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2020 Volume 48 Issue 5
2020, 48(5): 560-572
doi: 10.19756/j.issn.0253-3820.201024
Abstract:
Aptamers, short single stranded DNA or RNA, are selected through systematic evolution of ligands by exponential enrichment (SELEX), which can bind with target molecules with high affinity and high specificity. Proteins are vital functional macromolecules. Many SELEX techniques have been developed for screening of aptamers against proteins. In recent years, in order to obtain aptamers with high affinity and specificity, many SELEX methods have been developed to enhance selection efficiency, reduce cost and improve aptamer properties. Affinity characterization is also the key procedure in aptamer selection, which demonstrates if the obtained aptamer can satisfy the application requirement. In this review, the progress in aptamer screening for protein targets were summarized, and the optimization methods of nucleic acid library, improvement and development of screening methods and application of aptamers against proteins were mainly emphasized.
Aptamers, short single stranded DNA or RNA, are selected through systematic evolution of ligands by exponential enrichment (SELEX), which can bind with target molecules with high affinity and high specificity. Proteins are vital functional macromolecules. Many SELEX techniques have been developed for screening of aptamers against proteins. In recent years, in order to obtain aptamers with high affinity and specificity, many SELEX methods have been developed to enhance selection efficiency, reduce cost and improve aptamer properties. Affinity characterization is also the key procedure in aptamer selection, which demonstrates if the obtained aptamer can satisfy the application requirement. In this review, the progress in aptamer screening for protein targets were summarized, and the optimization methods of nucleic acid library, improvement and development of screening methods and application of aptamers against proteins were mainly emphasized.
2020, 48(5): 573-582
doi: 10.19756/j.issn.0253-3820.201101
Abstract:
Aptamer, as a novel recognition molecule, shows great potential in small molecule sensing analysis and detection. Selection of aptamers for small molecules is a pre-requisite for their application. This paper summarizes nearly 100 aptamers which are sequenced against 80 kinds of small molecular targets (e.g. toxins, antibiotics and hormones) from 2015 to 2019. Their equilibrium dissociation constants (KD) and determination methods are also summarized. In addition, the development and enhancements of systematic evolution of ligands by exponential enrichment (SELEX) methods based on graphene oxide, human genome, quartz crystal microbalance and in silico are briefly introduced.
Aptamer, as a novel recognition molecule, shows great potential in small molecule sensing analysis and detection. Selection of aptamers for small molecules is a pre-requisite for their application. This paper summarizes nearly 100 aptamers which are sequenced against 80 kinds of small molecular targets (e.g. toxins, antibiotics and hormones) from 2015 to 2019. Their equilibrium dissociation constants (KD) and determination methods are also summarized. In addition, the development and enhancements of systematic evolution of ligands by exponential enrichment (SELEX) methods based on graphene oxide, human genome, quartz crystal microbalance and in silico are briefly introduced.
2020, 48(5): 583-589
doi: 10.19756/j.issn.0253-3820.201173
Abstract:
Systematic evolution of ligands by exponential enrichment (SELEX) is a universal technology for aptamers’ screening. Accurate and efficient screening methods and strategy design are the key to successful screening of aptamers. This article briefly summarizes our works of capillary electrophoresis involving in the efficient screening of aptamers since 2007. The review also introduces the employment of capillary electrophoresis in pre-screening, screening and post-screening SELEX process, classification and screening strategies against different protein targets, multiple capillary electrophoresis screening modes, and aptamers screening for multi-scale targets. Finally, combined with the current research progress of aptamer screening in the world, some remaining issues are proposed.
Systematic evolution of ligands by exponential enrichment (SELEX) is a universal technology for aptamers’ screening. Accurate and efficient screening methods and strategy design are the key to successful screening of aptamers. This article briefly summarizes our works of capillary electrophoresis involving in the efficient screening of aptamers since 2007. The review also introduces the employment of capillary electrophoresis in pre-screening, screening and post-screening SELEX process, classification and screening strategies against different protein targets, multiple capillary electrophoresis screening modes, and aptamers screening for multi-scale targets. Finally, combined with the current research progress of aptamer screening in the world, some remaining issues are proposed.
