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Application of Aptamers in Food Safety
CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 39, Issue 6, June 2011 Online English edition of the Chinese language journal
Cite this article as: Chin J Anal Chem, 2011, 39(6), 925–933.
REVIEW
Application of Aptamers in Food Safety XU Dun-Ming1, WU Min1, ZOU Yuan2, ZHANG Qiang3, WU Cui-Chen2, ZHOU Yu1,*, LIU Xian-Jin3 1
Xiamen Entry-exit Inspection and Quarantine Bureau, Xiamen 361026, China College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China 3 Institute of Food Quality Safety and Detection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China 2
Abstract: Aptamers are all new ligands with high affinity for considerably diverse molecules ranging from large targets such as proteins, peptides, and complex molecules to drugs and small organic molecules as well as metal ions. These molecules are identified and selected through an in vitro process called SELEX (systematic evolution of ligands by exponential enrichment). Aptamers are widely used in different fields, including medical and pharmaceutical basic research, drug development, diagnosis, and therapy. Analytical and separation tools involving aptamers as molecular recognition and binding elements are another big field of application. The SELEX method has been improved over the last two decades in different ways – it has become more efficient and less time consuming and higher affinities of the aptamers and automation of the process have been achieved. In this review, a general background introduction, general principle of the SELEX, the development of aptamers by use of SELEX, and the application of aptamers in food safety are presented. Key Words: Aptamers; Systematic evolution of ligands by exponential enrichment; Food safety; Residue; Small organic molecules
1
Introduction
It is a milestone[1,2] in the history of molecular biology that the DNA double helix structure was proposed in 1953, and it marked the birth of modern molecular biology. Since then, research on large biological molecules such as nucleic acid has entered a brand-new era. In 1990, two independent research teams published their research papers on in vitro selection of the special target bound nucleic acid in Nature[3] and Science[4] respectively and named the nucleic acid “aptamer,” which combines the Latin word “aptus”(which means matching in English) and the Greek suffix “meros” and is translated into “Shiti,” “Shipeizi,” or “Hesuan Shiti” in Chinese. The aptamer selection technology SELEX[4], systematic evolution of ligands by exponential enrichment, is an integrated technology based on the combinatorial chemistry technology, the PCR technology and the gene clone sequencing technology, and so on. As the molecular biological technology has developed rapidly in recent years, the SELEX
technology has been optimized and many aptamer selection technologies[5,6] for different application purposes have been developed, which have greatly induced the rapid development of aptamer science. After a full 20 years of development, aptamer science has become incredibly sophisticated and its application in biology and iatrology has attracted great interest, e.g. molecular recognition[7,8], drug selection[7,8], target appraisal[8,9], gene expression regulation[9], medical imaging[9], clinical diagnosis[9,10], and disease curing[10]. Moreover, as a kind of target material bound molecules, aptamers also show great potential for application in the field of analytical science, especially in isolation and analysis of small molecular compounds. Food safety has been a matter of significant concern for the whole world in the 21st century, as it greatly affects the physical health of consumers. Since there are various types of food, many kinds of poisonous substances are found to be present in food. The amount of the substance present is extremely small and is usually in the range of ȝg, ng, or even
Received 26 December 2010; accepted 20 March 2011 * Corresponding author. Email: [email protected]; [email protected] This work was supported by the Science and Technology Foundation of Xiamen, China (No. 3502Z20092008), the Nature Science Foundation of Fujian, China (No.2008J0107), the Science and Technology Foundation of AQSIQ, China (No. 2010IK150), and the 973 Programme of China (2010CB 732400). Copyright © 2011, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(10)60447-1
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pg. Often, besides testing for a substance, testing for its derivatives and degradation products is also involved. Thus, it is required that the testing method be very accurate, quick, and convenient. Given the advantages of vast target molecules, strong specificity, high sensibility, low testing cost, safety, and reliability, aptamers are quite promising for applications in food safety testing. The paper presents a comprehensive description of the SELEX principle, the aptamer selection technology, and its growing application in food safety analyses.
2
General principle of SELEX
The general aim of the SELEX technology is to finally obtain the target aptamer[11] through several rounds of selections and exponential enrichment in the random ologonucleotide library (about 1015–1018 kinds of nucleic acid molecules), which is designed and synthesized according to the combinatory chemistry principle. As the aptamer selection technology, the SELEX course starts from the random ologonucleotide library (RNA or DNA) synthesized according to the combinatory chemistry principle. The nucleic acid molecules in the library are structurally characterized in that the two ends are fixed sequences and the middle is the random sequence. The random sequence (section) provides each nucleic acid molecule with different space structures and determines the capacity and diversity of the random ologonucleotide library. If the random sequence of ologonucleotide is composed of n bases theoretically, the library capacity will be 4n. If the artificial modification library is taken into consideration as well, the random sequence will be more diverse[6,12]. The SELEX technology is one of the molecular evolution engineering technologies and its technical process includes three courses similar to the Darwin evolution theory: spontaneous mutation, natural selection, and large proliferation[13]. The basic aptamer selection process with the SELEX technology is shown in Fig.1[6] and mainly consists of the following steps[14–16]: (1) Design and synthesize the largecapacity (1015–1018) single-stranded random oligonucleotide (DNA or RNA) sequence library with the molecular biology technology. Generally, the length of the sequence is around 15–60 bases. Binding sites are used for PCR proliferation and relevant primers of other enzymatic reaction. The random library is warmly bred with target molecules under the specified buffer system; (2) Separate the nucleic acid sequence binding target molecules from the sequence not binding target molecules with the specified isolation methods, e.g. centrifugation, membrane filtration, magnetic cell sorting, capillary electrophoresis, and column chromatography; (3) Conduct the reverse selection of the binding nucleic acid sequence isolated to eliminate the nonspecific binding nucleic acid sequence; (4) Conduct the PCR proliferation for the specific binding nucleic acid sequence acquired to form the
Fig.1 In vitro selection of target-specific aptamers using SELEX technology[6]
primary nucleic acid library. Repeat Step (2) and (3) with the sub-first level nucleic acid library generated. As conditions for each round of selection improve continuously, DNA or RNA molecules highly specifically binding target molecules increase exponentially and the sequences with low affinity are eliminated gradually until the dissociation constant of the nucleic acid library to target molecules reaches the target value; and (5) Obtain the clone-sequencing of the nucleic acid sequence acquired to appraise and test the binding specificity and the affinity of the sequence with its target molecules.
