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Figure 5. Schematic of cocaine sensing based on structure switching optical biosensing platform. (a) Structure switching of cocaine combining the two aptamer fragments; (b) hybridization and regeneration of the aptamer with optical fiber. Reproduced with the permission of [119]. Copyright 2016 Published by Elsevier B.V. Figure 5. Schematic of cocaine sensing based on structure switching optical biosensing platform. 3.3.3. Other Optical Aptasensors for Abused Drugs In addition to colorimetric and fluorescence-based aptasensors, chemiluminescence and SPR-based techniques were also used for abused drug detection. Different from other optical detection methods, in chemiluminescence, excitation energy comes from the chemical reaction itself. As the luminescence signal, which can be measured down to only a few emitted photons, is a highly detectable bio- or chemiluminescence technique, it has become favorable [167]. High sensitivity, wide calibration ranges, and simple instrumentation comprise the unique properties of chemiluminescence-based biosensor platforms. Several chemiluminescence-based aptasensors have been successfully applied to abused drug sensing [133-135]. All three studies were focused on the detection of cocaine. Yan et al. [134 used HRP, and alkaline phosphatase (ALP) to label different reporter DNA probes, and catalyzed chemiluminescence reactions were monitored. A relatively low LOD value of 3.2 nM was achieved with this straightforward strategy, proving its worth to be implemented in future studies for smal molecules. A similar approach was used by Li et al. [135], using the chemiluminescence signals of luminol-H)O-HRP-p-iodophenol (PIP) system. AuNPs were modified with magnetic microbeads (MB-AuNPs), and aptamers were immobilized on the surface of this conjugate. The complex was then hybridized with the signal DNA modified with HRP and in the presence of the cocaine, analyte and signal DNA were competed, forcing the MB-AuNPs to dissociate due to the aptamer structure switching. Thus, the chemiluminescence signal was obtained proportional to the cocaine present in the solution. With this method, 0.48 nM LOD value was obtained successfully. AA AitaAwa lls Alswrtenoannneatand sand linawinavnersnrs (BCT \ annenanrk <iace faniltatad he Give wt in chemiluminescence, excitation energy comes from the chemical reaction itself. As the luminescence

Figure 5 Schematic of cocaine sensing based on structure switching optical biosensing platform. (a) Structure switching of cocaine combining the two aptamer fragments; (b) hybridization and regeneration of the aptamer with optical fiber. Reproduced with the permission of [119]. Copyright 2016 Published by Elsevier B.V. Figure 5. Schematic of cocaine sensing based on structure switching optical biosensing platform. 3.3.3. Other Optical Aptasensors for Abused Drugs In addition to colorimetric and fluorescence-based aptasensors, chemiluminescence and SPR-based techniques were also used for abused drug detection. Different from other optical detection methods, in chemiluminescence, excitation energy comes from the chemical reaction itself. As the luminescence signal, which can be measured down to only a few emitted photons, is a highly detectable bio- or chemiluminescence technique, it has become favorable [167]. High sensitivity, wide calibration ranges, and simple instrumentation comprise the unique properties of chemiluminescence-based biosensor platforms. Several chemiluminescence-based aptasensors have been successfully applied to abused drug sensing [133-135]. All three studies were focused on the detection of cocaine. Yan et al. [134 used HRP, and alkaline phosphatase (ALP) to label different reporter DNA probes, and catalyzed chemiluminescence reactions were monitored. A relatively low LOD value of 3.2 nM was achieved with this straightforward strategy, proving its worth to be implemented in future studies for smal molecules. A similar approach was used by Li et al. [135], using the chemiluminescence signals of luminol-H)O-HRP-p-iodophenol (PIP) system. AuNPs were modified with magnetic microbeads (MB-AuNPs), and aptamers were immobilized on the surface of this conjugate. The complex was then hybridized with the signal DNA modified with HRP and in the presence of the cocaine, analyte and signal DNA were competed, forcing the MB-AuNPs to dissociate due to the aptamer structure switching. Thus, the chemiluminescence signal was obtained proportional to the cocaine present in the solution. With this method, 0.48 nM LOD value was obtained successfully. AA AitaAwa lls Alswrtenoannneatand sand linawinavnersnrs (BCT \ annenanrk <iace faniltatad he Give wt in chemiluminescence, excitation energy comes from the chemical reaction itself. As the luminescence