2020, 48(5): 590-600,684
doi: 10.19756/j.issn.0253-3820.191775
Abstract:
Aptamer is a type of single-stranded DNA or RNA that can specifically bind with target molecules, and has been widely used in the fields of biological separation analysis, clinical diagnosis, and disease targeted therapy, and so on. The development of aptamers is closely related to the progress of screening techniques. Based on the Systematic evolution of ligands by exponential enrichment (SELEX), a variety of in vitro aptamer screening techniques such as magnetic beads SELEX and capillary electrophoresis SELEX have been developed to obtain specific and high-affinity aptamers for target molecules. However, these methods have many disadvantages such as multiple screening rounds, long screening cycles, and large sample consumption, or have difficult in screening small molecules. The microfluidic chip has the characteristics of small size, high throughput, and easy integration, which can solve the above-mentioned problems of SELEX to a certain extent, and realize the rapid and high-throughput screening of aptamers. Based on the summary of SELEX and their key points, this paper focuses on the research progress of SELEX techniques based on microfluidic and microarray chips, and finally, the aspects of SELEX needing to be strengthened are summarized and prospected.
Aptamer is a type of single-stranded DNA or RNA that can specifically bind with target molecules, and has been widely used in the fields of biological separation analysis, clinical diagnosis, and disease targeted therapy, and so on. The development of aptamers is closely related to the progress of screening techniques. Based on the Systematic evolution of ligands by exponential enrichment (SELEX), a variety of in vitro aptamer screening techniques such as magnetic beads SELEX and capillary electrophoresis SELEX have been developed to obtain specific and high-affinity aptamers for target molecules. However, these methods have many disadvantages such as multiple screening rounds, long screening cycles, and large sample consumption, or have difficult in screening small molecules. The microfluidic chip has the characteristics of small size, high throughput, and easy integration, which can solve the above-mentioned problems of SELEX to a certain extent, and realize the rapid and high-throughput screening of aptamers. Based on the summary of SELEX and their key points, this paper focuses on the research progress of SELEX techniques based on microfluidic and microarray chips, and finally, the aspects of SELEX needing to be strengthened are summarized and prospected.
2020, 48(5): 601-607
doi: 10.19756/j.issn.0253-3820.191761
Abstract:
A DNA aptamer screening method for human IgG Fc fragments was established based on capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX). The purity, charged properties and non-adsorptivity of IgG, Fc and Fab were analyzed by capillary zone electrophoresis (CZE), and their affinity with ssDNA libraries was compared. The results showed that IgG formed significant complex with the ssDNA libraries, while Fc and Fab fragments exhibited weak affinity with ssDNA libraries. Therefore, the use of the Fab fragment as the reverse screening target had no significant effect on the sequence except for the specific binding of Fc. The aptamer screening strategy for Fc fragments was designed, the aptamers (Seq Fc1-3) were obtained under optimized incubation condition (10 mmol/L Na2HPO4-KH2PO4 (pH 7.17), 0.05 mmol/L Mg2+, incubated at 37℃ for 25 min) by three round of CE-SELEX, KD was 0.071-0.321 μmol/L, and their affinity and specificity were verified by AuNPs colorimetry.
A DNA aptamer screening method for human IgG Fc fragments was established based on capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX). The purity, charged properties and non-adsorptivity of IgG, Fc and Fab were analyzed by capillary zone electrophoresis (CZE), and their affinity with ssDNA libraries was compared. The results showed that IgG formed significant complex with the ssDNA libraries, while Fc and Fab fragments exhibited weak affinity with ssDNA libraries. Therefore, the use of the Fab fragment as the reverse screening target had no significant effect on the sequence except for the specific binding of Fc. The aptamer screening strategy for Fc fragments was designed, the aptamers (Seq Fc1-3) were obtained under optimized incubation condition (10 mmol/L Na2HPO4-KH2PO4 (pH 7.17), 0.05 mmol/L Mg2+, incubated at 37℃ for 25 min) by three round of CE-SELEX, KD was 0.071-0.321 μmol/L, and their affinity and specificity were verified by AuNPs colorimetry.