3
SELEX procedure modifications
The SELEX technology is characterized in that the molecule evolution course is transferred and conducted in vitro. However, in the actual selection of aptamer, SELEX is often limited by the selection environment and the technology itself, which impairs the selection result and efficiency. Thus, researchers have developed many different SELEX technologies in recent years for different purposes on the basis of the traditional SELEX technology, which have greatly promoted the application and development of the aptamer technology in various fields. 3.1
Capillary electrophoresis SELEX (CE-SELEX)
For the SELEX technology, it is crucial to isolate the target molecule-bound nucleic acid from the dissociating nucleic acid, which is also a difficulty to for development of the typical SELEX technology. Based on the fact that aptamers can bind target molecules to change their conformation and mass and thus their electrophoresis action obviously, Mendonsa and Bowser (2004) isolated[17] two kinds of nucleic acid molecules at different states by the capillary electrophoresis (CE) technology effectively. Application of
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CE has significantly promoted the SELEX selection efficiency, as it can dissociate the target molecule-aptamer compound from the never-binding DNA library rapidly and efficiently. With the CE-SELEX method, specific aptamers of target molecules will be available only after 2–4 rounds of selections. Based on ECEEM (equilibrium capillary electrophoresis of equilibrium mixtures), Drabovich et al (2005) acquired the aptamer recognizing the MutS protein only after three rounds of selections by making use of the aptamer mobility difference and collecting equilibrium mixtures at different time phases. However, a major defect of the CE-SELEX technology is the small sample injection volume (nL Level), which limits the number of DNA in the initial library. Thus, compared with the traditional SELEX technology, only libraries with 1013 DNA molecules are used for selection generally. The CE-SELEX technology greatly promotes the SELEX selection efficiency and shortens the SELEX selection course, and thus will become one of the major means for aptamer selection in the future[18]. 3.2
Tailored SELEX
Generally, the artificially synthesized oligonucleotide sequence consists of a random section in the middle and a fixed section of 15–25 nt known sequences at either end. Although the fixed section is convenient for PCR proliferation and in vitro transcription, but it is often involved in binding of aptamer and target molecules, which affects the function of later truncated active sequences. In addition, the fixed section may also be involved in annealing of the random section in the middle. Vater et al[19] established the tailored SELEX technology to overcome the aforementioned shortcomings and acquire RNA sequences binding target molecules directly and rapidly without any further truncation experiments. Its principle is to establish the random library, either end of which includes 4nt and 6nt fixed sequences to be connected with linker sequences. The single-stranded linking sequence at either end of the synthesized library consists of the T7 promoter sequence, bases capable of alkaline lysis, and sequences convenient for library proliferation. The synthesized single-stranded linker sequences are capable of annealing with the above linking sequences and fixed sequences of the library to form the bridge used for PCR proliferation. Thus, only the random library is involved in selection and the linking sequences and linker sequences at two ends are added in the means of linking and bridging after selection. After the PCR proliferation, sequences at two ends are removed through alkaline lysis and enter the next round of selection. Compared with traditional selection, the two steps of primer linking and primer removal through alkaline lysis after the PCR proliferation are added, and still it is the truncated fixed ologonucleotide sequence that enters the next circulation. With the tailored selection, short aptamers with an
extremely high affinity will be selected. Moreover, it is applicable in the automatic selection system. 3.3
Primer-free genomic SELEX
In 1999, Singer[20] and Gold[21] made the oligonucleotide library from their interested biological genes based on the SELEX technology principle, and the natural recognition sequences for bioactive molecules like protein, co-factor, amylase, and antibiotic can be selected from the oligonucleotide library. The genomic SELEX established by them provides a large number of experimental data to settle the problems of gene regulation and metabolism inside the cell[22]. As the primer sequences for PCR proliferation in genomic SELEX may match the middle genomic sequences, they interfere in binding of targets and genomic sequences. Wen et al[23] developed the primer-free genomic SELEX method in combination with the tailored SELEX principle. According to the method, primers are removed first from the genomic library before selection and then the selected gene fragments are added into the primer sequences for PCR proliferation through the hybridization-extension thermal circulation after the library and the target molecules are incubated. The primer-free genomic SELEX offers a new platform technology for research of genomic regulation. 3.4
Subtractive SELEX
In 2003, Wang et al[24] established the subtractive SELEX. According to its principle, nucleic acid molecules that can share known or unknown target molecules are eliminated first, and then the subtractive sub-level random library is put into solution containing target molecules for selection of specific aptamers. With the subtractive SELEX technology, specific aptamers of differentiated target molecules can be acquired by screening two groups of highly congenetic target molecule mixtures. The subtractive technology has its unique advantages in clinical diagnosis, target therapy and individualized tumor therapy. 3.5
Toggling SELEX
Toggling SELEX uses target proteins of different species to select the species crossing reactive aptamer. It is an improved SELEX technology developed by White et al (2001)[25]. They first selected the human thrombin and porcine thrombin mixture as the targets to screen the random RNA libraries, and then the enriched libraries were screened for human thrombin and porcine thrombin in round. After 13 rounds of selections, the aptamer[25] that is able to specifically bind both human thrombin and porcine thrombin was available. As human protein is often used in clinical drug research and development, but the animal model experiment has to be carried out during
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the activity testing, the crossing reactive aptamer will facilitate the preclinical evaluation of drug molecules on the animal model. In addition, for certain kinds of proteins, the conservative structure domain between species is often necessary for the proteins to exert their functions. With toggling SELEX, aptamers specific to the structure domain are available, so the functional activity of aptamers can be promoted. In addition, the method is also applicable to selection of aptamers for receptor family protein, ligand family protein, or structure-related protein targets, which have redundant or overlapping functions within the same species. 3.6
Expression cassette SELEX
When aptamers are applied in gene therapy, transfecting mammalian cells of the nucleic acid sequences have to undergo in vitro transcription. If the aptamer is inserted into tRNA expression box for transcription to generate chimeric tRNA, the transcriptive side-wing sequences often affect the space structure of the aptamer and thus reduce the biological activity of the aptamer. To eliminate the disadvantage, Martell et al (2002) inserted the aptamer of transcriptive factor E2F selected in the in vitro screening into tRNA, introduced the side wing into random section, and then selected sequences binging with E2F through in vitro selection with the SELEX flow. Thus, not only will the chimeric tRNA express the high-level aptamer in vitro, but it will also keep the aptamer activity. The expression box SELEX thus established laid foundation[26] for aptamer-based gene therapy. 3.7
FluMag SELEX
Bruno (1997)[27] selected chloroaromatic aptamers by first applying the affinity separation method based magnetic cell into selection. In 2003, when selecting aptamers for the hypothyroid transcriptive factor, Murphy et al[28] marked TTF1 with His-6 and then fixed it with the Ni-NTA magnetic cell. Thus, aptamers binding infectants can be reduced in selection. In 2005, Stoltenburgv et al[29] introduced fluorescent labeling for quantifying DNA and fixed target molecules with magnetic particles to facilitate separation, and thus established FluMag-SELEX. They established the method by taking streptavidin aptamer selection as an example. According to its basic procedure, streptavidin (as target molecules) is first covered with magnetic cells, and then incubated with the nucleic acid library; the unbinding nucleic acid is dissociated and removed, the binding nucleic acid is washed out, proliferation is conducted with the fluorescent labeling primer, polyacrylamide electrophoresis is carried out, and the normal stranded is reclaimed as the library for the next round of selection. As fluorescent quantification is adopted in FluMag-SELEX, isotope does not have to be used and the
amount of nucleic acid binding target molecules can be known directly; moreover, a small number of target molecules are used to reduce the cost of commercial purified target protein; the binding nucleic acid and the nonbinding nucleic acid can be separated rapidly and efficiently; and the operation is simple. These advantages of the FluMag-SELEX technology indicate that it is quite suitable for aptamer selection. 3.8
Automated SELEX
