Aptamers & Aptasensor and its application in clinical diagnostic By Elham Bazrafshan Professor Dadfarnia By Elham Bazrafshan Professor Dadfarnia

 Cancer attracts the worldwide attention with a marked increase in incidence and mortality Based on the global cancer statistics estimates, the number of new cancer cases and cancer deaths occurred in 2012 are about 14.1 million and 8.2 million, respectively. so early diagnosis and treatment is important to improve cancer patient survival. 1

 Aptamers are oligonucleotides, such as ribonucleic acid (RNA) and single-strand deoxyribonucleic acid (ssDNA) or peptide molecules that can bind to their targets with high affinity and specificity due to their specific three-dimensional structures.  Aptamers, derived from the Latin Aptos, meaning ‘‘to fit’’  Aptamers are oligonucleotides, such as ribonucleic acid (RNA) and single-strand deoxyribonucleic acid (ssDNA) or peptide molecules that can bind to their targets with high affinity and specificity due to their specific three-dimensional structures.  Aptamers, derived from the Latin Aptos, meaning ‘‘to fit’’ Introduction 2

 Aptamers have progressed since the in vitro selection process called Systematic Evolution of Ligands by Exponential enrichment (SELEX) was first reported by both Gold’s group and Szostak’s group in 1990  Their size is between 6 and 40 kDa, and sometimes they are particularly complex and complex with three-dimensional structures.  They are similar to antibodies  Aptamers have progressed since the in vitro selection process called Systematic Evolution of Ligands by Exponential enrichment (SELEX) was first reported by both Gold’s group and Szostak’s group in 1990  Their size is between 6 and 40 kDa, and sometimes they are particularly complex and complex with three-dimensional structures.  They are similar to antibodies 3

Comparison between APTAMER & ANTIBODIES  High stability of Aptamers  Production of Aptamers (synthesis/modification)  Low immunogenicity of Aptamers  Variety of target  High stability of Aptamers  Production of Aptamers (synthesis/modification)  Low immunogenicity of Aptamers  Variety of target 4

Comparison between APTAMER & ANTIBODIES Properties Aptamer Antibodies Dedication and hybrid very high high Sustainability good middle Synthesis/modification good middle The possibility of chemical changes yes no 5

Antibodies Aptamers 6

Systematic evolution of ligands by exponential enrichment (SELEX) o SELEX or in vitro selection is a technique used to isolate aptamers with high affinity for a given target from approximately combinatorial oligonucleotide libraries. 7

Systematic evolution of ligands by exponential enrichment (SELEX)  This method described primarily in 1990 (Ellington.A.D & Szostak.J.W.1990) 8

Polymerase chain reaction (PCR)  A technique that can be used to reproduce a specific molecule of DNA molecules in millions of times in a short period of time.  This piece of DNA may be:  A gene  Part of a chromosome  Or parts of the genome of an existing one.  A technique that can be used to reproduce a specific molecule of DNA molecules in millions of times in a short period of time.  This piece of DNA may be:  A gene  Part of a chromosome  Or parts of the genome of an existing one. 9

PCR  The basis of this method is very simple and is similar to the DNA replication reaction in living organisms by DNA polymerase enzyme.  In organisms, a set of several proteins and enzymes are involved in the DNA replication process, while in the PCR reaction only a certain type of heat-resistant DNA polymerase enzyme called Taq polymerase, along with buffers, magnesium chloride and nucleotides to reproduce DNA fragments Used.  The basis of this method is very simple and is similar to the DNA replication reaction in living organisms by DNA polymerase enzyme.  In organisms, a set of several proteins and enzymes are involved in the DNA replication process, while in the PCR reaction only a certain type of heat-resistant DNA polymerase enzyme called Taq polymerase, along with buffers, magnesium chloride and nucleotides to reproduce DNA fragments Used. 10

PCR 11

Advantage of Aptamers 12

Various application fields of Aptamers 13

o Start using Aptamers as Treatment was in 1990 In 2004, the approval by the Food and Drug Administration (FDA) of Macugen, 14