2020, 48(5): 608-614
doi: 10.19756/j.issn.0253-3820.191708
Abstract:
The colorimetric aptasensors based on magnetic particle and hybridization chain reaction (HCR) for detection of kanamycin was developed. Kanamycin, an aminoglycoside antibiotic, is extensively used to treat bacterial infections by interrupting protein synthesis. However, the abuse and overuse of kanamycin may cause serious side effects, such as kidney damage, dizziness and hearing loss and so on. In this study, a kind of Mbs@ds-DNA complexes was prepared. The kanamycin aptamer on the Mbs@ds-DNA complexes could bind to kanamycin with strong specificity, while the complementary strand of kanamycin aptamer was used as a signal probe to initiate hybrid chain reaction (HCR). The product of HCR (dsDNA) provided less stabilization without ssDNA sticky ends, and the gold nanoparticles (AuNPs) would aggregate and exhibit a color change from red to purple under the condition of high salt, as well the UV/vis absorption spectra was also changed. The data showed that this method has high sensitivity and specificity for the detection of kanamycin. The linear range was 1.6-32.0 nmol/L with a detection limit of 0.9 nmol/L (S/N=3). Additionally, the proposed aptasensor was successfully utilized for detection of kanamycin in milk and honey samples, with recovery of 96.0%-105.0%, showing good practicability. Aptamer could specifically recognize and bind to target molecules, and therefore this assay provided the reference value for the detection of other targets, showing good potential in the aspects of food safety control and prevention.
The colorimetric aptasensors based on magnetic particle and hybridization chain reaction (HCR) for detection of kanamycin was developed. Kanamycin, an aminoglycoside antibiotic, is extensively used to treat bacterial infections by interrupting protein synthesis. However, the abuse and overuse of kanamycin may cause serious side effects, such as kidney damage, dizziness and hearing loss and so on. In this study, a kind of Mbs@ds-DNA complexes was prepared. The kanamycin aptamer on the Mbs@ds-DNA complexes could bind to kanamycin with strong specificity, while the complementary strand of kanamycin aptamer was used as a signal probe to initiate hybrid chain reaction (HCR). The product of HCR (dsDNA) provided less stabilization without ssDNA sticky ends, and the gold nanoparticles (AuNPs) would aggregate and exhibit a color change from red to purple under the condition of high salt, as well the UV/vis absorption spectra was also changed. The data showed that this method has high sensitivity and specificity for the detection of kanamycin. The linear range was 1.6-32.0 nmol/L with a detection limit of 0.9 nmol/L (S/N=3). Additionally, the proposed aptasensor was successfully utilized for detection of kanamycin in milk and honey samples, with recovery of 96.0%-105.0%, showing good practicability. Aptamer could specifically recognize and bind to target molecules, and therefore this assay provided the reference value for the detection of other targets, showing good potential in the aspects of food safety control and prevention.
2020, 48(5): 615-622
doi: 10.19756/j.issn.0253-3820.191693
Abstract:
Capillary electrophoresis (CE) is a high-efficientcy method for aptamer selection, which exhibits many merits of high resolution, rapid separation, little sample consumption, and no immobilization. However, the influence of CE injection input on aptamer systematic evolution of ligands by exponential enrichment (SELEX) is controversial because the low injection volume may result in an insufficient capacity of the oligonucleotides library. In this research, Neuron-specific enolase (NSE) was used as a model protein to investigate the effects of library input capacity on aptamer selection for clarifying the validity of CE-SELEX. Four different concentrations of initial libraries (0.1, 1, 10 and 100 μmol/L) were utilized into aptamer selection. The affinity of the initial library and sub-libraries after three rounds of selection were evaluated. The results showed that there was no significant difference in the affinity of four different sub-libraries, and the aptamers with micromolar KD were all obtained after two rounds of selection. In addition, the sequences obtained via high concentration ssDNA library screening were more diverse, but the affinity of them was not enriched. The NSE aptamer Seq qN-01 obtained by two rounds of selection had a dissociation constant (KD) of (4.72±0.15) μmol/L, and also presented a good specificity. In conclusion, library input capacity could increase the sequence diversity but had no considerable effects on the efficiency of aptamer selection.