In 1998, Cox et al[30] created the first automatic selection working station based on the Beckmann Biomek 2000 Pipetting system and acquired the lysozyme aptamer successfully. The working station consists of a mechanical console, a thermal circulating instrument, an automatic magnetic cell separator, a multiscreen vacuum filter, an enzyme cooler, and an automatic liquid transfer tool. The selection flow includes: fixing the biotin target protein on magnetic cells with the mutual effect of streptavidin and biotin. Then the specifically binding sequences separation, and the RT-PCR proliferation and transcription are finished automatically with the set programs. Finally, the selected sequences are cloned to carriers for sequence testing and appraisal. With the automatic selection working station, the author finished 12 rounds of selections within two days. However, purified protein is required as the target by the automatic selection, which limits its application in proteomics[30]. Thus, Cox et al (2002)[31] made further improvement. They used the method of vitro gene transcription and encoding directly to generate the target protein. Then two cis-form mRNAs are transcripted and generated by introducing the T7 promoter and a BPL biotag at the end 5 of gene and the BPL gene and the T7 terminator at the end 3ƍ. After mRNAs are encoded, the BPL protein and the biotag labeling target protein are generated. When the dissociating biotic is added, the BPL biotag binds the biotic to the target protein in the covalent way, so it is convenient to fix the target protein on the streptavidin-covering magnetic cells. Eventually, the specific aptamer[31] is selected with the automatic selection working station. In 2005, Eulberg et al[32] established a semi-automatic selection platform to select the RNA aptamer of substance P. The system is based on RoboAmp 4200 E and integrates a super-filter, a fluorescence detector, and a self-quantitative PCR instrument, to monitor online the amount of nucleic acid binding target molecules and meet the requirements for selection in different buffer fluids and reagents under other strict selection conditions. These automatic platforms lay a smooth path for high-flux selection of aptamers. 3.9
Allosteric selection
The thought of allosteric selection originates from the
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allosteric ribozyme: the aptamer binds with the hammerhead ribozyme to generate the allosteric ribozyme; when the target molecules bind with the aptamer, the ribozyme conformation will be modified and the ribozyme activity will be partly activated or restrained. With the allosteric selection, structure domains related to aptamer are randomized and selection is carried out with the catalyzed activity of ribozyme to obtain the allosteric ribozyme responding to effectors. On the basis of the activity of the ribozyme lysis of substrate sequences, separation[33] of the binding aptamer from the dissociating aptamer can be realized easily. For example, Soukup et al (2000)[34] integrated the theophylline aptamer selected in vitro with the hammerhead ribozyme and the linking section of the aptamer and the ribozyme were randomized. Then, the theophylline was used as the target for second selection to obtain the ribozyme specific to theophylline. When the aptamer binds with the theophylline, the space structure of the linking section is modified to activate the ribozyme activity. After the linking section sequences are acquired, if the theophylline aptamer sequence is mutated randomly, it is quite easy to obtain the allosteric ribozyme[34] responding to the selected theophylline analogue. 3.10
Deconvolution selection
Generally, aptamer selection uses pure protein and low-molecular-mass pure matter as targets, while complex target SELEX uses several types of molecules as targets simultaneously to select the multimolecule aptamer without mutual interference. Morris et al (1998)[35] conducted 25 rounds of selections with the complex target SELEX method to obtain the ssDNA aptamer of the erythrocyte ghost, and proved through a photoaffinity crosslinking experiment that target molecules recognized by different aptamer groups are different. In addition, they applied Deconvolution SELEX to select aptamers for each target; the aptamer libraries acquired in the 25 rounds of selections mentioned earlier were used for PCR proliferation; ssDNA was purified and separated through PAGE, and then crosslinked and bound with complex targets; then products of the SDS-PAG separation and binding were transferred to the NC membrane, the crosslinking products were tested with autoradiography, and the appropriate Western blot band on the NC membrane was cut down and applied directly for PCR proliferation, so as to select and sequence the pure DNA molecules. Similar reports are also seen in selection[36,37] of complex target aptamers of human plasma and Matrix Gla Protein (MGP).
4
Application of aptamers in food safety
As the material living conditions are being improved continuously, people pay more and more attention to influences of poisonous substances on human health. However,
there are various types and components of poisonous and harmful substances discharged as industrial waste and the tested mass is extremely low. Existing analysis methods for some poisonous and harmful substances cannot meet the requirement for analysis of the existing poisonous substances, so it is necessary to develop new methods to test the poisonous and harmful substances. In virtue of the three dimensional structures, like hairpin, pseudoknot, bulge loop, and G-quartet, formed by the hydrogen bond, van Der Waals Force, hydrophobic interaction, and other molecular interactions, aptamers can identify the target specifically. Aptamers have many advantages: (1) Wide range of target molecules. The target molecules cover low-molecular-mass substances, including ATP, amino acid, nucleotide, and metal ion, and biological high-molecular-mass substances, including enzyme, growth factor, and cell adhesion molecules, and even complete virus, bacteria, and cell; (2) Strong affinity. Aptamers have strong binding strength with target molecules. The dissociation constant (Kd) can reach the ȝM–pM level; (3) High specificity. Aptamers can identify small changes of a cymene or hydroxyl of a target; (4) Convenient preparation and modification. Various necessary DNA sequences can be synthesized in vitro rapidly and flexibly with the chemical synthesis method. Moreover, exact site modification, such as fluorescent labeling and biotin labeling, can be realized. The biochemical characteristics of nucleic acid itself enable it to play a very important role in analysis of poisonous and harmful substances. 4.1
Inorganic components
As aptamers can identify some special ions, such as K+, Pb2+, and Hg2+, and form the specific secondary structure, heavy metallic sensors designed with aptamers are often used as important means for environment analysis and monitoring. Huang et al[38] used G-quadruplex formed by a section of ATP-binding aptamer to detect K+. G-quadruplex is a series of guanine stabilized by the interaction between hydrogen bonds and ions, so the folding degree of an aptamer relates much to the K+ content. The K+ sensor designed with aptamers can be used directly to measure the K+ content in human urine, which is used as an important index of kidney diseases. Liu et al[39] used the biological sensor formed by folding the ion-dependent aptamer to measure specifically the content of Pb2+ and Hg2+ in soil and pond samples. Its basic principle is that Hg2+ can induce a specific aptamer to form the hairpin structure and Pb2+ will make a section of specific thrombin recognizing aptamer being folded to form G-quadruplex. Metallic elements based on aptamer testing also include Zn2+ [40] and Ni2+ [41]. Ling et al[42] from Guangxi Normal University used the aptamer modified gold nanoparticle resonance scattering spectrum probe to measure the content of Pb2+, and the measuring limit is 0.03 nM.
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4.2
Biotoxins [43]
Tang et al adopted the SELEX technology to select the aptamer specifically recognizing the ricin target molecules from the random ologonucleotide library. The capillary electrophoresis technology as the separation means was introduced into the SELEX selection. The highly-efficient separation capability with the capillary electrophoresis technology reduced the selection period greatly. As the enzyme-linked immunity and dot blotting experiments rounded out, the ologonucleotide aptamer specifically recognizing the ricin target molecules can be obtained just after four rounds of selections. Tang et al[44] established the abrin analysis method based on the DNA aptamer technology. Its testing limit was 1 nM and the linear scope was 1–400 nM. Jiang et al[45] used the SELEX technology to select the aptamer specific to SEB. After 13 rounds of selections, the binding percentage of ssDNA library and SEB rises from 1.1% to 39.8%. The aptamer obtained had the strong specificity to SEB and had no specific binding with SPA and BSA. Jorge et al[46] applied the aptamer in testing of food mycotoxin and selected the aptamer of mycotoxin. Moreover, the ng-level mycotoxin can be tested in the wheat sample.
Table 1 Examples of antibiotics targets used for aptamer selections Target for aptamer selection
Type of aptamer
Determination limit
References
Kanamycin A Kanamycin B Streptomycin Neomycin Tobramycin Lividomycin Moenmycin A Chloramphenicol Tetracycline
RNA RNA RNA RNA RNA RNA RNA RNA RNA
< 300 nM 180 nM Not specified 100 nM 2–3 nM < 300 nM 300–400 nM 25–65 μM 1 μM
[42] [43] [44] [45] [46] [42, 47] [48] [49] [50]
Chemicals Fig.2 Specificity of aptasensors of tetracyclines
4.3
Antibiotics
Discovery and application of antibiotics is a great revolution and boon to mankind. Since then, humans have been provided with a strong weapon to battle with death. However, vast use and abuse of antibiotics has brought various poisonous side effects to human beings. There is ongoing research on new antibiotics. The SELEX technology is also widely used in analysis and testing of various antibiotics such as Kanamycin[47], Kanamycin B[48], Streptomycin[49], Neomycin[50], Tobramycin[51], [47,52] [53] Lividomycin , Moenmycin A , Chloramphenicol[54], and [55,56] Tetracycline . Details about the analysis are shown in Table 1. Kim (2010)[57] made the Tetracycline-electrochemical biosensor with the Tetracycline aptamer selected in their foregoing screening as the molecule recognition element. The specific binding of aptamer to Tetracycline is shown in the sensor signal output, which is indicated by the remarkable electric current reduction along with the increase of the Tetracycline content. The minimum testing limit for Tetracycline with the method is 10 nM and the linear testing scope is between 10 nM and 10 ȝM. As shown in Fig.2, in the aptamer electrochemical analysis of Tetracycline and other three structural analogs, there are remarkable differences between the responses to electric current changes by Tetracycline and the other three analogs, which indicate that the specificity of the Tetracycline aptamer-electrochemical biosensor to Tetracycline is quite ideal in the analysis.
