Analytical Platforms for Medical Diagnosis: A Study on the Performance and Recent Trends on Aptamer and Antibody Based Biosensors

Platforms for Medical Diagnosis: A Study on the Performance and Recent Trends on Aptamer and Antibody Based Biosensors. Antibodies and aptamers play a crucial role to improve the analytical performance of biosensors for medical applications. Besides their natural compatibility with many antigens and pathogens, their biochemical structure selectively binds the analyte providing high sensitivity and selectivity. Accordingly, this minireview presents the recent approaches in the field of immunosensors and aptasensors for clinical diagnoses with a focus on the key features of sensors over the conventional diagnostic assays and the trends in this field of knowledge (point-of-care devices for in situ applications, label-free bioreceptors, real-time monitoring of analytes and outstanding transduction techniques). Abbreviations: Enrichment;


Introduction
Advances in the field of molecular biology and chemistry have driven the studies in biosensing to an important and necessary level.
The increasing attention of the population to healthcare summed to the alterations in their alimentary and social habits significantly changed the needs for personal health. Miotto, et al. [1] mentioned that the current context of healthcare demands to "ensure that the right treatment is delivered to the right patient at the right time".
In this scenario, the study of biosensors has provided sufficient tools, especially in the last decade, to advise the science of sensitive, rapid and accurate medical diagnostics. Clark and Lyons [2] were Once biological molecules are irreplaceable agents in living beings to make humans and animals to perfectly function, not surprisingly, scientists and research companies devote maximum efforts to mimic the biochemical reactions that naturally occur in the nature. This is the basis of a biosensor. A biological element of recognition is attached to the surface of an electrode material to

Biosensors and Units of Biorecognition
Sensors are part of our daily lives, inserted in the most diverse equipment's with the most different functionalities. In general, a sensor is a device that transforms a certain physical or chemical property into an analytically measurable signal. In this way we can classify sensors where the variation of a biochemical property generates any signal, these devices we call biosensors, which can be defined according to IUPAC as being "device that uses specific biochemical reactions mediated by isolated enzymes, immune systems, tissues, organelles or whole cells to detect chemical compounds usually by electrical, thermal or optical signals" [4] A biosensor consists of two parts, one formed by the biological recognition element (receiver) and the other by the transducer, which can be electrochemical, optical, thermal, piezoelectric, capacitive and field effect. We can classify them, by the different methods of transduction, as well as according to the element's receptor. Here, we will classify them only this. Bioreceptors can be selective or not, but recognition element plays a crucial role in the overall biosensor performance and selectivity toward a particular analyte [5]. Temperature, pH, contaminants, ionic strength, type of solution (buffer solution, body fluids, water) are factors that determine the performance of the biosensors [6,7].

Aptamers / Aptasensors
Aptamers are short and single-stranded nucleic acids (DNA or RNA) with capacity to bind to target molecules with high affinity and specificity [8]. First introduced in 1990, the process of selecting an aptamer is called Systematic Evolution of Ligands by Exponential enrichment (SELEX), from a large oligonucleotide library [9,10]. Aptamers can be selected for a variety of targets, including small molecules, proteins, nucleic acids, microorganisms, cells, tissues, metal ions and chemical compounds [11][12][13]. With the advantages of small size, high binding affinity, good stability and easy synthesis, aptamers show potential for various applications, such as targeted therapy, detection and clinical diagnoses [14][15][16][17].
After selection and characterization, aptamers can be customized for developing sensors [18]. A large variety of aptamer-based biosensors (aptasensors) with various detection strategies have been developed and reported in the literature [19]. In comparison to antibodies, aptamers are smaller units containing oligonucleotides with sizes over 30 oligos [20].
They are similar to monoclonal antibodies in terms of binding affinities, being called synthetic antibodies [21] in addition to other advantages, such as chemical stability and regeneration of its threedimensional structure even after several cycles of denaturation/ renaturation [22]. Its small size allows a greater density of immobilized molecules. They are chemically synthesized, which allows the flexibility of the conformation of their two-dimensional structure, so it can be built for the detection of any antigen, from small molecules, heavy metals, protein, enzymes, microorganisms and cells, with the possibility of adjusting the sensitivity and selectivity [23][24][25][26][27][28].

Antibodies / Immunosensors
Antibodies (Abs) are proteins that can be employed as valuable tools in laboratory and clinics [29]. Antibodies include those secreted by a single clone of B lymphocytes, termed monoclonal antibodies (mAbs), and those produced by a mixture of various B lymphocyte clones, the polyclonal antibodies (pAbs) [30][31][32]. In 1975, Kohler and Milstein developed a system for the production of monoclonal antibodies. Abs demonstrate high affinity and specificity to target molecules and have been frequently selected for a wide variety of applications including immunodiagnoses, biomarker detection, immunological research and vaccine quality control [33][34][35]. Abs can be used to develop a variety of sensors (immunosensors) upon the formation of an antibody-antigen complex [36]. Immunosensors are based on antigen-antibody affinity, where an immunochemical reaction forms a very stable complex. Every protein has an isoelectric point (point where the global electrical charge is equal to zero) that varies according to the composition of the amino acids, thus determining the magnitude and polarity of that point at a specific pH [37].
One can assume that any protein (Ag), with charge Ch1, and its antibody pair with charge Ch2, the reaction of that system (AgAb) results in a global charge Ch3 which can be described by the