Capillary electrophoresis (CE) is a high-efficientcy method for aptamer selection, which exhibits many merits of high resolution, rapid separation, little sample consumption, and no immobilization. However, the influence of CE injection input on aptamer systematic evolution of ligands by exponential enrichment (SELEX) is controversial because the low injection volume may result in an insufficient capacity of the oligonucleotides library. In this research, Neuron-specific enolase (NSE) was used as a model protein to investigate the effects of library input capacity on aptamer selection for clarifying the validity of CE-SELEX. Four different concentrations of initial libraries (0.1, 1, 10 and 100 μmol/L) were utilized into aptamer selection. The affinity of the initial library and sub-libraries after three rounds of selection were evaluated. The results showed that there was no significant difference in the affinity of four different sub-libraries, and the aptamers with micromolar KD were all obtained after two rounds of selection. In addition, the sequences obtained via high concentration ssDNA library screening were more diverse, but the affinity of them was not enriched. The NSE aptamer Seq qN-01 obtained by two rounds of selection had a dissociation constant (KD) of (4.72±0.15) μmol/L, and also presented a good specificity. In conclusion, library input capacity could increase the sequence diversity but had no considerable effects on the efficiency of aptamer selection.
Selection and Identification of Common Aptamers Against Both Vibrio Harveyi and Vibrio Alginolyticus
2020, 48(5): 623-631
doi: 10.19756/j.issn.0253-3820.191663
Abstract:
Common sites are the sites of same or similar structure on different microorganisms. Some of the common sites can be shared by same genus of microorganisms, which can be used as the marker for the identification of the microorganism. Therefore, selection of common aptamers against these common sites is not only helpful to the analysis of the microorganisms, but also of great significance for the identification of the relative microorganisms. In the work, the common aptamers against both Vibiro harveyi and Vibiro alginolyticus were selected after 8 rounds of selection. The affinity of the enrichment pool of the aptamers increased by 24.1 times, and the analysis based on homologous tree showed that the sequences of the aptamer enriched pool showed obvious convergence, which showed a good selection efficiency. Two aptamers (C14 and C22) were proved for good affinity and specificity towards both V. harveyi and V. alginolyticus. The affinities between the two aptamers and V. harveyi were significantly stronger than those between the aptamers and V. alginolyticus. The affinity constants (K d) of the aptamer C14 against V. harveyi and V. alginolyticus were 55.76 and 82.88 nmol/L respectively. And the affinity constants of the aptamer C22 against V. harveyi and V. alginolyticus were 33.97 and 43.95 nmol/L respectively. Secondary structures of the two aptamers were mainly composed of stems and loops. The 3'-end structure of aptamer C22 seemed to be more complex than that of aptamer C14. Significant fluorescence could be found only on V. harveyi and V. alginolyticus after the aptamers marked with fluorescence group were mixed with V. harveyi, V. alginolyticus, Aeromonas hydrophila, Edwardsiella tarda and Escherichia coli, which showed that the two aptamers had good affinity and specificity towards the two target bacteria. This studies laid a foundation for the identification and detection of the two microorganisms as well as the aquatic pathogenic vibrios.
