Highly amplified detection of visceral adipose tissue-derived serpin (vaspin) using a cognate aptamer duo.
Ahmad Raston Nurul Hanun,Gu Man Bock
Biosensors & bioelectronics
A cognate aptamer duo for visceral adipose tissue-derived serpin (vaspin) which distinctively bind to two different sites on vaspin with high affinity and specificity were successfully developed by using graphene oxide-based systematic evolution of ligands by exponential enrichment (GO-SELEX), which offers immobilization-free screening of aptamers. The specific and simultaneous bindings of this aptamer duo (V1 and V49 aptamers) to the different sites of vaspin were confirmed by circular dichroism (CD) analysis and both sandwich-type surface plasmon resonance (SPR) and quantum dot labelled fluorescence imaging analysis (V1 aptamer serves as primary capturing aptamer and V49 aptamer as secondary signalling aptamer or vice versa). With this vaspin cognate aptamer duo on SPR platform, the detection of the target vaspin were improved to the limit of detection down to 3.5 ng/ml in buffer and 4.7 ng/ml in human serum samples. This cognate aptamer duo based biosensor could be utilized in the early diagnosis of type-2 diabetes.
10.1016/j.bios.2015.03.042
Nanomaterial based aptasensing of prostate specific antigen (PSA): Recent progress and challenges in efficient diagnosis of prostate cancer using biomedicine.
Farshchi Fatemeh,Hasanzadeh Mohammad
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie
Cancer is known to be one of the most major issues all around the world and is the most important cause of death. Prostate cancer is one of the most prevalent cancers among men, and the principal reason of death due to this cancer is the inappropriate detecting tools. Therefore, there is a great request for accurate diagnosis of prostate-specific antigen (PSA). Bio-analysis based on biomarkers might help to overcome this problem. Aptamers can be employed as high-affinity tools for cancer detection. The utilization of aptamer-based strategy in cancer investigation has demonstrated new horizons in biotechnology. The use of nanotechnology in biosensing is a serious development in this field. Advanced nanomaterials enhance the signal amplification in the biosensors, which also reduce the time required for diagnosis and analysis, they are also affordable, with high accuracy. In the present review (with 108 references), we discussed excellent features of the aptasensors on the sensitive and accurate monitoring of PSA biomarkers. Moreover, various types of nanomaterial-based aptasensors were surveyed for PSA detection (electrochemical, optical, piezoelectric, photoelectrochemical, electrochemiluminescent, and so forth). Furthermore, we reported the role of advanced nanomaterials, for instance graphene oxide, carbine nanotube, quantum dots, silica, gold, silver, and magnetic nanoparticles on the improvement of aptasensors of PSA. Finally, we discussed the advantages and limitations of different strategies on the early stage diagnosis of cancer. This article has been updated until July 2020.
10.1016/j.biopha.2020.110878
Aptamer-based nanobiosensors.
Kim Yeon Seok,Raston Nurul Hanun Ahmad,Gu Man Bock
Biosensors & bioelectronics
It has been more than two decades since aptamer and the systematic evolution of ligands by exponential enrichment (SELEX) method were discovered by Larry Gold and Andrew Ellington in 1990, respectively. Based on the various advantages of aptamers, they have become a potent rival of antibodies in therapeutics and bio-analysis. Especially, the recent advances in aptamer biosensor application are remarkable due to its intrinsic properties of aptamers as nucleic acids and target induced conformational changes, in addition to the introduction of graphene oxide-based easy and simple immobilization-free screening method even for dual aptamers. In addition, the incorporation of various nanomaterials such as metallic nanoparticles, carbon materials, and functional nanospheres in aptasensors has facilitated the improvement of analytical performance and commercial application of aptasensors. In this review, recent prominent reports on aptasensors utilizing nanomaterials were introduced to understand the principle of aptamer-based biosensors and provide an insight for new strategies of aptasensors and the application of various nanomaterials. The perspective on aptamer-based biosensors and diagnostics was also discussed in view of technology and market.
10.1016/j.bios.2015.06.040
Development of Dual-Aptamers for Constructing Sandwich-Type Pancreatic Polypeptide Assay.
Qin Shiya,Chen Nandi,Yang Xiaohai,Wang Qing,Wang Kemin,Huang Jin,Liu Jianbo,Zhou Maogui
ACS sensors
Pancreatic polypeptide (PP) is a specific biomarker of nonfunctional pancreatic neuroendocrine tumors (NF-pNETs). Clinical significance of PP inspires researchers to make great efforts in developing sensitive and specific sensors. However, there is no existing biosensor for detecting PP that combines facility and functionality. Addressing this challenge, a pair of aptamers which could be used to develop a sandwich assay for PP is reported. First, several high affinity aptamers are screened through graphene oxide-based SELEX, and appropriate dual-aptamers which could bind to different epitopes of PP are identified through fluorescence assays. Then the feasibility of the dual-aptamers for constructing the sandwich assay is validated via dynamic light scattering. This sandwich assay shows considerable sensitivity and specificity. The above results imply that the dual-aptamers have the potential toward developing novel sensors for PP in clinical samples.