Key Features on the Performance of Biosensors
The most important characteristics of a biosensor are its selectivity, reproducibility, stability, sensitivity and linearity.
The combination of these parameters has been the focus of many researchers specially in the last decade to develop high performance devices for diagnosing molecules of medical interests.
These features can be defined as follows:

Traditional Analytical Techniques for Diseases Diagnosis
Diagnosis, detection and prognosis techniques have been studied for several years and many methods for fault detection and diagnosis have been developed [45]. Molecular diagnostics assays use in vitro biological techniques for detection. Polymerase chain reaction (PCR) and quantitative PCR are performed to detect and amplify a genetic material (DNA or RNA) from a specific organism, for instance, a virus [46,47]. The advantages of PCR include the high sensitivity, quick performance and the ability to detect lesscommon organisms. On the other hand, its disadvantages include the supply costs, machinery fees and training expenses [48,49]. At present, PCR assay is regarded worldwide to as the most accurate and reliable test to detect active COVID-19 infections [50,51].
Immunoassays, such as enzyme-linked immunoassays (ELISA) and point-of-care (POC) techniques can be used for detection of antigens or specific antibodies [52]. Currently, immunoassays play a prominent role in the analysis of many clinical laboratory analytes such as proteins [53]. A broad variety of tests detecting specific SARS-CoV-2 antigens and IgA, IgM and/or IgG antibodies were developed [54,55]. Although the classic immunoassays can provide very sensitive and accurate diagnoses, many of them possess some important limitations: high cost, they are time consuming, demand sophisticated equipment and high skilled staff [56].

Recent Trends in Biosensors for Detection of Analytes of Medical Interest
It is worthy notable that the field of biosensing through

Point-of-Care Biosensors
Point-of-care diagnoses collect several unquestionable advantages over traditional laboratory setups. Not surprisingly, the golden characteristic refers to the possibility of running the test wherever the patient is, on-demand and onsite [57]. It makes the sensors amenable for bedside monitoring, analysis in pharmacies or even by the user himself. Consequently, this kind of device tends to gain increasing visibility in the market. Eguilaz et al. [58] highlight that these devices are even more relevant in resource-

Label-Free Detection
The Zhang and Liu [64] mentioned that the success of using DNA in label-free devices based on optical biosensors has inspired the same approach in the aptamer field. However, the authors explain that aptamers can fold DNA and hide its bases, providing slow kinetics of target binding, especially when the target is a small molecule. Therefore and since aptamers possess lower affinity to small molecules (KD around low micromolar units) than DNA (KD approximately in picomolar or low nanomolar), the detection of aptasensors tends to be more challenging, justifying the efforts on label-free sensing to enhance its analytical response. On the other hand, numerous works can be easily found in the field of labelfree immunosensors for detection of analytes for highly sensitive diagnoses [65][66][67][68].

Real-Time Measurements
The key point of real-time biosensing is the necessity of the sensor to rapidly recognize the target molecule. If so, the output signal will be registered by the transducer source in short time intervals and a variation in its magnitude could be notable as illustrated in Figure 1. This need makes some important well recognized techniques such as ELISA and Luminex assay to fail as real-time methods for in vivo applications, since they require laborious and pre-defined longtime steps [69]. Cohen et al. [69] highlight that ELISA, for instance, depends on diffusion processes concerning the interaction between antibodies and antigens in a non-mixed solution, which is associated to a low binding equilibrium constant and makes the response time longer. Typically, this technique requires approximately 3 hours to be performed [70,71].
In this regard, Shengnan, et al. [72] reported the construction of an aptasensor for the real-time detection of vascular endothelial growth factor, one of the most important cytokines present in cancer patients (with average concentration of 434 pg/mL). The authors achieved a LOD of 0.1 pg/mL within a linear detection window from 2 pg/mL to 500 pg/mL. The mechanism of recognition was based on a Chronoamperometry test at the positive redox peak potential of ferrocene-labeled aptamer for 5,000 seconds. fabrication of biosensors are CV [76,77], EIS [78][79][80], potentiometry [81,82] and amperometry [83,84]. When real-time performance is required, time-based assays (such as chronoamperometry, chronocoulometry and chronopotentiometry) well fits medical applications.

Efficiency of Immunosensors and Aptasensors
Cesewski and Johnson [85]  In this regard, Table 1 contains a list of recent researches in the literature of biosensors for medical applications using antibodies and aptamers as bioreceptors. It is worthy notable that these biomolecules facilitate the biosensing of analytes at concentrations as low as some femtograms per milliliter [86][87][88][89][90]. Note: *NPs = Nanoparticles Regardless the obvious different protocols used to attach antibodies and aptamers to the different transducer substrates, it is worthy notable that the sensitivity of these devices are really high. Besides, in this recent literature is not rare to observe a trend in using label-free molecules to optimize the fabrication step and to allow accessible in-situ measurements [91][92][93][94][95][96][97][98]. Nonetheless, it is also evident that many authors have employed electrochemical techniques to ensure accuracy and high performance of biosensors, corroboration the previous discussion brought to this minireview in the section "Recent trends in biosensors for detection of analytes of medical interest".

Conclusion
With the increasing humans needs for accurate, fast and It is believed that this specific application demands advanced technologies, especially to shorten the detection time, since early diagnoses are essential for the administration of first aids and precise medications that can enhance the chances of cure and survival of patients (especially those who have less access to health centers). The main challenges in the area still seem to be related to the commercial viability of these devices. Likewise, quite possibly, the prospect of advances in technology is likely to be based on the study of alternative materials and methods to make immunosensors and aptensensors increasingly simple and inexpensive.