TET: Tetracycline; OTC: Oxytetracycline; DOX: Doxycycline; DCF: Diclofenac
4.4
Organic dyes
Since the SELEX technology was proposed in 1990, it has been applied in the analysis of organic dyes. The testing limit with the DNA aptamer is 100–600 μM[3], while the testing limit with the RNA aptamer is 33–46 μM[58]. According to the report by Grate and Wilson[59], the limit for testing the malachite green with the RNA aptamer could reach 1μmol/L. The testing limit was 190 nM for testing Sulforhodamine B with the DNA aptamer by Wilson and Szostak[60]. Brochstedt et al[61] studied the aptamer testing technology for 4,4’-Methylenedianiline, and the testing limit was 0.15–15 μM. Sara et al[62] conducted a semiquantitive testing of the malachite green inside the fish texture and the concealed malachite green. As it rounded out, the RNA aptamer could be used to replace the antibody and applied for testing for chemical residuals in food. Application of the SELEX technology in the industrial dye testing developed continuously and the testing sensitivity is basically superior to that of the liquid tandem mass spectrum method. 4.5
Agonists
Dope is a preparation to activate or enhance the central nervous system activity and includes benzedrine, cocaine, caffeine, other xanthine drugs, nicotinamide drugs, and synthetic anorexics. Doping will produce many direct hazards
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to human physical and mental health. Use of different kinds or different dosages of dopes will result in different degrees of harm to human body. Application of SELEX in dope analysis is becoming popular. In 1997 Mannironi et al[63] reported the research on dopamine with aptamer, whose testing limit could reach 2.8 μM. There are many research reports on the application of SELEX in cocaine analysis. Baker et al (2006) established an electrochemical biosensor to test cocaine. They assembled an MB labeling aptamer on a gold electrode surface of ~1 mm2. Without cocaine, the aptamer formed a double-stranded arm and MB at the end point was away from the gold electrode; with cocaine, the aptamer would form three double-stranded arms and MB will approach the gold electrode to generate the electric signal variation and serve the purpose of qualitative and quantitative cocaine testing[64]. Recently, an electrically excited chemical luminescent aptamer sensor has been developed successfully to test cocaine[65]. According to its principle, a hydrosulphonylmodified cocaine aptamer undergoes Ru(bpy)2(dcbpy)NHS(Fc) modification again and is fixed on the gold particle surface; the gold particles and the gold electrode are connected via a rigid nucleic acid segment. Without cocaine, the aptamer can form a segment of double-stranded section to make Fc away from the gold particle surface and no chemical luminescent signal is produced. After cocaine is added, the aptamer will form three double-stranded sections to make Fc close to the gold particle surface and chemical luminescent signals are produced. The chemical luminescent signals keep a record of constant pressure in the case of 0.1 M tripropylamine. The cocaine testing limit can reach 1 nM. The method provides important reference for high-sensitivity testing of low-molecular-mass substances. Zhang et al (2008) cut the cocaine-recognizing aptamer into two segments. With the existence of cocaine, the two segments can be reassembled into the functional cocaine aptamer and form the complex with cocaine. If gold nanoparticles and a salt solution of certain concentration are added, the gold nanoparticles will gather and their color will change. Without the existence of cocaine, the gold nanoparticles will not gather easily and still keep red[66] after the gold nanoparticles and the certain-concentration salt solution are added, as the nitrogenous bases are fully exposed in the double-segment DNA molecules. 4.6
Microorganisms in food
Bhunia et al[67] conducted the specific testing of Listeria in foods such as beef and chicken by using an aptamer and an antibody-functional optical fiber biosensor. The lower testing limit was 103 CFU mL–1. Hye et al[68] established a method to test E. coli with an aptamer and RT-PCR. The linear scope was 101-107 CFU mL–1 and the lower testing limit was 10 E.coli mL–1. E. Torres-Chavolla and E.C. Alociljac[69] have
made the comprehensive retrospect and prospect for application of aptamer biosensors in microorganism testing. Since SELEX appeared, some oligonucleotide aptamers aiming at virus, bacteria, and other pathogen active proteins have been selected, which open new ways for diagnosis and control of epidemic diseases. It is believed that the SELEX technology will play a more important role in the rapid testing of pathogen microorganisms in the near future.
5
Future prospects
As primary molecule recognizing elements, aptamers have a specific recognition capability and a strong binding force to its targets. They offer many advantages, such as low molecular mass, simplicity for synthesis and modification, and relative stability, so they have wide application. It is especially notable that aptamers have the high recognition and binding capability to low-molecular-mass substances as well, which enables separation, purification, analysis, and testing of low-molecular-mass substances. As Wang et al[70] pointed out, although research on aptamers and their selection methods are still at the starting stage, their application fields have developed from testing sensors to the aptamer drug Macuge used to cure the wet type senile macular degeneration. Currently, application of the aptamer technology has just started in the field of analysis of low-molecular-mass substances. There is much to be developed and studied deeply, which can be summarized into the following three aspects. First, the molecular characteristics of aptamers have to be improved. Currently, research on this aspect mainly focuses on the aptamer stability enhancement, e.g. 2-Aminopyrimidine modification, 2-fluoropyrimidine modification, 2-Dimethoxy nucleotide modification, Spiegelmer, and locked nucleic acid. As the aptamer molecule stability is enhanced, the in vivo and in vitro application space of aptamer will be expanded, especially the resistance of low-molecular-mass substances to the complex systems or adverse environments during the analysis and testing. In the future, improvement of the molecular characteristics of aptamers may be in terms of their resistance to ion intensity, temperature, acidity-alkalinity, and organic reagents. Second, aptamers have to incorporated in other analysis technologies. Aptamer molecules are quite small and can be modified and fixed easily. After aptamers bind with target molecules, their space structure may be changed remarkably. The structural and functional characteristics of aptamers may be the ideal molecule recognizing element used for development of biosensors and other analysis technologies. Currently, aptamers are incorporated in many technologies and methods to expand the scope of analysis, e.g. fluorescence technology, nano technology, surface enhanced Raman scattering (SERS), surface plasmon resonance (SPR), magnetic resonance imaging (MRI), affinity chromatography,
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capillary electrophoresis, colorimetry, and electrochemistry. Aptamers are currently an important subject of research, and therefore, in the future, there surely will be more technologies and methods to be integrated with aptamers and hence, they will have a very wide application A general approach to tailoring aptamer sequences into functional subunits to design target-induced light-switching excimer sensors for rapid, sensitive, and selective detection of important molecules in complex biological fluids has been developed[71]. In the presence of target molecules, two aptamer fragments are induced to self-assemble to form aptamer-target complex and bring two pyrene molecules in close proximity to form an excimer, resulting in fluorescent switching from ~400 nm to 485 nm. With an anti-cocaine sensor, as low as 1 ȝM of cocaine can be detected using steady-state fluorescence assays and more importantly low picomole levels of target can be directly visualized with naked eyes. Because the excimer has a long fluorescence lifetime, time-resolved measurements were used to directly detect as low as 5 ȝM cocaine in urine samples quantitatively without any sample pretreatment[71]. In addition, research on the molecular mechanism for binding of aptamers to low-molecular-mass substances will also be a subject in the future. The molecular recognition mechanism has always been an important subject of research. Although they have a simple structure, aptamers can bind closely with vast target molecules, which undoubtedly makes them an ideal research model to study molecular interactions and may even offer the reference for the target of ligand molecule “customization.”