10.1021/acssensors.6b00836
An aptamer based fluorometric microcystin-LR assay using DNA strand-based competitive displacement.
Chinnappan Raja,AlZabn Razan,Abu-Salah Khalid M,Zourob Mohammed
Mikrochimica acta
The high-affinity region of a truncated aptamer was applied to the development of a sensitive method for the determination of microcystin-LR (MC-LR) using competitive displacement and molecular beacons. In this assay, the fluorophore and quencher labelled complementary sequences of the aptamer are hybridized with the truncated aptamer to form a fluorophore-quencher pair. In the presence of MC-LR, the aptamer duplex dissociates, and the fluorophore-quencher pair is separated. This turn leads to an increase in the yellow fluorescence which is best measured at excitation/emission wavelengths of 555/580 nm. One of the truncated aptamers showed a 50-fold increase in the affinity (0.93 nM) compared to the wild type aptamer (50 nM). The truncated sequence shows considerable cross-reactivity with L congeners but none with other congeners. The assay works in 0.5 to 200 nM MC-LR concentration range. It was applied to spiked tap water samples and gave recoveries around 95 ± 5%. Graphical abstract Schematic representation of a method for determination of microcystin-LR via fluorescence that is induced by competitive displacement of complementary DNA strands in a truncated dsDNA aptamer.
10.1007/s00604-019-3504-8
Development of a FRET-based fluorescence aptasensor for the detection of aflatoxin B1 in contaminated food grain samples.
RSC advances
The present study aimed to develop an aptamer-based FRET detection strategy for the specific and sensitive detection of AFB1 in contaminated food grains. The study comprises generation of ssDNA aptamers against AFB1 by whole-cell SELEX and their application in a FRET-based platform utilizing graphene oxide (GO) and quantum dots (QDs). The generated aptamers were characterized to determine their specificity and sensitivity using indirect ELISA where AFB1-OVA was used as a coating antigen. Among the aptamers generated, the ATB1 aptamer showed good reactivity and selectivity against AFB1. This aptamer was further characterized to determine its secondary structure and KD value, which was found to be 5.9 kcal mol. The characterized aptamers were conjugated onto Cd/Se quantum dots to develop a fluorimetric system for the detection of aflatoxin B1 using a graphene oxide platform. The presence of graphene oxide quenches the fluorescence ability of the quantum dots due to π-π stacking interactions between the aptamer and GO. Upon target addition, the aptamer forms a complex with aflatoxin B1 thereby restoring the fluorescence intensity. The developed assay shows a linear response from 0.002 μg μl to 0.2 μg μl with a detection limit of 0.004 μg μl for the AFB1 standard toxin and showed no cross-reactivity with other closely related mycotoxins. To validate the reliability of the developed method, several field samples spiked with AFB1 were included in this study and the results obtained were cross verified using a standard commercial AFB1 kit. In conclusion, the developed method may find good utility in routine food testing laboratories for risk assessment of AFB1.
10.1039/c8ra00317c
Selection and characterization of DNA aptamers against Staphylococcus aureus enterotoxin C1.
Huang Yukun,Chen Xiujuan,Duan Nuo,Wu Shijia,Wang Zhouping,Wei Xinlin,Wang Yuanfeng
Food chemistry
Enterotoxins from pathogenic bacteria are known as the main reason that can cause the bacterial foodborne diseases. In this study, aptamers that bound to Staphylococcus aureus enterotoxin C1 (SEC1) with high affinity and selectivity were generated in vitro by twelve rounds of selection based on magnetic separation technology, with a low-level dissociation constant (Kd) value of 65.14 ± 11.64 nmol/L of aptamer C10. Aptamer-based quantification of SEC1 in the food sample by a graphene oxide (GO)-based method was implemented to investigate the potential of the aptamer against SEC1 with a limit of detection of 6 ng/mL. On the basis of this work, biosensors using the selected SEC1 aptamers as new molecular recognition elements could be applied for innovative determinations of SEC1.
10.1016/j.foodchem.2014.06.039
Highly adaptable and sensitive FRET-based aptamer assay for the detection of Salmonella paratyphi A.