Common sites are the sites of same or similar structure on different microorganisms. Some of the common sites can be shared by same genus of microorganisms, which can be used as the marker for the identification of the microorganism. Therefore, selection of common aptamers against these common sites is not only helpful to the analysis of the microorganisms, but also of great significance for the identification of the relative microorganisms. In the work, the common aptamers against both Vibiro harveyi and Vibiro alginolyticus were selected after 8 rounds of selection. The affinity of the enrichment pool of the aptamers increased by 24.1 times, and the analysis based on homologous tree showed that the sequences of the aptamer enriched pool showed obvious convergence, which showed a good selection efficiency. Two aptamers (C14 and C22) were proved for good affinity and specificity towards both V. harveyi and V. alginolyticus. The affinities between the two aptamers and V. harveyi were significantly stronger than those between the aptamers and V. alginolyticus. The affinity constants (K d) of the aptamer C14 against V. harveyi and V. alginolyticus were 55.76 and 82.88 nmol/L respectively. And the affinity constants of the aptamer C22 against V. harveyi and V. alginolyticus were 33.97 and 43.95 nmol/L respectively. Secondary structures of the two aptamers were mainly composed of stems and loops. The 3'-end structure of aptamer C22 seemed to be more complex than that of aptamer C14. Significant fluorescence could be found only on V. harveyi and V. alginolyticus after the aptamers marked with fluorescence group were mixed with V. harveyi, V. alginolyticus, Aeromonas hydrophila, Edwardsiella tarda and Escherichia coli, which showed that the two aptamers had good affinity and specificity towards the two target bacteria. This studies laid a foundation for the identification and detection of the two microorganisms as well as the aquatic pathogenic vibrios.
2020, 48(5): 632-641
doi: 10.19756/j.issn.0253-3820.191694
Abstract:
Aptamer is oligonucleotide with high affinity and specificity toward the target obtained via systematic evolution of ligands by exponential enrichment (SELEX). However, due to the complexity of environmental factors and the unknown influence on the selection and application of aptamers, the optimization of multiple environmental factors through conventional experiments requires a huge amount of work and samples, so there are few systematic studies. Capillary electrophoresis (CE)-SELEX has become a highly efficient technology for aptamers screening because of the advantages of CE in microscale, high efficiency and low cost. In this study, Human apo-transferrin (A-TF) was used as a model to study the effects of factors based on CE, including length of ssDNA library, incubation temperature, type and pH of incubation buffer, metal ions etc. The results showed that the shorter ssDNA library had higher affinity with the target protein, the incubation temperature, type and pH of incubation buffer affected the formation of complex, and the low concentrations of K＋, Ca2+ and Mg2+ metal ions promoted the formation of complexes. The A-TF aptamer Seq A3 obtained by three rounds of selection had a dissociation constant (KD) of 0.476 μmol/L, and its affinity and specificity were verified by CE-laser induced fluorescence in human serum environment. The results indicated that Seq A3 could specifically recognize A-TF in serum.
Aptamer is oligonucleotide with high affinity and specificity toward the target obtained via systematic evolution of ligands by exponential enrichment (SELEX). However, due to the complexity of environmental factors and the unknown influence on the selection and application of aptamers, the optimization of multiple environmental factors through conventional experiments requires a huge amount of work and samples, so there are few systematic studies. Capillary electrophoresis (CE)-SELEX has become a highly efficient technology for aptamers screening because of the advantages of CE in microscale, high efficiency and low cost. In this study, Human apo-transferrin (A-TF) was used as a model to study the effects of factors based on CE, including length of ssDNA library, incubation temperature, type and pH of incubation buffer, metal ions etc. The results showed that the shorter ssDNA library had higher affinity with the target protein, the incubation temperature, type and pH of incubation buffer affected the formation of complex, and the low concentrations of K＋, Ca2+ and Mg2+ metal ions promoted the formation of complexes. The A-TF aptamer Seq A3 obtained by three rounds of selection had a dissociation constant (KD) of 0.476 μmol/L, and its affinity and specificity were verified by CE-laser induced fluorescence in human serum environment. The results indicated that Seq A3 could specifically recognize A-TF in serum.