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Cite this article as: Chin J Anal Chem, 2011, 39(6), 925–933.
REVIEW
Application of Aptamers in Food Safety XU Dun-Ming1, WU Min1, ZOU Yuan2, ZHANG Qiang3, WU Cui-Chen2, ZHOU Yu1,*, LIU Xian-Jin3 1
Xiamen Entry-exit Inspection and Quarantine Bureau, Xiamen 361026, China College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China 3 Institute of Food Quality Safety and Detection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China 2
Abstract: Aptamers are all new ligands with high affinity for considerably diverse molecules ranging from large targets such as proteins, peptides, and complex molecules to drugs and small organic molecules as well as metal ions. These molecules are identified and selected through an in vitro process called SELEX (systematic evolution of ligands by exponential enrichment). Aptamers are widely used in different fields, including medical and pharmaceutical basic research, drug development, diagnosis, and therapy. Analytical and separation tools involving aptamers as molecular recognition and binding elements are another big field of application. The SELEX method has been improved over the last two decades in different ways – it has become more efficient and less time consuming and higher affinities of the aptamers and automation of the process have been achieved. In this review, a general background introduction, general principle of the SELEX, the development of aptamers by use of SELEX, and the application of aptamers in food safety are presented. Key Words: Aptamers; Systematic evolution of ligands by exponential enrichment; Food safety; Residue; Small organic molecules
1
Introduction
It is a milestone[1,2] in the history of molecular biology that the DNA double helix structure was proposed in 1953, and it marked the birth of modern molecular biology. Since then, research on large biological molecules such as nucleic acid has entered a brand-new era. In 1990, two independent research teams published their research papers on in vitro selection of the special target bound nucleic acid in Nature[3] and Science[4] respectively and named the nucleic acid “aptamer,” which combines the Latin word “aptus”(which means matching in English) and the Greek suffix “meros” and is translated into “Shiti,” “Shipeizi,” or “Hesuan Shiti” in Chinese. The aptamer selection technology SELEX[4], systematic evolution of ligands by exponential enrichment, is an integrated technology based on the combinatorial chemistry technology, the PCR technology and the gene clone sequencing technology, and so on. As the molecular biological technology has developed rapidly in recent years, the SELEX
technology has been optimized and many aptamer selection technologies[5,6] for different application purposes have been developed, which have greatly induced the rapid development of aptamer science. After a full 20 years of development, aptamer science has become incredibly sophisticated and its application in biology and iatrology has attracted great interest, e.g. molecular recognition[7,8], drug selection[7,8], target appraisal[8,9], gene expression regulation[9], medical imaging[9], clinical diagnosis[9,10], and disease curing[10]. Moreover, as a kind of target material bound molecules, aptamers also show great potential for application in the field of analytical science, especially in isolation and analysis of small molecular compounds. Food safety has been a matter of significant concern for the whole world in the 21st century, as it greatly affects the physical health of consumers. Since there are various types of food, many kinds of poisonous substances are found to be present in food. The amount of the substance present is extremely small and is usually in the range of ȝg, ng, or even
Received 26 December 2010; accepted 20 March 2011 * Corresponding author. Email: [email protected]; [email protected] This work was supported by the Science and Technology Foundation of Xiamen, China (No. 3502Z20092008), the Nature Science Foundation of Fujian, China (No.2008J0107), the Science and Technology Foundation of AQSIQ, China (No. 2010IK150), and the 973 Programme of China (2010CB 732400). Copyright © 2011, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(10)60447-1
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pg. Often, besides testing for a substance, testing for its derivatives and degradation products is also involved. Thus, it is required that the testing method be very accurate, quick, and convenient. Given the advantages of vast target molecules, strong specificity, high sensibility, low testing cost, safety, and reliability, aptamers are quite promising for applications in food safety testing. The paper presents a comprehensive description of the SELEX principle, the aptamer selection technology, and its growing application in food safety analyses.
2
General principle of SELEX
The general aim of the SELEX technology is to finally obtain the target aptamer[11] through several rounds of selections and exponential enrichment in the random ologonucleotide library (about 1015–1018 kinds of nucleic acid molecules), which is designed and synthesized according to the combinatory chemistry principle. As the aptamer selection technology, the SELEX course starts from the random ologonucleotide library (RNA or DNA) synthesized according to the combinatory chemistry principle. The nucleic acid molecules in the library are structurally characterized in that the two ends are fixed sequences and the middle is the random sequence. The random sequence (section) provides each nucleic acid molecule with different space structures and determines the capacity and diversity of the random ologonucleotide library. If the random sequence of ologonucleotide is composed of n bases theoretically, the library capacity will be 4n. If the artificial modification library is taken into consideration as well, the random sequence will be more diverse[6,12]. The SELEX technology is one of the molecular evolution engineering technologies and its technical process includes three courses similar to the Darwin evolution theory: spontaneous mutation, natural selection, and large proliferation[13]. The basic aptamer selection process with the SELEX technology is shown in Fig.1[6] and mainly consists of the following steps[14–16]: (1) Design and synthesize the largecapacity (1015–1018) single-stranded random oligonucleotide (DNA or RNA) sequence library with the molecular biology technology. Generally, the length of the sequence is around 15–60 bases. Binding sites are used for PCR proliferation and relevant primers of other enzymatic reaction. The random library is warmly bred with target molecules under the specified buffer system; (2) Separate the nucleic acid sequence binding target molecules from the sequence not binding target molecules with the specified isolation methods, e.g. centrifugation, membrane filtration, magnetic cell sorting, capillary electrophoresis, and column chromatography; (3) Conduct the reverse selection of the binding nucleic acid sequence isolated to eliminate the nonspecific binding nucleic acid sequence; (4) Conduct the PCR proliferation for the specific binding nucleic acid sequence acquired to form the
Fig.1 In vitro selection of target-specific aptamers using SELEX technology[6]
primary nucleic acid library. Repeat Step (2) and (3) with the sub-first level nucleic acid library generated. As conditions for each round of selection improve continuously, DNA or RNA molecules highly specifically binding target molecules increase exponentially and the sequences with low affinity are eliminated gradually until the dissociation constant of the nucleic acid library to target molecules reaches the target value; and (5) Obtain the clone-sequencing of the nucleic acid sequence acquired to appraise and test the binding specificity and the affinity of the sequence with its target molecules.
