Nano-Aptasensor: Strategies and Categorizing

The rapid detection of agents, toxins, and disease markers is beyond the scope of traditional sampling and detection methods; however, it is possible to solve this problem using biosensors.1 Aptamers are short fragments of oligonucleotides, peptides, and peptide nucleic acids (PNAs), which, specifically and with high affinity, bind to various biological, organic, and inorganic targets.2 Aptamers act like pharmaceutical analogues for monoclonal antibodies and have high binding affinity with target molecules. They also have high potential for sensitive and accurate detection.3 Over the past two decades, nano-biosensors have been widely used in various research and applied fields, including tracking bio-terrorist attacks, clinical diagnoses, drug discovery, human health development, and bio-research, increasingly consolidating their position. The important parts of biosensors include the bio-receptor, signal transducer, signal amplifier, and processor (Figure 1). Biosensors are highly diverse. An aptamer used in a bio-receptor is called an aptasensor, and it is called a nanoaptasensor if nano-methods and nano-materials are used in the construction of the aptasensor.4-9

human body liquids. Dr. Leland C. Clark first developed the concept of biosensing, and a biosensor is sometimes called a Clark electrode. 18 In the beginning of the 1990s, scientists began to think about using such sensors as tools. In addition to the issue of tracking analytes, ways to measure and quantify analytes were also sought. To this day, every sensor used to track, detect, and measure bioanalytes is considered a biosensor. However, scientists such as Professor Kissinger essentially disagree with this idea and consider biosensors as the only measurement tool comprised of biocomponents. 18 Some go even further and do not consider nanoparticles or quantum dots transmitted to cells or tissues to transmit the signals of intracellular interactions and extracellularly track the signal resulting from its labeling as biosensors. They also believe that the new generation biosensors are those made by researchers using biomaterial and components such as enzymes, proteins, cells, intracellular organelles, tissues, etc. to detect analytes that may not necessarily be of biological components. 18 Widely diverse methods have been used to create biosensors, yet despite the apparent variety and differences, they are all comprised of sensing element parts (bioreceptor), a transducer (detector), and a processor.
Transducer: The transducer is the second part of the biosensor that converts the chemical or physicochemical signals generated through the binding of the sensing element to an analyte into a measurable output signal. This part of the biosensor can send the type and amount of reaction with different signals to the processor using different physicochemical methods and changes (electrochemical, optical, mass, or thermal before and after the reaction). 20,21 Processor: Using one of the signal reading methods, this part receives (directly or indirectly), amplifies, and sends the signal from the changes in the bioreceptor. The message may be sent by changing the visible (changing the color of the sensors determining the shelf life of food products) or invisible frequency (e.g., electromagnetic waves) or by changing the 0 and 1 (zero/one) algorithms in the computer field.
Bioreceptor: A bioreceptor is a biological-element (antibodies, nucleic acids, enzymes, cells, and other biomaterials) which can selectively react with only a specific substance. The analyte molecules are transferred to the receptor in the following three ways: micro/nanofluidic or microcurrent concentration-dependent systems, filtration, and selection-dependent methods. 22 The bioreceptor part is of particular importance and is considered the major component of the biosensor, because it basically performs the biorecognition act. Bioreceptors used in biosensors include enzymes, antibodies, cell receptors, nucleic acids of DNA or RNA, microorganisms or whole cells, synthetic tissues, and receptors. 22,23 Aptamer: The term aptamer is derived from Latin and Greek words (aptos) meaning "to fit". Aptamers are short fragments of oligonucleotides, peptides, and PNAs, which bind various biological, organic, and mineral targets specifically and with high affinity. Because of their folding ability, aptamers are able to form second and third structures that are expected to bind target structures at the picomole or nanomole unit. [24][25][26] The growing number of aptamer-related studies ( Figure  2) indicates the importance of these macromolecules. Compared with monoclonal antibodies, these biomolecules not only have the advantages of antibodies, but also overcome many of their weaknesses. 27 These macromolecules have higher temperatures, greater environmental stability, and the ability to be denatured and renaturated. 28 The production and detection of aptamers are far simpler, less costly, and faster than the production of monoclonal antibodies. 29 They have an extraordinary repeatability, 5,6 less immunogenicity, 7,8 a variety of targets, and can be synthesized against a wide range of substances, molecules, and even toxic and non-immunogenic ions. 5 Some of the characteristics of these magic molecules along with antibodies are presented in Table 1.