2020, 48(5): 642-649
doi: 10.19756/j.issn.0253-3820.201004
Abstract:
Calreticulin (CRT) is closely related to cancer, and is considered to be a novel biomarker for invasion and metastasis of breast cancer, as well as an indicator of staging and prognosis. Here, we screened and identified aptamers of CRT for the first time with capillary electrophoresis-based systematic evolution of ligands by exponential enrichment (CE-SELEX). The high affinity and specificity Apt 23 was screened and verified by CE isothermal titration calorimetry and gold nanoparticles-based colorimetric assay. By using FAM-labeled Apt 23 (FAM-Apt 23) as an affinity probe, CRT was dected in the linear concentration range of 1-1000 nmol/L in serum by laser-induced fluorescence detector with a detection limit of 0.5 nmol/L. FAM-Apt 23 could also be used as an immunofluorescence imaging probe, which could be bound to the surface of breast cancer cells 4T1 with overexpressed CRT. Preliminary studies have shown that Apt 23 could be used as aptamer probes for recognition and detection of CRT.
Calreticulin (CRT) is closely related to cancer, and is considered to be a novel biomarker for invasion and metastasis of breast cancer, as well as an indicator of staging and prognosis. Here, we screened and identified aptamers of CRT for the first time with capillary electrophoresis-based systematic evolution of ligands by exponential enrichment (CE-SELEX). The high affinity and specificity Apt 23 was screened and verified by CE isothermal titration calorimetry and gold nanoparticles-based colorimetric assay. By using FAM-labeled Apt 23 (FAM-Apt 23) as an affinity probe, CRT was dected in the linear concentration range of 1-1000 nmol/L in serum by laser-induced fluorescence detector with a detection limit of 0.5 nmol/L. FAM-Apt 23 could also be used as an immunofluorescence imaging probe, which could be bound to the surface of breast cancer cells 4T1 with overexpressed CRT. Preliminary studies have shown that Apt 23 could be used as aptamer probes for recognition and detection of CRT.
2020, 48(5): 650-661
doi: 10.19756/j.issn.0253-3820.191647
Abstract:
Grouper iridovirus is a nucleocytoplasmic DNA virus, which not only causes great economic losses in mariculture but also seriously threatens the global biodiversity. To further elucidate the molecular mechanism of grouper iridovirus pathogenesis, and provide the new theoretical basis for the prevention, diagnosis and treatment of grouper iridovirus disease, in this study, we generated four single-stranded DNA aptamers against SGIV-Gx infected cells using systematic evolution of ligands by exponential enrichment (SELEX). Then, the properties of aptamers were characterized. Firstly, each aptamer exhibited high specificity and affinity to grouper iridovirus Guangxi strain (SGIV-Gx)-infected cells. Secondly, aptamers exerted no cytotoxic effects, and aptamer LYGV3 appeared to inhibit SGIV-Gx infection in vitro. The target molecules of LYGV1, LYGV2, LYGV3, LYGV4 could be directly or indirectly related to surface proteins anchored on the membranes of SGIV-Gx infected cells. The targets of aptamers (LYGV1, LYGV2, LYGV3, LYGV4) appeared on the cells surface at 2, 4, 8, and 6 hour post infection.
Grouper iridovirus is a nucleocytoplasmic DNA virus, which not only causes great economic losses in mariculture but also seriously threatens the global biodiversity. To further elucidate the molecular mechanism of grouper iridovirus pathogenesis, and provide the new theoretical basis for the prevention, diagnosis and treatment of grouper iridovirus disease, in this study, we generated four single-stranded DNA aptamers against SGIV-Gx infected cells using systematic evolution of ligands by exponential enrichment (SELEX). Then, the properties of aptamers were characterized. Firstly, each aptamer exhibited high specificity and affinity to grouper iridovirus Guangxi strain (SGIV-Gx)-infected cells. Secondly, aptamers exerted no cytotoxic effects, and aptamer LYGV3 appeared to inhibit SGIV-Gx infection in vitro. The target molecules of LYGV1, LYGV2, LYGV3, LYGV4 could be directly or indirectly related to surface proteins anchored on the membranes of SGIV-Gx infected cells. The targets of aptamers (LYGV1, LYGV2, LYGV3, LYGV4) appeared on the cells surface at 2, 4, 8, and 6 hour post infection.