3
SELEX procedure modifications
The SELEX technology is characterized in that the molecule evolution course is transferred and conducted in vitro. However, in the actual selection of aptamer, SELEX is often limited by the selection environment and the technology itself, which impairs the selection result and efficiency. Thus, researchers have developed many different SELEX technologies in recent years for different purposes on the basis of the traditional SELEX technology, which have greatly promoted the application and development of the aptamer technology in various fields. 3.1
Capillary electrophoresis SELEX (CE-SELEX)
For the SELEX technology, it is crucial to isolate the target molecule-bound nucleic acid from the dissociating nucleic acid, which is also a difficulty to for development of the typical SELEX technology. Based on the fact that aptamers can bind target molecules to change their conformation and mass and thus their electrophoresis action obviously, Mendonsa and Bowser (2004) isolated[17] two kinds of nucleic acid molecules at different states by the capillary electrophoresis (CE) technology effectively. Application of
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CE has significantly promoted the SELEX selection efficiency, as it can dissociate the target molecule-aptamer compound from the never-binding DNA library rapidly and efficiently. With the CE-SELEX method, specific aptamers of target molecules will be available only after 2–4 rounds of selections. Based on ECEEM (equilibrium capillary electrophoresis of equilibrium mixtures), Drabovich et al (2005) acquired the aptamer recognizing the MutS protein only after three rounds of selections by making use of the aptamer mobility difference and collecting equilibrium mixtures at different time phases. However, a major defect of the CE-SELEX technology is the small sample injection volume (nL Level), which limits the number of DNA in the initial library. Thus, compared with the traditional SELEX technology, only libraries with 1013 DNA molecules are used for selection generally. The CE-SELEX technology greatly promotes the SELEX selection efficiency and shortens the SELEX selection course, and thus will become one of the major means for aptamer selection in the future[18]. 3.2
Tailored SELEX
Generally, the artificially synthesized oligonucleotide sequence consists of a random section in the middle and a fixed section of 15–25 nt known sequences at either end. Although the fixed section is convenient for PCR proliferation and in vitro transcription, but it is often involved in binding of aptamer and target molecules, which affects the function of later truncated active sequences. In addition, the fixed section may also be involved in annealing of the random section in the middle. Vater et al[19] established the tailored SELEX technology to overcome the aforementioned shortcomings and acquire RNA sequences binding target molecules directly and rapidly without any further truncation experiments. Its principle is to establish the random library, either end of which includes 4nt and 6nt fixed sequences to be connected with linker sequences. The single-stranded linking sequence at either end of the synthesized library consists of the T7 promoter sequence, bases capable of alkaline lysis, and sequences convenient for library proliferation. The synthesized single-stranded linker sequences are capable of annealing with the above linking sequences and fixed sequences of the library to form the bridge used for PCR proliferation. Thus, only the random library is involved in selection and the linking sequences and linker sequences at two ends are added in the means of linking and bridging after selection. After the PCR proliferation, sequences at two ends are removed through alkaline lysis and enter the next round of selection. Compared with traditional selection, the two steps of primer linking and primer removal through alkaline lysis after the PCR proliferation are added, and still it is the truncated fixed ologonucleotide sequence that enters the next circulation. With the tailored selection, short aptamers with an
extremely high affinity will be selected. Moreover, it is applicable in the automatic selection system. 3.3
Primer-free genomic SELEX
In 1999, Singer[20] and Gold[21] made the oligonucleotide library from their interested biological genes based on the SELEX technology principle, and the natural recognition sequences for bioactive molecules like protein, co-factor, amylase, and antibiotic can be selected from the oligonucleotide library. The genomic SELEX established by them provides a large number of experimental data to settle the problems of gene regulation and metabolism inside the cell[22]. As the primer sequences for PCR proliferation in genomic SELEX may match the middle genomic sequences, they interfere in binding of targets and genomic sequences. Wen et al[23] developed the primer-free genomic SELEX method in combination with the tailored SELEX principle. According to the method, primers are removed first from the genomic library before selection and then the selected gene fragments are added into the primer sequences for PCR proliferation through the hybridization-extension thermal circulation after the library and the target molecules are incubated. The primer-free genomic SELEX offers a new platform technology for research of genomic regulation. 3.4
Subtractive SELEX
In 2003, Wang et al[24] established the subtractive SELEX. According to its principle, nucleic acid molecules that can share known or unknown target molecules are eliminated first, and then the subtractive sub-level random library is put into solution containing target molecules for selection of specific aptamers. With the subtractive SELEX technology, specific aptamers of differentiated target molecules can be acquired by screening two groups of highly congenetic target molecule mixtures. The subtractive technology has its unique advantages in clinical diagnosis, target therapy and individualized tumor therapy. 3.5
Toggling SELEX
Toggling SELEX uses target proteins of different species to select the species crossing reactive aptamer. It is an improved SELEX technology developed by White et al (2001)[25]. They first selected the human thrombin and porcine thrombin mixture as the targets to screen the random RNA libraries, and then the enriched libraries were screened for human thrombin and porcine thrombin in round. After 13 rounds of selections, the aptamer[25] that is able to specifically bind both human thrombin and porcine thrombin was available. As human protein is often used in clinical drug research and development, but the animal model experiment has to be carried out during
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the activity testing, the crossing reactive aptamer will facilitate the preclinical evaluation of drug molecules on the animal model. In addition, for certain kinds of proteins, the conservative structure domain between species is often necessary for the proteins to exert their functions. With toggling SELEX, aptamers specific to the structure domain are available, so the functional activity of aptamers can be promoted. In addition, the method is also applicable to selection of aptamers for receptor family protein, ligand family protein, or structure-related protein targets, which have redundant or overlapping functions within the same species. 3.6
Expression cassette SELEX
When aptamers are applied in gene therapy, transfecting mammalian cells of the nucleic acid sequences have to undergo in vitro transcription. If the aptamer is inserted into tRNA expression box for transcription to generate chimeric tRNA, the transcriptive side-wing sequences often affect the space structure of the aptamer and thus reduce the biological activity of the aptamer. To eliminate the disadvantage, Martell et al (2002) inserted the aptamer of transcriptive factor E2F selected in the in vitro screening into tRNA, introduced the side wing into random section, and then selected sequences binging with E2F through in vitro selection with the SELEX flow. Thus, not only will the chimeric tRNA express the high-level aptamer in vitro, but it will also keep the aptamer activity. The expression box SELEX thus established laid foundation[26] for aptamer-based gene therapy. 3.7
FluMag SELEX
Bruno (1997)[27] selected chloroaromatic aptamers by first applying the affinity separation method based magnetic cell into selection. In 2003, when selecting aptamers for the hypothyroid transcriptive factor, Murphy et al[28] marked TTF1 with His-6 and then fixed it with the Ni-NTA magnetic cell. Thus, aptamers binding infectants can be reduced in selection. In 2005, Stoltenburgv et al[29] introduced fluorescent labeling for quantifying DNA and fixed target molecules with magnetic particles to facilitate separation, and thus established FluMag-SELEX. They established the method by taking streptavidin aptamer selection as an example. According to its basic procedure, streptavidin (as target molecules) is first covered with magnetic cells, and then incubated with the nucleic acid library; the unbinding nucleic acid is dissociated and removed, the binding nucleic acid is washed out, proliferation is conducted with the fluorescent labeling primer, polyacrylamide electrophoresis is carried out, and the normal stranded is reclaimed as the library for the next round of selection. As fluorescent quantification is adopted in FluMag-SELEX, isotope does not have to be used and the
amount of nucleic acid binding target molecules can be known directly; moreover, a small number of target molecules are used to reduce the cost of commercial purified target protein; the binding nucleic acid and the nonbinding nucleic acid can be separated rapidly and efficiently; and the operation is simple. These advantages of the FluMag-SELEX technology indicate that it is quite suitable for aptamer selection. 3.8
Automated SELEX