Rm Renuka,Maroli Nikhil,J Achuth,Ponmalai Kolandaivel,K Kadirvelu
Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy
Here we demonstrate a facile and versatile fluorescence resonance energy transfer (FRET) based aptasensor for rapid detection of Salmonella paratyphi A. The assay shows a detection limit up to 10 cfu·mL with no cross-reactivity with other bacterial species. Less than 8% of inter-assay coefficient variance and recovery rate between 85 and 102% attests the assay reliability. The advantages of FRET-based aptamer assay over the conventional immunoassay formats such as ELISA are the specificity, speed, reliability, and simplicity of the assay. The ssDNA aptamers specific towards pathogenic Salmonella paratyphi A were generated via whole-cell SELEX. The aptamer was conjugated onto quantum dot (QD) that served as the molecular beacon and graphene oxide (GO) was used as a fluorescence quencher. Thus the proposed method enables detection of target pathogen using FRET-based assay. Further interaction of aptamer with pathogen protein DNA gyrase was explored using classical molecular dynamics simulation.
10.1016/j.saa.2020.118662
Label-Free Direct Detection of Saxitoxin Based on a Localized Surface Plasmon Resonance Aptasensor.
Ha Su-Ji,Park Jin-Ho,Lee Bobin,Kim Min-Gon
Toxins
Seafood is an emerging health food, and interest in improving the quality of seafood is increasing. Saxitoxin (STX) is a neurotoxin produced by marine dinoflagellates that is accumulated in seafood. It can block the neuronal transmission between nerves and muscle cell membranes, resulting in the disturbance of neuromuscular transmission and subsequent voluntary muscle paralysis. Here, we developed a new aptamer for the detection of STX using graphene oxide-systematic evolution of ligands by exponential enrichment (GO-SELEX). Furthermore, we confirmed sensitivity and selectivity of the developed aptamer specific to STX using a localized surface plasmon resonance (LSPR) sensor. The sensing chip was fabricated by fixing the new STX aptamer immobilized on the gold nanorod (GNR) substrate. The STX LSPR aptasensor showed a broad, linear detection range from 5 to 10,000 μg/L, with a limit of detection (LOD) of 2.46 μg/L (3σ). Moreover, it was suitable for the detection of STX (10, 100, and 2000 μg/L) in spiked mussel samples and showed a good recovery rate (96.13-116.05%). The results demonstrated that the new STX aptamer-modified GNR chip was sufficiently sensitive and selective to detect STX and can be applied to real samples as well. This LSPR aptasensor is a simple, label-free, cost-effective sensing system with a wide detectable range.
10.3390/toxins11050274
Fabrication of MERS-nanovesicle biosensor composed of multi-functional DNA aptamer/graphene-MoS nanocomposite based on electrochemical and surface-enhanced Raman spectroscopy.
Sensors and actuators. B, Chemical
Middle East respiratory syndrome coronavirus (MERS-CoV) is one of the most harmful viruses for humans in nowadays. To prevent the spread of MERS-CoV, a valid detection method is highly needed. For the first time, a MERS-nanovesicle (NV) biosensor composed of multi-functional DNA aptamer and graphene oxide encapsulated molybdenum disulfide (GO-MoS) hybrid nanocomposite was fabricated based on electrochemical (EC) and surface-enhanced Raman spectroscopy (SERS) techniques. The MERS-NV aptamer was designed for specifically binding to the spike protein on MERS-NVs and it is prepared using the systematic evolution of ligands by exponential enrichment (SELEX) technique. For constructing a multi-functional MERS aptamer (MF-aptamer), the prepared aptamer was connected to the DNA 3-way junction (3WJ) structure. DNA 3WJ has the three arms that can connect the three individual functional groups including MERS aptamer (bioprobe), methylene blue (signal reporter) and thiol group (linker) Then, GO-MoS hybrid nanocomposite was prepared for the substrate of EC/SERS-based MERS-NV biosensor construction. Then, the assembled multifunctional (MF) DNA aptamer was immobilized on GO-MoS. The proposed biosensor can detect MERS-NVs not only in a phosphate-buffered saline (PBS) solution (SERS LOD: 0.176 pg/ml, EIS LOD: 0.405 pg/ml) but also in diluted 10% saliva (SERS LOD: 0.525 pg/ml, EIS LOD: 0.645 pg/ml).
10.1016/j.snb.2021.131060
Selection and Characterization of DNA Aptamers for Constructing Aptamer-AuNPs Colorimetric Method for Detection of AFM1.