The following benefits are mentioned for aptamers in comparison with antibodies 30-33 : Apta-biosensors: As noted above, a biosensor is a sensor in which biological components are used to detect a particular analyte. 34 Aptamers are used for detection proposes according to different strategies, and aptamers can be classified accordingly. The apta-biosensors can be classified into labelfree, label-dependent, or nano-apta-biosensor groups. Labelfree aptasensors do not need a special label to detect an analyte, and the signal is directly amplified and recorded. 35 Sensors in this category can also be classified according to the type of message transmission system, such as: Surface plasmon resonance aptasensors: Surface plasmon resonance (SPR) is a reference optical method. The resonance angle of the gold disk surface has been changed and is reported with the resonance unit (Ru) by stabilizing a ligand on the surface of a chip of plasmon-containing metals (for example, gold) and the introduction of the flow of liquid containing the target ligand at a constant and specified speed due to the ligand-target interaction. These units are increased proportionally to the concentration of the reactive component in the liquid phase. 36 Quartz crystal microbalance aptasensors: Quartz crystal microbalance (QCM) is another technology used in combination with aptamers to detect various factors. In this method, a quartz piece is placed between two electrodes, and biomolecules (for example, an aptamer) are stabilized on electrodes. The quartz crystal frequency changes of this system are observed at the same time using a frequency meter; if the target molecule binds the bioreceptor, the mass changes and the frequency decreases. 36 Surface acoustic wave aptabiosensors: Sensors that work based on short-acoustic waves have recently been used in combination with aptamers. In some studies, a sensitive surface is engraved using lithography. When the aptamer is stabilized by juxtaposing the analyte with a biochip, the acoustic-wave changes are recorded by a computer, and tracking and measurement are reported as compared to the base state. 37,38 Cantilever aptasensors: There is a vibrational thin striped metal piece in this type of aptasensor, the upper plate of which is uniformly coated with aptamers, which bind to an electrochip from its narrow tip. These aptasensors are mainly used in the gas phase, but they are also used in solutions in practice and to measure the frequency variation of the quartz oscillator plate that occurs due to the surface mass absorption of the analyte in the oscillator. These sensors are subjected to the analyte, and the analyte-aptamer binding changes the pressure due to a mass increase in the vibration of the cantilever plate, which is transmitted to the electrochip and forms the basis of the tracking and measurement processes. 39 Label-dependent aptasensors: To visualize and track the outcome of the aptamer-target reaction in this type of aptasensor, the analyte needs to be labeled. If the target component is labeled, the signal should be reported indirectly. That is, the analyte concentration is obtained by calculating the light or color changes and comparing the results with the base state. Some of these apta-biosensors include: Optical aptasensors: Aptamers have been used as bioreceptors in biosensors based on optical signals which are often detectable using methods of measuring fluorescence signals, chemiluminescence, or optical methods with higher wavelengths (known as colorimetry).

Fluorescence-based aptasensors:
Fluorescence-based aptasensors use fluorescence labels for ultimate tracking. In this method, for example, fluorescence caused by fluorescein isothiocyanate is altered by the establishment of an aptamertarget interaction which can be tracked and recorded. In a simple form, however, the aptamer should be labeled using both fluorescence and quencher. That is, the presence of a fluorophore-quencher pair is obligatory. The aptamer must have a definite shape; for example, it must be like a hairpin or consist of complementary components, and opened when binds the analyte, loop or a double-strand, and the signal is

Characteristics Antibody Aptamer
Binding affinity μM-pM intervals μM-pM intervals Production Process • The presence of contamination in the production process may affect the quality of the final product.
• It is difficult to produce antibodies against a toxic substance that is not tolerated by an animal.
• To produce antibodies against non-immunogenic targets, these targets should be conjugated or changed in some way. • Small size allows the aptamer to easily enter into biological structures.
• It is sensitive to kidney filtration and requires more modifications, such as PEG.
on or off ( Figure 3). One study used a fluorescence-based aptasensor sample, and the Förster resonance energy transfer of cocaine was successfully detected at a detection limit ranging from 100 nmol to 900 µmol. 40,41 Chemiluminescence aptasensor: Luminescence is a general term meaning that light is caused to be emitted from certain compounds based on the process of changing energy levels in a molecule. When the molecular is excited, it becomes energetic and releases its energy as light after returning to the base state. If the luminescence is caused by the excitation energy of a chemical or electrochemical reaction, it is called chemiluminescence. Nano-aptasensors, which produce or enhance the signal using a chemiluminescence process, have good competitive advantages because of their molecular nature. 15,42,43 Colorimetric aptasensors: Materials or labels are either found in nature or made by researchers; in the presence of an analyte or a change in concentration, they cause color changes in the environment. These changes can be detected using colorimetric or photometric instruments or even the naked eye.
