2.2.1. Nanodiagnostics

In general, nanodiagnosis is considered as the design and development of analytical and imaging systems that allow for the detection of disease or abnormal cell function in early stages, both in vivo and in vitro [1].

Nanomaterials can be used for in vivo diagnosis, being used as contrast agents to visualize tissue structures inside the human body and to delimit healthy vs. pathological tissues. To this end, NPs are designed with different contrast properties for different modalities, such as computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), single photon emission computed tomography (SPECT) or fluorescence imaging. NPs will be designed to target specific tissues and generate the contrast. Then, to illustrate the applications, some of these examples are described below (Figure 2) [7].

**Figure 2.** Current nanoparticle (NP) involvement in the multiple applications of nanomedicine.

In the case of CT, X-ray imaging takes advantage of tissue-specific attenuation to generate contrast on X-rays screenings, i.e., bone generates more contrast than soft tissue due to a higher relative electron density in the bone. To increase the contrast of these soft tissues, elements such as iodine or barium, which have a high electron density, were used, but to increase the low sensitivity, NPs were developed as contrast agents [7]. Among these NPs, AuNPs, which have a high electron density, stand out [10]. AuNPs have directional ligands like folic acid to bind to different tissue structures through their corresponding receptor composed of other types of materials that have a high atomic number are also suitable for CT. NP-based CT imaging technologies may change the way clinical diagnosis based on CT is performed [11]. In the case of iodine or barium, the doses required are very high, the contrast agents are usually non-specific and do not bind to cellular biomarkers or accumulate in tissues of interest, so the aim is to design NPs with high atomic number materials conjugated with targeting molecules that allow for different cell types to be specifically marked in vivo [7].

In the MRI example, contrast agents based on biocompatible NPs have advantages over the conventional contrast, such as the ability to adapt their size, shape, composition, circulation time, target cells, and optical and physical properties to optimize the images [7].

There are "smart" NPs that are activated by certain stimuli, such as pH, temperature, redox reactions, ions, proteases or light. These NPs respond to a change in the tumour microenvironment (TME) and allow for the selection of the diagnostic and therapeutic mechanism, which is highly relevant in oncology, because the TME regulates the progression of the tumour and its metastasis. In the case of MRI, probes of these "smart" NPs have been designed that are sensitive to pH, since it is a very important physiological parameter and its deregulation might be a biomarker of cancer. Additionally, hypoxia in the TME results in the production of lactic acid and therefore in acidic conditions, which also constitute a Damage-Associated Molecular Pattern (DAMP). Other probes of these types of NPs used in MRI are the temperature sensitive ones, since in tumours, differences in temperature between tissues are very common [7].

NPs could also be used for in vitro diagnosis, i.e., the detection of molecules, cells and tissues outside the human body. In this case, the function of NPs is to identify unique biological molecules in biological fluids that are associated with the health of patients and are useful for diagnosis. In this case, NPs are coated with ligands and biomolecules to allow for bio-recognition of biological molecules in such fluids [7]. Following the example of AuNPs, in this case they are modified with ligands that bind to a specific complementary protein, causing the agglutination of these NPs, which can be observed colorimetrically [12]. This knowledge has also been used in the detection of colorimetric DNA. The AuNPs diagnostic technique is used in the clinic to analyse patient samples [13]. Hence, AuNPs also serve as biosensors, conjugated with antibodies against signalling proteins, such as anti-CA15-3-HRP, to test CA15-3, which is an important tumour biomarker for breast cancer follow-up. The use of magnetic NPs as proximity sensors in MRI is known as diagnostic magnetic resonance imaging (DMR) [2,14,15].

Another example is the use of QD as fluorescence markers in proteins or nucleic acid assays, such as the detection of antigen surface epitopes [16]. Organic and inorganic polymer NPs have been used in intracellular detection applications. An example is silica NPs carrying fluorophores for intracellular detection of oxygen, pH or metal ion levels [17].