Four methods are proposed for identifying Aptamer-bound molecules  1-Sandwitch technique  2- De-quenching Fluorescent Signals in Aptamer Beacons  3- Method of Fluorescence Anisotropy  4-Modulation of Fluorescent emission by signaling Aptamer  1-Sandwitch technique  2- De-quenching Fluorescent Signals in Aptamer Beacons  3- Method of Fluorescence Anisotropy  4-Modulation of Fluorescent emission by signaling Aptamer 15

Four methods are proposed for identifying Aptamer- bound molecules  1. Sandwich technique This method of using Aptamer is similar to that of Elisa. A monoclonal antibody immobilized and then labeled with an Aptamar marker.  2- De-quenching Fluorescent Signals in Aptamer Beacons Molecular beacons include a nucleotide sequence that has complementary sequences on both sides. These two end sequences together give a base pair and cause the oligonucleotide to become a hair pin. At the two ends of these oligonucleotides, there is both a fluorescent substance and a quencher material. If this marker is not attached, the target in the creation of the structure of the Stem-loop, the fluorescent substance is placed in front of the quencher, and thus no light is emitted And so they can create beacons that are specific to the second or third structure of the Aptamers  1. Sandwich technique This method of using Aptamer is similar to that of Elisa. A monoclonal antibody immobilized and then labeled with an Aptamar marker.  2- De-quenching Fluorescent Signals in Aptamer Beacons Molecular beacons include a nucleotide sequence that has complementary sequences on both sides. These two end sequences together give a base pair and cause the oligonucleotide to become a hair pin. At the two ends of these oligonucleotides, there is both a fluorescent substance and a quencher material. If this marker is not attached, the target in the creation of the structure of the Stem-loop, the fluorescent substance is placed in front of the quencher, and thus no light is emitted And so they can create beacons that are specific to the second or third structure of the Aptamers 16

Four types of methods are proposed for identifying Aptamer-bound molecules  3- Method of Fluorescence Anisotropy In this method, an uneven change in the degree of fluorescence of the Aptamar marker is observed. One of the problems with this method is that between the two strong and weak optical signals, we need to identify the weaker optical signal. Although the Aptamer connection is made to small molecules, it is less recognizable as the amount of light that does not emit a small region. There is a limitation of the Aptamer 's minimum size for this purpose.  4. Modulation of Fluorescent emission by signaling Aptamer When the Aptamers connect to their ligands, they undergo space deformation. In studies conducted at the Ellington University labs, it has been determined that the intensity of fluorescence irradiated after connecting Aptamer to The ligand is detectable and can be shown by connecting Aptamers to their targets.  3- Method of Fluorescence Anisotropy In this method, an uneven change in the degree of fluorescence of the Aptamar marker is observed. One of the problems with this method is that between the two strong and weak optical signals, we need to identify the weaker optical signal. Although the Aptamer connection is made to small molecules, it is less recognizable as the amount of light that does not emit a small region. There is a limitation of the Aptamer 's minimum size for this purpose.  4. Modulation of Fluorescent emission by signaling Aptamer When the Aptamers connect to their ligands, they undergo space deformation. In studies conducted at the Ellington University labs, it has been determined that the intensity of fluorescence irradiated after connecting Aptamer to The ligand is detectable and can be shown by connecting Aptamers to their targets. 17

Aptamer -Based Sensor  A biosensor that is based on Aptamers as a recognition element is called an aptasensor.  The capabilities of these biosensors include simultaneous measurement of hundreds of different molecular species, high sensitivity, high specificity, Fast analysis, very simple chemical principles that can be easily automated.  A biosensor that is based on Aptamers as a recognition element is called an aptasensor.  The capabilities of these biosensors include simultaneous measurement of hundreds of different molecular species, high sensitivity, high specificity, Fast analysis, very simple chemical principles that can be easily automated. 18

Aptasensors 1. Electrochemical aptasensor 2. optical biosensors 3. mass-sensitive biosensors. 1. Electrochemical aptasensor 2. optical biosensors 3. mass-sensitive biosensors. Aptasensors can be constructed through a variety of methodologies, including: 19