Nano-aptabiosensors: A nano-biosensor is referred to as a sensing system which has, at least, a nanostructure in its construction. The use of nanomaterials greatly increases the sensitivity of this system. The application of nanotechnology in molecular, atomic, and subatomic detection is a relatively new field of science that is also known as nano-detection. Nanosensors which are based on biological sciences or applied in biological sciences are called nano-biosensors. However, this definition is not universally accepted, and some apply the use of bio-elements in the preparation of nano-sensors only as naming criteria. If an aptamer is used as a bioreceptor in the biosensor, it is called a nano-aptasensor. 9 Aptamers are compatible with different types of electrochemical, acoustic, fluorescence, and piezoelectric signals, and, while possessing excellent biodetection, they also have the ability to bind and combine with organic, inorganic, and mineral components as well as quantum dots and other components. They also play an important role in detecting biological agents in combination with these nanomaterials. The use of nano-aptasensors has led to the detection of very low levels of chemical (toxins and ions) and bio-contaminants (viruses, fungi, parasites, bacteria, molecules, and macromolecules) in the agricultural, food, and pharmaceutical industries as well as in military, police, and judicial areas. 36,44,45 The detectability of some of these sensors reaches 1 nmol in 15 minutes while reducing the sample size by 10 μL. 46 Nanomaterials are at the center of attention in most studies because of their characteristics, including their unique size-dependent optical properties, shape and chemical composition, high surface energy level, high surface-volume ratio, and adjustable surface properties. The contribution of nanomaterials and aptamers has improved signal detection, increased sensitivity, increased selectivity, extended linearity, and, in general, facilitates the better detection of various chemical and biological agents. In the last 10 years, nanoaptasensors have been used and investigated based on different sensing strategies such as colorimetric, fluorescence, electrochemical, current changes, surface properties changes, electromagnetic spectrum changes, etc, in various industrial, hygiene-health, defense-security, and research fields. 47 The use of metal nanoparticles in nano-aptasensors: Metal nanoparticles may account for the highest number of nanomaterials used in sensors (Table 2). Gold, iron oxides, and silver nanoparticles are the most common metal nanoparticles used in this field. 47 Metal nanoparticles exhibit vast change in their properties, i.e. size and distance, and have a high surface-volume ratio which makes them suitable options to be used as colorimetric nano-biosensors. 48 The appropriate properties of sensors, detectors, and reporters are usually heavily size-dependent. In addition, the combination of metal nanoparticles (gold-silver) or special shapes of nanomaterials (gold nanorods) consist of two SPR bands proportional to their length-to-width ratios, which in turn paves the way for their more widespread use. 49 These properties have led to the use of metal nanomaterials in the fabrication of nanobiosensors based on various sensing methods ( Table 2). [50][51][52] Use of quantum dots for the production of nano-aptasensors: Quantum dots are defined as small semiconductor crystals. An electromagnetic field emits light at different wavelengths by controlling the dimensions of the quantum dots. Since quantum dots can produce light with particular wavelengths, these fine crystals are used in optical devices. In this field, quantum dots can be used to make infrared detectors and light emitting diodes. [53][54][55][56][57][58] The use of nanotubes in the production of nano-aptasensors: Single-walled and multi-walled carbon nanotubes, due to their unique mechanical and electronic properties, have recently been used in the variety of applications, including their use as highly-precise and fast sensors for detection of substances at very low concentrations. These sensors are highly sensitive in detecting very small concentrations of gas molecules at room temperature. These nanomaterials have been used in different fields due to their high electrical conductivity. These properties have important implications for detection applications. Maehashi et al used a sample of nano-aptasensors mixed with single-walled carbon nanotubes to detect immunoglobulin E (IgE), which has a nanomolar detection limit. 71 The use of nano-tools in the production of nano-aptasensors: Studies on nano-tools are part of the up-to-date scientific research in the world. Research results published in Biosensor and Bioelectronics in 2009 reported a biosensor gold electrode surface that was modified in such a way that gold nanoparticles were deposited electrochemically on a smooth electrode surface. The electrode was then immersed in a bath containing para-benzoquinone, chitosan, glucose oxidase, and ionic liquid to complete the preparation of enzyme electrode. The response time of this amperometric biosensor for glucose was less than 5 seconds. Compared with smooth gold electrode modification (without the use of gold nanoparticles), sensitivity was increased 2.5 times, and the detection limit was improved 20 times. 72 Given the breadth and variety of strategies for using nano-aptasensors, the classification and grouping of these nano-tools is essential in spite of their complexity. 47

Classification of Nano-Aptasensors
Like other bio-sensing groups, nano-aptasensors are divided into different types based on detection methods and strategies. According to the reporting method, they can be divided into colorimetric, fluorescence, mass spectrometry sensors, etc.