In 1998, Cox et al[30] created the first automatic selection working station based on the Beckmann Biomek 2000 Pipetting system and acquired the lysozyme aptamer successfully. The working station consists of a mechanical console, a thermal circulating instrument, an automatic magnetic cell separator, a multiscreen vacuum filter, an enzyme cooler, and an automatic liquid transfer tool. The selection flow includes: fixing the biotin target protein on magnetic cells with the mutual effect of streptavidin and biotin. Then the specifically binding sequences separation, and the RT-PCR proliferation and transcription are finished automatically with the set programs. Finally, the selected sequences are cloned to carriers for sequence testing and appraisal. With the automatic selection working station, the author finished 12 rounds of selections within two days. However, purified protein is required as the target by the automatic selection, which limits its application in proteomics[30]. Thus, Cox et al (2002)[31] made further improvement. They used the method of vitro gene transcription and encoding directly to generate the target protein. Then two cis-form mRNAs are transcripted and generated by introducing the T7 promoter and a BPL biotag at the end 5 of gene and the BPL gene and the T7 terminator at the end 3ƍ. After mRNAs are encoded, the BPL protein and the biotag labeling target protein are generated. When the dissociating biotic is added, the BPL biotag binds the biotic to the target protein in the covalent way, so it is convenient to fix the target protein on the streptavidin-covering magnetic cells. Eventually, the specific aptamer[31] is selected with the automatic selection working station. In 2005, Eulberg et al[32] established a semi-automatic selection platform to select the RNA aptamer of substance P. The system is based on RoboAmp 4200 E and integrates a super-filter, a fluorescence detector, and a self-quantitative PCR instrument, to monitor online the amount of nucleic acid binding target molecules and meet the requirements for selection in different buffer fluids and reagents under other strict selection conditions. These automatic platforms lay a smooth path for high-flux selection of aptamers. 3.9
Allosteric selection
The thought of allosteric selection originates from the
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allosteric ribozyme: the aptamer binds with the hammerhead ribozyme to generate the allosteric ribozyme; when the target molecules bind with the aptamer, the ribozyme conformation will be modified and the ribozyme activity will be partly activated or restrained. With the allosteric selection, structure domains related to aptamer are randomized and selection is carried out with the catalyzed activity of ribozyme to obtain the allosteric ribozyme responding to effectors. On the basis of the activity of the ribozyme lysis of substrate sequences, separation[33] of the binding aptamer from the dissociating aptamer can be realized easily. For example, Soukup et al (2000)[34] integrated the theophylline aptamer selected in vitro with the hammerhead ribozyme and the linking section of the aptamer and the ribozyme were randomized. Then, the theophylline was used as the target for second selection to obtain the ribozyme specific to theophylline. When the aptamer binds with the theophylline, the space structure of the linking section is modified to activate the ribozyme activity. After the linking section sequences are acquired, if the theophylline aptamer sequence is mutated randomly, it is quite easy to obtain the allosteric ribozyme[34] responding to the selected theophylline analogue. 3.10
Deconvolution selection
Generally, aptamer selection uses pure protein and low-molecular-mass pure matter as targets, while complex target SELEX uses several types of molecules as targets simultaneously to select the multimolecule aptamer without mutual interference. Morris et al (1998)[35] conducted 25 rounds of selections with the complex target SELEX method to obtain the ssDNA aptamer of the erythrocyte ghost, and proved through a photoaffinity crosslinking experiment that target molecules recognized by different aptamer groups are different. In addition, they applied Deconvolution SELEX to select aptamers for each target; the aptamer libraries acquired in the 25 rounds of selections mentioned earlier were used for PCR proliferation; ssDNA was purified and separated through PAGE, and then crosslinked and bound with complex targets; then products of the SDS-PAG separation and binding were transferred to the NC membrane, the crosslinking products were tested with autoradiography, and the appropriate Western blot band on the NC membrane was cut down and applied directly for PCR proliferation, so as to select and sequence the pure DNA molecules. Similar reports are also seen in selection[36,37] of complex target aptamers of human plasma and Matrix Gla Protein (MGP).
4
Application of aptamers in food safety
As the material living conditions are being improved continuously, people pay more and more attention to influences of poisonous substances on human health. However,
there are various types and components of poisonous and harmful substances discharged as industrial waste and the tested mass is extremely low. Existing analysis methods for some poisonous and harmful substances cannot meet the requirement for analysis of the existing poisonous substances, so it is necessary to develop new methods to test the poisonous and harmful substances. In virtue of the three dimensional structures, like hairpin, pseudoknot, bulge loop, and G-quartet, formed by the hydrogen bond, van Der Waals Force, hydrophobic interaction, and other molecular interactions, aptamers can identify the target specifically. Aptamers have many advantages: (1) Wide range of target molecules. The target molecules cover low-molecular-mass substances, including ATP, amino acid, nucleotide, and metal ion, and biological high-molecular-mass substances, including enzyme, growth factor, and cell adhesion molecules, and even complete virus, bacteria, and cell; (2) Strong affinity. Aptamers have strong binding strength with target molecules. The dissociation constant (Kd) can reach the ȝM–pM level; (3) High specificity. Aptamers can identify small changes of a cymene or hydroxyl of a target; (4) Convenient preparation and modification. Various necessary DNA sequences can be synthesized in vitro rapidly and flexibly with the chemical synthesis method. Moreover, exact site modification, such as fluorescent labeling and biotin labeling, can be realized. The biochemical characteristics of nucleic acid itself enable it to play a very important role in analysis of poisonous and harmful substances. 4.1
Inorganic components
As aptamers can identify some special ions, such as K+, Pb2+, and Hg2+, and form the specific secondary structure, heavy metallic sensors designed with aptamers are often used as important means for environment analysis and monitoring. Huang et al[38] used G-quadruplex formed by a section of ATP-binding aptamer to detect K+. G-quadruplex is a series of guanine stabilized by the interaction between hydrogen bonds and ions, so the folding degree of an aptamer relates much to the K+ content. The K+ sensor designed with aptamers can be used directly to measure the K+ content in human urine, which is used as an important index of kidney diseases. Liu et al[39] used the biological sensor formed by folding the ion-dependent aptamer to measure specifically the content of Pb2+ and Hg2+ in soil and pond samples. Its basic principle is that Hg2+ can induce a specific aptamer to form the hairpin structure and Pb2+ will make a section of specific thrombin recognizing aptamer being folded to form G-quadruplex. Metallic elements based on aptamer testing also include Zn2+ [40] and Ni2+ [41]. Ling et al[42] from Guangxi Normal University used the aptamer modified gold nanoparticle resonance scattering spectrum probe to measure the content of Pb2+, and the measuring limit is 0.03 nM.
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4.2
Biotoxins [43]
Tang et al adopted the SELEX technology to select the aptamer specifically recognizing the ricin target molecules from the random ologonucleotide library. The capillary electrophoresis technology as the separation means was introduced into the SELEX selection. The highly-efficient separation capability with the capillary electrophoresis technology reduced the selection period greatly. As the enzyme-linked immunity and dot blotting experiments rounded out, the ologonucleotide aptamer specifically recognizing the ricin target molecules can be obtained just after four rounds of selections. Tang et al[44] established the abrin analysis method based on the DNA aptamer technology. Its testing limit was 1 nM and the linear scope was 1–400 nM. Jiang et al[45] used the SELEX technology to select the aptamer specific to SEB. After 13 rounds of selections, the binding percentage of ssDNA library and SEB rises from 1.1% to 39.8%. The aptamer obtained had the strong specificity to SEB and had no specific binding with SPA and BSA. Jorge et al[46] applied the aptamer in testing of food mycotoxin and selected the aptamer of mycotoxin. Moreover, the ng-level mycotoxin can be tested in the wheat sample.
Table 1 Examples of antibiotics targets used for aptamer selections Target for aptamer selection
Type of aptamer
Determination limit
References
Kanamycin A Kanamycin B Streptomycin Neomycin Tobramycin Lividomycin Moenmycin A Chloramphenicol Tetracycline
RNA RNA RNA RNA RNA RNA RNA RNA RNA
< 300 nM 180 nM Not specified 100 nM 2–3 nM < 300 nM 300–400 nM 25–65 μM 1 μM
[42] [43] [44] [45] [46] [42, 47] [48] [49] [50]
Chemicals Fig.2 Specificity of aptasensors of tetracyclines
4.3
Antibiotics
Discovery and application of antibiotics is a great revolution and boon to mankind. Since then, humans have been provided with a strong weapon to battle with death. However, vast use and abuse of antibiotics has brought various poisonous side effects to human beings. There is ongoing research on new antibiotics. The SELEX technology is also widely used in analysis and testing of various antibiotics such as Kanamycin[47], Kanamycin B[48], Streptomycin[49], Neomycin[50], Tobramycin[51], [47,52] [53] Lividomycin , Moenmycin A , Chloramphenicol[54], and [55,56] Tetracycline . Details about the analysis are shown in Table 1. Kim (2010)[57] made the Tetracycline-electrochemical biosensor with the Tetracycline aptamer selected in their foregoing screening as the molecule recognition element. The specific binding of aptamer to Tetracycline is shown in the sensor signal output, which is indicated by the remarkable electric current reduction along with the increase of the Tetracycline content. The minimum testing limit for Tetracycline with the method is 10 nM and the linear testing scope is between 10 nM and 10 ȝM. As shown in Fig.2, in the aptamer electrochemical analysis of Tetracycline and other three structural analogs, there are remarkable differences between the responses to electric current changes by Tetracycline and the other three analogs, which indicate that the specificity of the Tetracycline aptamer-electrochemical biosensor to Tetracycline is quite ideal in the analysis.