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Figure 1. The systematic evolution of ligands by exponential enrichment (SELEX) cycle. SELEX starts with a random nucleic acid aptamer library which is used to initiate the SELEX cycle (top arrow entering cycle). The library is incubated with the target, and the target is washed to remove and discard unbound aptamers (right arrow exiting cycle) before the bound aptamers are eluted and amplified by PCR. The amplified sequences seed the next round of SELEX. Typically, around 12 SELEX cycles are performed before sequencing and aptamer characterization (left arrow exiting cycle). Reproduced with permission of [33]. Copyright 2017 Published by MDPI. high-affinity binders are enriched. Finally, the resulting pool is sequenced, and the selected aptamers are characterized for their binding affinity. Since the first introduction of the SELEX method in 1990 by two independent groups [24,25], a large number of aptamers have been developed for various targets ranging from small molecules [26,27] to proteins [28,29], whole cells [30], and tissues [31,32]. entering cycle). The library is incubated with the target, and the target is washed to remove and discard
Figure 1. The systematic evolution of ligands by exponential enrichment (SELEX) cycle. SELEX starts with a random nucleic acid aptamer library which is used to initiate the SELEX cycle (top arrow entering cycle). The library is incubated with the target, and the target is washed to remove and discard unbound aptamers (right arrow exiting cycle) before the bound aptamers are eluted and amplified by PCR. The amplified sequences seed the next round of SELEX. Typically, around 12 SELEX cycles are performed before sequencing and aptamer characterization (left arrow exiting cycle). Reproduced with permission of [33]. Copyright 2017 Published by MDPI. high-affinity binders are enriched. Finally, the resulting pool is sequenced, and the selected aptamers are characterized for their binding affinity. Since the first introduction of the SELEX method in 1990 by two independent groups [24,25], a large number of aptamers have been developed for various targets ranging from small molecules [26,27] to proteins [28,29], whole cells [30], and tissues [31,32]. entering cycle). The library is incubated with the target, and the target is washed to remove and discard
Figure 2. Main advantages of aptamers compared to antibodies.
Figure 2. Main advantages of aptamers compared to antibodies.
Figure 3. Typical optical detection for both light transmitted through liquid sample platforms (i.e., cuvette, well plate) and light reflected from solid sample platforms (i.e., test strip, cassette) using (a) colorimetric assays, (b) fluorescence-based assays, (c) bio- and chemiluminescence assays, and (d) scattering-based assays. Reproduced with the permission of [106]. Copyright 2016 Published by The Royal Society of Chemistry. Due to their high performance in terms of sensitivity, selectivity, response time, and operational simplicity, optical aptasensors have been widely used for the detection of abused drugs. Optical biosensors typically use four different signal detection principles: colorimetric, fluorescence-based, chemi-/bioluminescence-based, and scattering-based sensing (Figure 3) [117,118]. These systems can be formed in two formats which are solution-based and flat substrate-based assays. The literature survey of the detection of abused drugs is abundant of optical biosensors, using colorimetric [119-127], fluorescence-based [86,128-132], chemiluminescence-based [133-135], and SPR-based techniques [136]. The results from the literature survey are summarized in Table 52. well plate) and light reflected from solid sample platforms (i.e., test strip, cassette) using (a) colorimetric
Figure 3. Typical optical detection for both light transmitted through liquid sample platforms (i.e., cuvette, well plate) and light reflected from solid sample platforms (i.e., test strip, cassette) using (a) colorimetric assays, (b) fluorescence-based assays, (c) bio- and chemiluminescence assays, and (d) scattering-based assays. Reproduced with the permission of [106]. Copyright 2016 Published by The Royal Society of Chemistry. Due to their high performance in terms of sensitivity, selectivity, response time, and operational simplicity, optical aptasensors have been widely used for the detection of abused drugs. Optical biosensors typically use four different signal detection principles: colorimetric, fluorescence-based, chemi-/bioluminescence-based, and scattering-based sensing (Figure 3) [117,118]. These systems can be formed in two formats which are solution-based and flat substrate-based assays. The literature survey of the detection of abused drugs is abundant of optical biosensors, using colorimetric [119-127], fluorescence-based [86,128-132], chemiluminescence-based [133-135], and SPR-based techniques [136]. The results from the literature survey are summarized in Table 52. well plate) and light reflected from solid sample platforms (i.e., test strip, cassette) using (a) colorimetric
Figure 4. Lateral flow based detection of cocaine. (a) DNA sequences and linkages in cocaine aptamer-linked nanoparticle aggregates. Test of the cocaine-sensing lateral flow device with varying concentrations of cocaine in buffer solution (b) and in undiluted human blood serum (c). Coc = cocaine, Ade = adenosine. Reproduced with the permission of [109]. Copyright 2006 Published by Wiley-VCH.