Electrochemical Aptasensor  An electrochemical analysis is an attractive platform, because it offers high sensitivity, compatibility with novel microfabrication technologies, and low cost.  Therefore, various electrochemical Aptasensors have been fabricated using several techniques  To enhance the sensitivity, are used as a material for signal transduction such as methylene blue (MB), ferrocence, ruthenium complexes, and Fe(CN) 6 4-/3-  An electrochemical analysis is an attractive platform, because it offers high sensitivity, compatibility with novel microfabrication technologies, and low cost.  Therefore, various electrochemical Aptasensors have been fabricated using several techniques  To enhance the sensitivity, are used as a material for signal transduction such as methylene blue (MB), ferrocence, ruthenium complexes, and Fe(CN) 6 4-/3- 20

Electrochemical Aptasensor 21

Fluorescence-Based Optical Aptasensor  Aptamers have been used as bio-probes in optical sensors based primarily on the incorporation of a fluorophore or a nanoparticle.  In the case of fluorescence detection, the simplest format is to label the Aptamers with both a quencher and a fluorophore.  Aptamer beacons can change into two or more conformations, such as hairpin shape or a hybridization form, by target binding.  Aptamers have been used as bio-probes in optical sensors based primarily on the incorporation of a fluorophore or a nanoparticle.  In the case of fluorescence detection, the simplest format is to label the Aptamers with both a quencher and a fluorophore.  Aptamer beacons can change into two or more conformations, such as hairpin shape or a hybridization form, by target binding. 22

Fluorescence-Based Optical Aptasensor 23

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 Exosomes, a kind of extracellular membrane vesicles ( nm), are actively secreted by various living cells types.  acting as cellular messengers for delivering information between cells.  Mucin 1 (MUC1), a large cell-surface-associated glycoprotein, is applied as a diagnostic marker in cancer, and is expected to be used as a therapeutic target for cancer  Exosomes, a kind of extracellular membrane vesicles ( nm), are actively secreted by various living cells types.  acting as cellular messengers for delivering information between cells.  Mucin 1 (MUC1), a large cell-surface-associated glycoprotein, is applied as a diagnostic marker in cancer, and is expected to be used as a therapeutic target for cancer 25

 In this study, selected the mucin 1 (MUC1) protein as a carcinoma biomarker in human breast cancer MCF-7 cells.  Indeed, demonstrated for the first time that MUC1 was highly expressed on the surface of exosomes secreted by MCF-7 cells compared to normal breast cell  Therefore, detected exosomes secreted by MCF-7 cells using the specific interaction between MUC1 and its oligonucleotide aptasensor of hairpin structure ， which can be applied effectively for recognizing MUC1 protein on exosomes secreted by MCF-7 cells  In this study, selected the mucin 1 (MUC1) protein as a carcinoma biomarker in human breast cancer MCF-7 cells.  Indeed, demonstrated for the first time that MUC1 was highly expressed on the surface of exosomes secreted by MCF-7 cells compared to normal breast cell  Therefore, detected exosomes secreted by MCF-7 cells using the specific interaction between MUC1 and its oligonucleotide aptasensor of hairpin structure ， which can be applied effectively for recognizing MUC1 protein on exosomes secreted by MCF-7 cells 26

In the aptasensor, the TAMRA was luminophore. On the contrary, Dabcyl was quenching group. 27

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 Mucin 1 (MUC1), it is the most common tumor marker of breast cancer  The Aptamer S2.2, a targeting ligand for MUC1 antigen  The cyanine (Cy5)-tagged S2.2-functionalized silicon nanodot (SiND-S2.2-Cy5) Aptasensor for specifically targeting MUC1.  the SiNDs can quench the fluorescence of Cy5 in Cy5-tagged S2.2 effectively, and the fluorescence of Cy5 can be restored in the presence of MUC1.  Mucin 1 (MUC1), it is the most common tumor marker of breast cancer  The Aptamer S2.2, a targeting ligand for MUC1 antigen  The cyanine (Cy5)-tagged S2.2-functionalized silicon nanodot (SiND-S2.2-Cy5) Aptasensor for specifically targeting MUC1.  the SiNDs can quench the fluorescence of Cy5 in Cy5-tagged S2.2 effectively, and the fluorescence of Cy5 can be restored in the presence of MUC1. 29