TET: Tetracycline; OTC: Oxytetracycline; DOX: Doxycycline; DCF: Diclofenac
4.4
Organic dyes
Since the SELEX technology was proposed in 1990, it has been applied in the analysis of organic dyes. The testing limit with the DNA aptamer is 100–600 μM[3], while the testing limit with the RNA aptamer is 33–46 μM[58]. According to the report by Grate and Wilson[59], the limit for testing the malachite green with the RNA aptamer could reach 1μmol/L. The testing limit was 190 nM for testing Sulforhodamine B with the DNA aptamer by Wilson and Szostak[60]. Brochstedt et al[61] studied the aptamer testing technology for 4,4’-Methylenedianiline, and the testing limit was 0.15–15 μM. Sara et al[62] conducted a semiquantitive testing of the malachite green inside the fish texture and the concealed malachite green. As it rounded out, the RNA aptamer could be used to replace the antibody and applied for testing for chemical residuals in food. Application of the SELEX technology in the industrial dye testing developed continuously and the testing sensitivity is basically superior to that of the liquid tandem mass spectrum method. 4.5
Agonists
Dope is a preparation to activate or enhance the central nervous system activity and includes benzedrine, cocaine, caffeine, other xanthine drugs, nicotinamide drugs, and synthetic anorexics. Doping will produce many direct hazards
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to human physical and mental health. Use of different kinds or different dosages of dopes will result in different degrees of harm to human body. Application of SELEX in dope analysis is becoming popular. In 1997 Mannironi et al[63] reported the research on dopamine with aptamer, whose testing limit could reach 2.8 μM. There are many research reports on the application of SELEX in cocaine analysis. Baker et al (2006) established an electrochemical biosensor to test cocaine. They assembled an MB labeling aptamer on a gold electrode surface of ~1 mm2. Without cocaine, the aptamer formed a double-stranded arm and MB at the end point was away from the gold electrode; with cocaine, the aptamer would form three double-stranded arms and MB will approach the gold electrode to generate the electric signal variation and serve the purpose of qualitative and quantitative cocaine testing[64]. Recently, an electrically excited chemical luminescent aptamer sensor has been developed successfully to test cocaine[65]. According to its principle, a hydrosulphonylmodified cocaine aptamer undergoes Ru(bpy)2(dcbpy)NHS(Fc) modification again and is fixed on the gold particle surface; the gold particles and the gold electrode are connected via a rigid nucleic acid segment. Without cocaine, the aptamer can form a segment of double-stranded section to make Fc away from the gold particle surface and no chemical luminescent signal is produced. After cocaine is added, the aptamer will form three double-stranded sections to make Fc close to the gold particle surface and chemical luminescent signals are produced. The chemical luminescent signals keep a record of constant pressure in the case of 0.1 M tripropylamine. The cocaine testing limit can reach 1 nM. The method provides important reference for high-sensitivity testing of low-molecular-mass substances. Zhang et al (2008) cut the cocaine-recognizing aptamer into two segments. With the existence of cocaine, the two segments can be reassembled into the functional cocaine aptamer and form the complex with cocaine. If gold nanoparticles and a salt solution of certain concentration are added, the gold nanoparticles will gather and their color will change. Without the existence of cocaine, the gold nanoparticles will not gather easily and still keep red[66] after the gold nanoparticles and the certain-concentration salt solution are added, as the nitrogenous bases are fully exposed in the double-segment DNA molecules. 4.6
Microorganisms in food
Bhunia et al[67] conducted the specific testing of Listeria in foods such as beef and chicken by using an aptamer and an antibody-functional optical fiber biosensor. The lower testing limit was 103 CFU mL–1. Hye et al[68] established a method to test E. coli with an aptamer and RT-PCR. The linear scope was 101-107 CFU mL–1 and the lower testing limit was 10 E.coli mL–1. E. Torres-Chavolla and E.C. Alociljac[69] have
made the comprehensive retrospect and prospect for application of aptamer biosensors in microorganism testing. Since SELEX appeared, some oligonucleotide aptamers aiming at virus, bacteria, and other pathogen active proteins have been selected, which open new ways for diagnosis and control of epidemic diseases. It is believed that the SELEX technology will play a more important role in the rapid testing of pathogen microorganisms in the near future.
5
Future prospects
As primary molecule recognizing elements, aptamers have a specific recognition capability and a strong binding force to its targets. They offer many advantages, such as low molecular mass, simplicity for synthesis and modification, and relative stability, so they have wide application. It is especially notable that aptamers have the high recognition and binding capability to low-molecular-mass substances as well, which enables separation, purification, analysis, and testing of low-molecular-mass substances. As Wang et al[70] pointed out, although research on aptamers and their selection methods are still at the starting stage, their application fields have developed from testing sensors to the aptamer drug Macuge used to cure the wet type senile macular degeneration. Currently, application of the aptamer technology has just started in the field of analysis of low-molecular-mass substances. There is much to be developed and studied deeply, which can be summarized into the following three aspects. First, the molecular characteristics of aptamers have to be improved. Currently, research on this aspect mainly focuses on the aptamer stability enhancement, e.g. 2-Aminopyrimidine modification, 2-fluoropyrimidine modification, 2-Dimethoxy nucleotide modification, Spiegelmer, and locked nucleic acid. As the aptamer molecule stability is enhanced, the in vivo and in vitro application space of aptamer will be expanded, especially the resistance of low-molecular-mass substances to the complex systems or adverse environments during the analysis and testing. In the future, improvement of the molecular characteristics of aptamers may be in terms of their resistance to ion intensity, temperature, acidity-alkalinity, and organic reagents. Second, aptamers have to incorporated in other analysis technologies. Aptamer molecules are quite small and can be modified and fixed easily. After aptamers bind with target molecules, their space structure may be changed remarkably. The structural and functional characteristics of aptamers may be the ideal molecule recognizing element used for development of biosensors and other analysis technologies. Currently, aptamers are incorporated in many technologies and methods to expand the scope of analysis, e.g. fluorescence technology, nano technology, surface enhanced Raman scattering (SERS), surface plasmon resonance (SPR), magnetic resonance imaging (MRI), affinity chromatography,
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capillary electrophoresis, colorimetry, and electrochemistry. Aptamers are currently an important subject of research, and therefore, in the future, there surely will be more technologies and methods to be integrated with aptamers and hence, they will have a very wide application A general approach to tailoring aptamer sequences into functional subunits to design target-induced light-switching excimer sensors for rapid, sensitive, and selective detection of important molecules in complex biological fluids has been developed[71]. In the presence of target molecules, two aptamer fragments are induced to self-assemble to form aptamer-target complex and bring two pyrene molecules in close proximity to form an excimer, resulting in fluorescent switching from ~400 nm to 485 nm. With an anti-cocaine sensor, as low as 1 ȝM of cocaine can be detected using steady-state fluorescence assays and more importantly low picomole levels of target can be directly visualized with naked eyes. Because the excimer has a long fluorescence lifetime, time-resolved measurements were used to directly detect as low as 5 ȝM cocaine in urine samples quantitatively without any sample pretreatment[71]. In addition, research on the molecular mechanism for binding of aptamers to low-molecular-mass substances will also be a subject in the future. The molecular recognition mechanism has always been an important subject of research. Although they have a simple structure, aptamers can bind closely with vast target molecules, which undoubtedly makes them an ideal research model to study molecular interactions and may even offer the reference for the target of ligand molecule “customization.”
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