Figure 4. Lateral flow based detection of cocaine. (a) DNA sequences and linkages in cocaine aptamer-linked nanoparticle aggregates. Test of the cocaine-sensing lateral flow device with varying concentrations of cocaine in buffer solution (b) and in undiluted human blood serum (c). Coc = cocaine, Ade = adenosine. Reproduced with the permission of [109]. Copyright 2006 Published by Wiley-VCH.
Figure 6. Electrochemical (Path A), photoelectrochemical (Path B), and surface plasmon resonance (SPR) (Path C) analysis of cocaine. Reproduced with the permission of [125]. Copyright 2009 Published by American Chemical Society. a 2g, Seeeeemene: A Ey Ree eR a se Se eS Se, Se Se RT | FN developed and very low LOD values for cocaine, such as 10 pM, were achieved with this technique. SPR is an optical—electrical techno interaction of light with the electrons of a O8y biomolecule thickness or mass on the surface monitoring with a small sample amount samples [171,172], to detect small molecu molecule causes a too small change in t ne re SPR as an analytical technique usually use me fractive index al nanostructu based on the change in the refractive index due to the metal surface which is directly proportional to the mass of the 7,118]. This method provides rapid, label-free, real-time 7]. Even though SP es with this method R has been thoroughly used for biological still remains as a challenge as the aptamer for analysis. Thus, aptasensors adapting res as a signal enhancement tool [140,173]. In work conducted by Golub et al. [136], electrochemical, photoelectrochemical, and SPR detection of cocaine was successfully carried out using two cocaine aptamer subunits. One subunit is attached to an SPR-active Au surface, and the other subunit is labeled with either platinum nanoparticles (PtNPs), CdSNPs, or AuNPs, depending on the method to be used. AuNPs were used for SPR-based detection mode (Figure 6). In the presence of cocaine, a supramolecular complex was formed between the AuNP-cocaine aptamer and the cocaine on the Au surface, enabling the SPR monitoring of cocaine according to the reflectance changes resulting from the electronic coupling between the localized plasmon of the Au-NPs and the surface plasmon wave. A low range of LOD values, 1 to 10 nM, was achieved using this technique.
Figure 6. Electrochemical (Path A), photoelectrochemical (Path B), and surface plasmon resonance (SPR) (Path C) analysis of cocaine. Reproduced with the permission of [125]. Copyright 2009 Published by American Chemical Society. a 2g, Seeeeemene: A Ey Ree eR a se Se eS Se, Se Se RT | FN developed and very low LOD values for cocaine, such as 10 pM, were achieved with this technique. SPR is an optical—electrical techno interaction of light with the electrons of a O8y biomolecule thickness or mass on the surface monitoring with a small sample amount samples [171,172], to detect small molecu molecule causes a too small change in t ne re SPR as an analytical technique usually use me fractive index al nanostructu based on the change in the refractive index due to the metal surface which is directly proportional to the mass of the 7,118]. This method provides rapid, label-free, real-time 7]. Even though SP es with this method R has been thoroughly used for biological still remains as a challenge as the aptamer for analysis. Thus, aptasensors adapting res as a signal enhancement tool [140,173]. In work conducted by Golub et al. [136], electrochemical, photoelectrochemical, and SPR detection of cocaine was successfully carried out using two cocaine aptamer subunits. One subunit is attached to an SPR-active Au surface, and the other subunit is labeled with either platinum nanoparticles (PtNPs), CdSNPs, or AuNPs, depending on the method to be used. AuNPs were used for SPR-based detection mode (Figure 6). In the presence of cocaine, a supramolecular complex was formed between the AuNP-cocaine aptamer and the cocaine on the Au surface, enabling the SPR monitoring of cocaine according to the reflectance changes resulting from the electronic coupling between the localized plasmon of the Au-NPs and the surface plasmon wave. A low range of LOD values, 1 to 10 nM, was achieved using this technique.