Colorimetric nano-aptasensors: The use of nano-aptasensors in colorimetric-based strategies involves a wide range of methods. In a simple example, variations in environmental colors due to the presence or absence of an analyte and the subsequent accumulation or diffusion of nanoparticles were considered as signals. Using this method, adenosine triphosphate was detected with a detection limit of 0.3 to 20 μm during several minutes. 60 With another method, researchers labeled aptamers using quantum dots. Considering the presence or absence of the target molecule, they measured the color variation caused by the quantum dots and reported it as an indicator of the presence of the target molecule. For example, Liu et al were able to detect cocaine to a final limit of 10 to 20 μM. 59 Wang et al presented a more sensitive and precise method for detecting small molecules by stabilizing single-stranded DNA aptamers on gold plates in SPR. In this method, the aptamer structural changes caused by the binding of the target agent led to the binding of the complementary strands of DNA labeled with gold nanoparticles, which in turn amplified the SPR signal. The detection limit of this method was estimated to range from 1 micromole to one nanomole. 61 Gold nanomaterials are often used in colorimetric nanoaptasensors, so aptamar's thiol derivatives covalently bind so aptamers derivatives immobilised on nanosurface covalently (au-s). These derivatives are more expensive than the nonmanipulated types. To overcome this problem, Wang et al used genetic nano manipulated aptamers to develop an nano detection method that can detect adenosine tri-phosphate (2 μL) at at 37°C during a period of less than 1 hour a detection limit of 2 μmol, with a naked eye. 70 Chen et al increased the adenosine detection limit by up to 10 nM using a similar method. 69 In a colorimetric nano-aptasensor sample, they detected a protein target with a detection limit of up to 0.83 nM using non-manipulated gold nanoparticles (73). Wang et alused the same technique to increase the thrombin detection limit to 9 nm. 74 Jana et al also achieved similar results using the same method. 75 Xu et al detected thrombin with a detection limit of 2.5 nanomolar using nano-aptasensors based on dry-reagent strip gold nanoparticles. The author stated that thrombin was detected in human plasma up to a dilution rate of 0.6 pmol. 76 Fluorescence nano-aptasensors: Fluorescent nanomaterials, including gold nanoparticles, silver crystals, and quantum dots, have a lot of potential for sensing applications. Fluorescent nanomaterials, especially quantum dots, have many advantages over traditional fluorescent materials, including optical stability, chemical stability, limited emissions, and extensive excitation. 77 Researchers can use various luminescence methods, including chemiluminescence, transient energy luminescence, and delayed luminescence. Each of these scientific fields can have an impact on the promotion of nano-aptasensors. Emission and quenching act as on and off switches and make the sensor externally controllable. Song et al provided a sample of a fluorescent nano-aptasensor that was capable of detecting the amp up to 0.1 μM. 78 Mass spectrometry-based nano-aptasensors: Nanomaterials are used in mass spectrometry to detect various agents. Fe 3 O 4 , gold, TiO 2 nanoparticles, etc. are more noticeable with this method. Gold nanoparticles (13 nm particle size) together with an aptamer were used to detect adenosine triphosphate and made it possible to detect this substance to about 3-48 μm. 79 Nano-aptasensors can also be classified based on the type of transducer, such as the field-effect transistor aptasensor.
