design of disposable and inexpensive tests. Moreover, magnetic interactions are not affected by surface charges, pH, ionic strength, or temperature, being thus compatible with most biochemical processes [10].

Unlike inorganic NPs, organic NPs have enhanced structural flexibility and biocompatibility, while being biodegradable. Liposomes constitute an attractive type of organic NPs for efficient DNA-probe labeling and for signal amplification. This is commonly achieved by filling liposome particles with dye and fluorophore molecules, which amplify the response signal and are able to yield quantitative results. Another way of using liposomes in biosensing is in conjunction with resistive techniques. As such, negatively charged liposomes, upon binding to immobilized DNA chains (which are also negative), form giant negatively charged surfaces that repel the target DNA chain, leading to shifts in the electrochemical response [24].

In the last years, chemistry research has rendered a range of new structures based on carbon allotropes. The most promising for biosensing purposes seem to be carbon nanotubes (CNTs), both in the form of single- (SWCNT) or multi-walled CNTs (MWCNTs), depending on the number of cylindrical layers, with unique electronic properties and enlarged surface area for DNA immobilization. They also possess high electrical conductivity (similar to copper and much higher than in polymers), physical robustness, and chemical inertness. Each nanotube may act as an individual nanoelectrode, with sufficient free space between neighboring nanotubes preventing the overlap of their diffusion layers, therefore yielding high S/N values and hence improved detection limits [77]. By providing high sensitivity, they are amenable to PCR-free detection. Their production is sometimes unacceptably irreproducible for ultrasensitive detection, but this has been circumvented by using cheap CNT arrays for multiple biological targets as a way of averaging out between different batches [78].

The recent advances in nucleic-acid synthesis and modification processes and the discovery of nucleic acids with catalytic and regulatory activities have prompted the development of nanoengineered nucleic-acid analogues with new and improved abilities for biorecognition and diagnostic purposes. Among them are aptamers; they are synthetic nucleic acids able to interact with molecular or cellular targets with high specificity and sensitivity for their ability to fold into many tertiary conformations. Aptamers can be generated by “Systematic Evolution of Ligands by Exponential Enrichment” (SELEX), a combinatorial procedure that starts with a pool of candidate nucleic-acid molecules to generate a nucleic-acid library [79]. Compared to antibodies, nucleic-acids can be synthesized in a more reproducible way, have longer shelf lives, and can be reversibly denatured without loss of activity. A remarkable characteristic of these probes for biosensing is that they do not require prior knowledge about the molecular differences between the specific target and nonspecific ones. As shown in cancer diagnosis, the DNA sequences from the DNA library that bind the cell-surface markers of a cancer cell can be determined by comparison with those that bind a healthy (control) cell. In addition, detection occurs before the corresponding antibody against that cancer has been produced [80]. This process is obviously attractive for application to the diagnosis of infectious diseases as well. The high selectivity and sensitivity achieved with aptamers permits eliminating sample pretreatment and is thus promising for POC applications [53]. The inability to distinguish the fluorescent signal from labeled and unlabeled probes is a common problem in microfluidic devices, since labeled probes that did not bind targets cannot be washed out from the microchannels. Different fluorescent labels can be used to tag the probe and the target, with the detection proceeding via fluorescence resonance energy transfer (FRET) upon the occurrence of the bioaffinity reaction. However, this procedure is unpractical in bioanalysis owing to the cumbersome dual labeling procedure [81]. In the case of DNA detection, this can be circumvented with the use of molecular beacons (MBs), which can be considered a particular type of aptamers. MBs are single-stranded oligonucleotides with a hairpin (stem-and-loop) structure, labeled with a fluorophore in one extremity of the chain and a fluorescence quencher in the other extremity. The close proximity between the extremities prevents fluorescence emission, but when a hybridization event occurs with a complimentary chain, the structure becomes linearized and hence fluorescence arises. In this way, target labeling is unnecessary. Another type of synthetic nucleic-acid analogues is constituted by peptide nucleic acids (PNAs), which are in which the sugar-phosphate backbone is replaced by a peptidic structure. When used as probes in nucleic-acid recognition systems, they allow very selective and sensitive hybridization in low ionic-strength media, while having high thermal stability [82]. For being electrically uncharged, PNAs are suitable to promote the occurrence of biochemical events triggered by the formation of the negatively charged PNA/single-stranded DNA hybrid, i.e., a kind of “on/off” processes.

Biosensing schemes reported in the literature employing at least one of the nanotechnology-based structures described above are depicted in Table 2, together with the transduction mechanism employed and performance quantification.