three-electrode system which includes a reference electrode that is commonly made from Ag/AgCl, a working electrode that can be either carbon, gold or graphene-made, and a counter or auxiliary 2lectrode, which generally is a Pt electrode [176]. Electrochemical biosensor platforms can be divided into four major categories in terms of their measurement principles: amperometric, impedimetric, conductometric, and potentiometric (Figure 7) [177]. However, amperometric [11,91,178-193] and impedimetric [11,91,180-185,187,188,190,191,194,195] electrochemical sensor systems have been more commonly used for determination of abused drugs. The results from the literature survey are summarized in Table 53. Amperometric techniques are commonly used due to high sensitivity and wide linear range and have different measurement modes, such as chronoamperometry, cyclic voltammetry (CV) and differential pulse voltammetry (DPV) [139,196]. DPV and CV methods have been one of the most widely used techniques for the determination of abused drugs. In the CV method, a varying potential is applied to the working electrode, and while the potential in the forward direction is terminated the potential in the reverse direction is started scanning. This method provides information about whether the reaction taking place in the working electrode is reversible [197]. DPV is a technique where an increased potential is applied to the system, and the current is measured before and after the pulse. The obtained difference in the peaks is directly proportional to the concentration of target molecules [197]. In the impedimetric biosensors, electrochemical impedance spectroscopy (EIS) is used for measurements. EIS is a technique for determining electrode kinetics and binding characteristics of the analyte on the electrode surface with the application of an alternative current at different frequencies to the system [198]. As a result of the measurements, electrochemical spectrum data are obtained and the impedance values are calculated accordingly [199,200]. Ta tha. laa FARTS SPARKS OLAALKEA AR AmIGAl ATTA CGCARGAKS FRAGA BEARA Ait se FARA MAGE SZAMMAR I
three-electrode system which includes a reference electrode that is commonly made from Ag/AgCl, a working electrode that can be either carbon, gold or graphene-made, and a counter or auxiliary 2lectrode, which generally is a Pt electrode [176]. Electrochemical biosensor platforms can be divided into four major categories in terms of their measurement principles: amperometric, impedimetric, conductometric, and potentiometric (Figure 7) [177]. However, amperometric [11,91,178-193] and impedimetric [11,91,180-185,187,188,190,191,194,195] electrochemical sensor systems have been more commonly used for determination of abused drugs. The results from the literature survey are summarized in Table 53. Amperometric techniques are commonly used due to high sensitivity and wide linear range and have different measurement modes, such as chronoamperometry, cyclic voltammetry (CV) and differential pulse voltammetry (DPV) [139,196]. DPV and CV methods have been one of the most widely used techniques for the determination of abused drugs. In the CV method, a varying potential is applied to the working electrode, and while the potential in the forward direction is terminated the potential in the reverse direction is started scanning. This method provides information about whether the reaction taking place in the working electrode is reversible [197]. DPV is a technique where an increased potential is applied to the system, and the current is measured before and after the pulse. The obtained difference in the peaks is directly proportional to the concentration of target molecules [197]. In the impedimetric biosensors, electrochemical impedance spectroscopy (EIS) is used for measurements. EIS is a technique for determining electrode kinetics and binding characteristics of the analyte on the electrode surface with the application of an alternative current at different frequencies to the system [198]. As a result of the measurements, electrochemical spectrum data are obtained and the impedance values are calculated accordingly [199,200]. Ta tha. laa FARTS SPARKS OLAALKEA AR AmIGAl ATTA CGCARGAKS FRAGA BEARA Ait se FARA MAGE SZAMMAR I