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An Aptamer Cross-Linked Hydrogel as a Colorimetric Platform for Visual Detection 31

Hydrogel by Aptamer to identify cocaine 32

Changes in the color of the hydrogel due to the presence of cocaine molecules 33

Conclusions  Aptamar is an appropriate alternative to antibody and is very stable under severe and difficult conditions. Because Aptamers are oligo nucleic acid and can easily be produced at high purity levels and modified by simple chemical reactions, aptamers have a great deal of power in a wide range of applications. In addition, aptamers are non-immune and non-immune materials that can be used in medicine for the diagnosis and treatment of diseases, medicine, biological imaging.  Aptamers stand as great candidates in medical sciences for development of biosensors and targeted drug delivery systems because of their unique properties such as high affinity and selectivity towards their targets, inexpensiveness and in vitro synthesis, high stability and small size.  Aptamar is an appropriate alternative to antibody and is very stable under severe and difficult conditions. Because Aptamers are oligo nucleic acid and can easily be produced at high purity levels and modified by simple chemical reactions, aptamers have a great deal of power in a wide range of applications. In addition, aptamers are non-immune and non-immune materials that can be used in medicine for the diagnosis and treatment of diseases, medicine, biological imaging.  Aptamers stand as great candidates in medical sciences for development of biosensors and targeted drug delivery systems because of their unique properties such as high affinity and selectivity towards their targets, inexpensiveness and in vitro synthesis, high stability and small size. 34

References  Danesh, N.M.; Yousefi2, F.; Alinezhad, M.; Abnous, K.; Taghdisi, M. Birjand University of Medical Sciences. 2018; 24 (4):  Zhang, J.; Zhao, H.; Shi, J.; Liu, W.; Zhang,K. Sensors and Actuators B  Zhang, Y.; Guo, S.; Huang, H.; Mao, G.; Ji, X.; He, Z. Analytica Chimica Acta  Song, K.; Lee, S.; Ban, C. sensors. 2012, 12,  Saberian1, M.; Asgari1, D.; Hamzeiy, H. Journal of Police Medicine  Zhu, Z.; Wu, C.; Liu, H.; Zou, Y.; Zhang, X.; Kang, H.; James Yang, C.; Tan, W. Angew. Chem. Int. 2010, 49, 1052 – 1056  Danesh, N.M.; Yousefi2, F.; Alinezhad, M.; Abnous, K.; Taghdisi, M. Birjand University of Medical Sciences. 2018; 24 (4):  Zhang, J.; Zhao, H.; Shi, J.; Liu, W.; Zhang,K. Sensors and Actuators B  Zhang, Y.; Guo, S.; Huang, H.; Mao, G.; Ji, X.; He, Z. Analytica Chimica Acta  Song, K.; Lee, S.; Ban, C. sensors. 2012, 12,  Saberian1, M.; Asgari1, D.; Hamzeiy, H. Journal of Police Medicine  Zhu, Z.; Wu, C.; Liu, H.; Zou, Y.; Zhang, X.; Kang, H.; James Yang, C.; Tan, W. Angew. Chem. Int. 2010, 49, 1052 –

And science always goes forward and surprising techniques and techniques are created, and the least that can be done is to get to know their principles. Perhaps the time has come for the technology gap between societies to fall enough so that an Iranian can also be in Iran. It is a technology manufacturer, not a consumer. The End And science always goes forward and surprising techniques and techniques are created, and the least that can be done is to get to know their principles. Perhaps the time has come for the technology gap between societies to fall enough so that an Iranian can also be in Iran. It is a technology manufacturer, not a consumer. The End