2020, 48(5): 662-669
doi: 10.19756/j.issn.0253-3820.201030
Abstract:
An aptamer affinity column (AAC) was applied to specific identification and enrichment of aflatoxin B1 (AFB1). Aptamer was used as a novel recognition element for the high affinity and strong specificity of target. By optimizing the loading, washing and eluting conditions of AFB1 in the sample, AFB1 aptamer affinity column exhibited a good sample capacity (334.6±18.2) ng. The content of AFB1 lotus seeds was detected by photo chemical reactor for enhanced high-performance liquid chromatography combined with photo chemical reactor for enhanced detection and fluorescence detection (HPLC-PCD-FLD) and AAC clean-up, with LOD of 0.05 ng/mL and excellent recoveries of 91.8%-108.6%. Meanwhile, the AAC could be reused at least twenty times. This study provides a new method for the enrichment and purification of trace toxins in complex matrix such as food, agricultural products and traditional Chinese medicines, and has a good application prospect.
An aptamer affinity column (AAC) was applied to specific identification and enrichment of aflatoxin B1 (AFB1). Aptamer was used as a novel recognition element for the high affinity and strong specificity of target. By optimizing the loading, washing and eluting conditions of AFB1 in the sample, AFB1 aptamer affinity column exhibited a good sample capacity (334.6±18.2) ng. The content of AFB1 lotus seeds was detected by photo chemical reactor for enhanced high-performance liquid chromatography combined with photo chemical reactor for enhanced detection and fluorescence detection (HPLC-PCD-FLD) and AAC clean-up, with LOD of 0.05 ng/mL and excellent recoveries of 91.8%-108.6%. Meanwhile, the AAC could be reused at least twenty times. This study provides a new method for the enrichment and purification of trace toxins in complex matrix such as food, agricultural products and traditional Chinese medicines, and has a good application prospect.
2020, 48(5): 670-675
doi: 10.19756/j.issn.0253-3820.191676
Abstract:
The biolayer interferometry (BLI), one promising biosensing technique for real-time, high-sensitive detection of analytes, is a new surface-sensitive spectrometric technique in which the change of biolayer thickness is reflected by measuring the interferometric phase deflections. In our previous research, we selected an ssDNA aptamer, 39 nt, with definite interaction characteristics for recombinant human erythropoietin-α (EPO-α). In this work, a new aptameric BLI assay of EPO-α based on this aptamer 39 nt was established. Firstly, the biotinylated aptamer 39 nt was immobilized onto the optical fiber streptavidin biosensor tip, and the analytes in microliter volume were quickly diffused via high-speed oscillation at 2200 r/min. Then, the key parameters such as the orientation of aptamer sequence, the concentration for immobilization, and the overcome of nonspecific adsorption were investigated and optimized to exhibit the affinity recognition of aptamers, and it was found that the 3’-end biotinylated aptamer 39 nt with 50 nmol/L provided the best sensitivity, and the addition of Tween 20 and bovine serum albumin (BSA) could efficiently avoid nonspecific adsorption effect. Besides, towards the original designed disposable biosensor tip, the affinity between EPO-α and aptamer 39 nt for ca. 10 times was maintained by optimizing the regeneration condition. Furthermore, a signal amplification approach of a sandwiched format for detection of EPO-αvia wheat germ agglutinin (WGA) was established, with the linear range of 10-200 nmol/L, and a limit of detection of 5 nmol/L. This assay was further applied to the measurement of EPO-α in 50% human plasma with recoveries of 86.7%-104.2% and RSD less than 11%, providing a satisfied result. The BLI sensing system developed here provides a helpful way for practical applications for label-free protein interaction and detection.