Figure 8. Aptasensor systems designing with fragments on Au surface with MCE as (a) Au/Cx5S/MCE, (b) Au/Cy5S/MCE, (c) Au/Cy3S/MCE. Reproduced with the permission of [183]. Copyright 2012 Published by Elsevier B.V. SEONG NA: RRL NANA RAS Re RSENS INNER ENR I emmtehige Mitte h Ney OER ENA NEE OCR poate Connecting the aptamer in its correct conformation on the electrode surface is crucial for the measurements in electrochemical aptasensor platforms. According to a study by Zhang et al. [194], a synthesized cocaine aptamer was divided into two fragments, and these fragments were called Cx and Cy. 3’ and 5’. Terminates of these fragments were modified with thiol groups, and three different aptasensor systems were designed with these fragments on the gold electrode (Au) surface with mercaptoethanol (MCE) as Au/Cx5S/MCE, Au/Cy3S/MCE, and Au/Cy5S/MCE. The Cy aptamer and cocaine were added into the Au/Cx5S/MCE system, while Cx and cocaine were added into the Au/Cy3S/MCE and Au/Cy5S/MCE systems. Impedance measurements of these systems were conducted in a ferricyanide redox probe solution, and the best results were obtained with Au/Cx5S/MCE modification in response to suitable immobilization of the aptamer and conformation of the supramolecular complex (Figure 8). In another study conducted by Shen et al. for the detection of cocaine, a similar strategy was followed, and the aptamer was divided into two fragments: Co35 and Co3B [187]. The Co3S fragment was attached on the gold electrode via a thiol group, and 3’ terminal of Co3B was labeled with biotin. In the presence of cocaine, the fragments were joined to capture the analyte. Then, streptavidin was added onto the electrode surface and was attached on the biotin terminal of the Co3B fragment, producing single-strand DNAs with rolling circle amplification in the presence of nucleotides and phi29 DNA polymerase. An important amplification for the determination of cocaine was achieved with streptavidin-alkaline phosphatase (ST-AP) and «-naphthyl phosphate «-NP) addition to the surface. The LOD value of suggested aptasensor system was determined as .3 nM, and this platform was successfully used for cocaine detection in urine samples.
Figure 8. Aptasensor systems designing with fragments on Au surface with MCE as (a) Au/Cx5S/MCE, (b) Au/Cy5S/MCE, (c) Au/Cy3S/MCE. Reproduced with the permission of [183]. Copyright 2012 Published by Elsevier B.V. SEONG NA: RRL NANA RAS Re RSENS INNER ENR I emmtehige Mitte h Ney OER ENA NEE OCR poate Connecting the aptamer in its correct conformation on the electrode surface is crucial for the measurements in electrochemical aptasensor platforms. According to a study by Zhang et al. [194], a synthesized cocaine aptamer was divided into two fragments, and these fragments were called Cx and Cy. 3’ and 5’. Terminates of these fragments were modified with thiol groups, and three different aptasensor systems were designed with these fragments on the gold electrode (Au) surface with mercaptoethanol (MCE) as Au/Cx5S/MCE, Au/Cy3S/MCE, and Au/Cy5S/MCE. The Cy aptamer and cocaine were added into the Au/Cx5S/MCE system, while Cx and cocaine were added into the Au/Cy3S/MCE and Au/Cy5S/MCE systems. Impedance measurements of these systems were conducted in a ferricyanide redox probe solution, and the best results were obtained with Au/Cx5S/MCE modification in response to suitable immobilization of the aptamer and conformation of the supramolecular complex (Figure 8). In another study conducted by Shen et al. for the detection of cocaine, a similar strategy was followed, and the aptamer was divided into two fragments: Co35 and Co3B [187]. The Co3S fragment was attached on the gold electrode via a thiol group, and 3’ terminal of Co3B was labeled with biotin. In the presence of cocaine, the fragments were joined to capture the analyte. Then, streptavidin was added onto the electrode surface and was attached on the biotin terminal of the Co3B fragment, producing single-strand DNAs with rolling circle amplification in the presence of nucleotides and phi29 DNA polymerase. An important amplification for the determination of cocaine was achieved with streptavidin-alkaline phosphatase (ST-AP) and «-naphthyl phosphate «-NP) addition to the surface. The LOD value of suggested aptasensor system was determined as .3 nM, and this platform was successfully used for cocaine detection in urine samples.
Table 1. SWOT analysis on aptasensors for abused drug detection. Supplementary Materials: The following are available online at http://www.mdpi.com/2079-6374/9/4/118/s1, Table S1: Binding affinity and post-SELEX optimization of aptamers selected for abused drugs, Table S2: Literature survey of optical aptasensors for abused drugs, Table S3: Literature survey of electrochemical aptasensors for abused drugs.
Table 1. SWOT analysis on aptasensors for abused drug detection. Supplementary Materials: The following are available online at http://www.mdpi.com/2079-6374/9/4/118/s1, Table S1: Binding affinity and post-SELEX optimization of aptamers selected for abused drugs, Table S2: Literature survey of optical aptasensors for abused drugs, Table S3: Literature survey of electrochemical aptasensors for abused drugs.
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