Feld-effect transistor aptasensor: Field-effect transistor (FET) aptasensor are a series of transistors which control the current by an electric field. Given that only one carrier (free electron or electron cavity) is involved in the electrical current in these transistors, they can be considered as unipolar transistors that are placed against bipolar transistors. FETs are sensitive to chemical changes in which the analyteactive coating interaction results in a change in the intensity of the source current. The analyte-active coating interaction is similar to the potentiometric ion-selective sensors in terms of its chemical property. Over the past decade, researchers have offered nano-aptasensors using aptamers and field-effect transistors which were capable of precisely detecting and measuring analytes such as immunoglobulin E or pathogenic bacteria, in small dimensions, small sample sizes, and with high sensitivity. This sensing method can be very efficient. 71,80 Classification of Nano-Aptasensors Based on Type of Transducer Physical or chemical changes resulting from the aptameranalyte attachment or detachment should be converted by a transducer to a transmittable signal. By converting signals based on different strategies, this section of sensor attempts to establish a quantitative relationship between the amount of analyte presence and the comprehensible signal for the processor of the nano-aptabiosensor. 81 Electrochemical nano-aptasensors: In addition to being highly sensitive to and compatible with modern nanostructure and miniatorized system, electrochemical nano-aptasensors are more coherent than other similar ones. Some of these electrochemical nano-aptasensors are electrochemical impedance spectroscopy (EIS), potentiometric, ion selective electrode (ISE), electrochemiluminescence (ECL), closing velocity (CV), and differential pulse voltammetry (DPV) nano-aptasensors. 15,16,34,82,83 Capacitance measurement electrochemical nano-aptasensor: Changes are made in the capacitance of a capacitor in the signal transducer section of this type of nano-aptasensor. In fact, the basis for the formation of these sensors is the convergence and accumulation of basic and applied sciences in the fields of chemistry, biology, and physics that finally come to serve nano-biotechnology. The basis of this type of biosensor is the change in dielectric properties of an electrode surface. Previous research has shown that such kind of biosensor can be used to detect the analyte-element binding stabilized on the surface of an electrode. 84 The binding of an analyte to a bio-element stabilized in a capacitor can directly change the capacitor capacity without the need for labeling. 85 In their study, Rashidiani et al detected nanomolar dilutions of botulinum neurotoxin type A light chain in an aptamer binding in a capacitive cell using an LCR meter ( Figure 4). Another disadvantage associated with aptasensors is related to their nature. That is, the application of these materials requires a high level of skill, while most diagnostic laboratories do not have professional scientific cadres, and the use of these sensors in home detection tools makes the problem more complicated. 59 Using this type of sensor in research and applied fields is a step toward the widespread use of this technology.

Conclusions
New approaches to the production of biosensors, in addition to the sharp reduction in detection limits, detection process times, test costs, and the need for user experience and skill, have increased the sensitivity, biocompatibility, and user friendliness of biosensors and the shelf life of materials and reagents. 15,16 Some of the primary biosensors were the size of a workroom, but recently, a mass spectrometer with the size of a suitcase and biosensors measuring a few cubic centimeters in size have surprised many consumers. 18 It should be explicitly stated that their advantages give nano-aptasensors the potential to be used in analyzing devices (biosensors, disease diagnosis, rapid detection of pesticides and risk factors), research areas (chemical and biochemical analysis, and the discovery and measurement of new drugs), and industrial areas (food industry, defense and security fields, and reagents). 27 This has made it a tempting market opportunity. Nano-sensors are inherently smaller (albeit not always) and more sensitive than other sensors, and in combination with aptamers, their sensitivity and specificity are significantly enhanced. In conclusion, because nano-aptasensors reduce the purchasing and operating costs, their use as arrays and masses can be cost-effective and can also be used extensively in parts. The military and national security sector also needs very sensitive sensors that can be used widely to monitor threats. In the medical field, there is also a need for highly sensitive sensors such as lab-on-a-chip (LOC) devices that can detect the smallest symptoms of a disease. Under optimal conditions, nano-aptasensors should have a high binding affinity to the target (low detection limit), high specificity (low interference), wide dynamic range, fast response time, and a high shelf-life. The results of tests on aptasensors in detecting various analytes (protein, peptide, small molecules, metal ions, and whole cells, etc) indicate high repeatability, excellent accuracy, and ease of use. The dissociation constant (KD) of aptamer-target has been reported as ranging from micromole to picomole, 47 but have not yet been fully commercialized in clinical and laboratory detection fields. One reason for the absence of aptamer-based detection devices may be the sensitivity of the conditions for their application and their lack of user-friendliness for commercialization and entry into the market. 59 However, the use of simple techniques such as capacitor capacity measurement in aptamer-based sensors can be an effective step in the enhancement of the user-friendliness of this type of sensor. Indicators that make this type of apta-biosensor superior include high sensitivity and low detection limit, short testing times, higher detection speed, the application of lower analyte volumes, lower price, miniaturization potential, and mass production. 83