The biolayer interferometry (BLI), one promising biosensing technique for real-time, high-sensitive detection of analytes, is a new surface-sensitive spectrometric technique in which the change of biolayer thickness is reflected by measuring the interferometric phase deflections. In our previous research, we selected an ssDNA aptamer, 39 nt, with definite interaction characteristics for recombinant human erythropoietin-α (EPO-α). In this work, a new aptameric BLI assay of EPO-α based on this aptamer 39 nt was established. Firstly, the biotinylated aptamer 39 nt was immobilized onto the optical fiber streptavidin biosensor tip, and the analytes in microliter volume were quickly diffused via high-speed oscillation at 2200 r/min. Then, the key parameters such as the orientation of aptamer sequence, the concentration for immobilization, and the overcome of nonspecific adsorption were investigated and optimized to exhibit the affinity recognition of aptamers, and it was found that the 3’-end biotinylated aptamer 39 nt with 50 nmol/L provided the best sensitivity, and the addition of Tween 20 and bovine serum albumin (BSA) could efficiently avoid nonspecific adsorption effect. Besides, towards the original designed disposable biosensor tip, the affinity between EPO-α and aptamer 39 nt for ca. 10 times was maintained by optimizing the regeneration condition. Furthermore, a signal amplification approach of a sandwiched format for detection of EPO-αvia wheat germ agglutinin (WGA) was established, with the linear range of 10-200 nmol/L, and a limit of detection of 5 nmol/L. This assay was further applied to the measurement of EPO-α in 50% human plasma with recoveries of 86.7%-104.2% and RSD less than 11%, providing a satisfied result. The BLI sensing system developed here provides a helpful way for practical applications for label-free protein interaction and detection.
2020, 48(5): 676-684
doi: 10.19756/j.issn.0253-3820.191728
Abstract:
Aflatoxin B1 (AFB1) is a toxic metabolite produced by fungi or molds, which can cause serious harm to human health. Based on the classic gold nanoparticles (AuNPs)-based colorimetric method, an AuNPs-aptasensor applicable for two different lengths of AFB1 aptamers determining AFB1 in corn oil was established in this study. The sensitivity of the AuNPs-aptasensor was further improved by optimizing the concentration of aptamer and NaCl and the reaction temperature. Under the optimal conditions, the limits of detection of AuNPs-aptasensor based on 50 (B50) and 80 (A80) base aptamers were 13.55 ng/mL and 10.56 ng/mL, respectively, with the linear range of 20-1000 ng/mL. Moreover, the AuNPs-aptasensor had good selectivity and the recoveries were 102.4%-104.9% and 98.1%-109.1%, respectively. The results indicated that AuNPs-aptasensor could be utilized as an efficient tool for AFB1 identification in corn oil sample. The successful development of AuNPs-aptasensor provided a powerful support for rapid on-site detection and efficient screening of AFB1 in food and showed the potential for construction of biosensors based on other targets and aptamers.
Aflatoxin B1 (AFB1) is a toxic metabolite produced by fungi or molds, which can cause serious harm to human health. Based on the classic gold nanoparticles (AuNPs)-based colorimetric method, an AuNPs-aptasensor applicable for two different lengths of AFB1 aptamers determining AFB1 in corn oil was established in this study. The sensitivity of the AuNPs-aptasensor was further improved by optimizing the concentration of aptamer and NaCl and the reaction temperature. Under the optimal conditions, the limits of detection of AuNPs-aptasensor based on 50 (B50) and 80 (A80) base aptamers were 13.55 ng/mL and 10.56 ng/mL, respectively, with the linear range of 20-1000 ng/mL. Moreover, the AuNPs-aptasensor had good selectivity and the recoveries were 102.4%-104.9% and 98.1%-109.1%, respectively. The results indicated that AuNPs-aptasensor could be utilized as an efficient tool for AFB1 identification in corn oil sample. The successful development of AuNPs-aptasensor provided a powerful support for rapid on-site detection and efficient screening of AFB1 in food and showed the potential for construction of biosensors based on other targets and aptamers.