Foods (Basel, Switzerland)
Aflatoxin M1 (AFM1), one of the most toxic mycotoxins, is a feed and food contaminant of global concern. To isolate the ssDNA aptamer of AFM1, synthesized magnetic graphene oxide nanomaterials, 12 rounds of subtractive systematic evolution of ligands by exponential enrichment (SELEX) selection were carried out. As a result, 24 candidate aptamers were selected, and their sequence similarity exceeded 97%. Their binding affinity and specificity were further examined by fluorescence and biofilm interferometry (BLI) methods. One aptamer (Apt-5) against AFM1 with a high affinity and specificity was isolated and demonstrated to be the optimal aptamer, whose dissociation constant reached the nanomolar level, Kd = 8.12 ± 1.51 nM. Additionally, molecular docking studies were used to predict the possible binding sites and mechanisms of the two. Based on Apt-5, an unlabeled aptamer-AuNPs colorimetric method was established to detect AFM1 in milk with a linear range of 0.078-10 ng/mL, and the actual detection limit was 0.078 ng/mL. These results demonstrated that this detection technique could be useful for the quantitative determination of AFM1 in milk and dairy products.
10.3390/foods11121802
A highly sensitive DNA aptamer-based fluorescence assay for sarcosine detection down to picomolar levels.
Özyurt Canan,Canbay Zeynep Çelik,Dinçkaya Erhan,Evran Serap
International journal of biological macromolecules
Sarcosine is an amino acid derivative, which is considered as a key metabolite in various metabolic processes. Therefore, simple and sensitive detection methods are needed for further understanding its metabolic role and diagnostic value. In this study, we developed a novel method that meets the need for practical and sensitive detection in a complex medium mimicking urine conditions. For this aim, we selected sarcosine-specific DNA aptamers using graphene oxide-assisted systemic evolution of ligands by exponential enrichment (GO-SELEX). The candidate aptamers were labeled with 6-carboxyfluorescein (6-FAM) at their 5' ends. Two aptamers, namely 9S and 13S produced a significant fluorescence signal upon sarcosine binding. Both aptamers enabled a sensitive analysis with a detection limit of 0.5 pM. The linear detection ranged between 5 pM and 50 μM for 9S aptamer, while 13S aptamer enabled a wider linear detection range between 5 pM and 500 μM. The aptamer-based assay allowed rapid detection with no need for chemical derivatization of sarcosine and sophisticated instruments. Moreover, the aptamer-based assay was free of interference from urea and human serum albumin.
10.1016/j.ijbiomac.2019.02.030
Screening and development of DNA aptamers as capture probes for colorimetric detection of patulin.
Wu Shijia,Duan Nuo,Zhang Weixiao,Zhao Sen,Wang Zhouping
Analytical biochemistry
Patulin (PAT) is a kind of mycotoxin that has serious harmful impacts on both food quality and human health. A high-affinity ssDNA aptamer that specifically binds to patulin was generated using systemic evolution of ligands by exponential enrichment (SELEX) assisted by graphene oxide (GO). After 15 rounds of positive and negative selection, a highly enriched ssDNA pool was sequenced and the representative sequences were subjected to binding assays to evaluate their affinity and specificity. Of the eight aptamer candidates tested, the sequence PAT-11 bound to patulin with high affinity and excellent selectivity with a dissociation constant (Kd) of 21.83 ± 5.022 nM. The selected aptamer, PAT-11, was subsequently used as a recognition element to develop a detection method for patulin based on an enzyme-chromogenic substrate system. The colorimetric aptasensor exhibited a linear range from 50 to 2500 pg mL(-1), and the limit of detection was found to be 48 pg mL(-1). The results indicated that GO-SELEX technology was appropriate for the screening of aptamers against small-molecule toxins, offering a promising application for aptamer-based biosensors.
10.1016/j.ab.2016.05.024
Highly sensitive detection of 25-HydroxyvitaminD by using a target-induced displacement of aptamer.
Lee Bang Hyun,Nguyen Van Thuan,Gu Man Bock
Biosensors & bioelectronics
For the prevention of 25-HydroxyvitaminD deficiency, in this study, aptamers which can bind to 25-HydroxyvitaminD with high specificity and affinity, were successfully developed by using immobilization-free, graphene oxide-based systemic evolution of ligands by exponential enrichment (GO-SELEX) method. The 9 sequences including VDBA14 aptamer were obtained out of 16 aptamer candidates, based on the specificity and affinity of the aptamers confirmed by both the gold nanoparticles (AuNPs)-based colorimetric assay and the isothermal titration calorimetry (ITC) method. Among them, the aptamer, VDBA14, developed in this study was found to show a great affinity to 25-HydroxyvitaminD, with 11nM of its Kd value. Moreover, the circular dichroism (CD) analysis data indicated the target-induced displacement of the aptamer VDBA14clearly. In addition, this target-induced change of the aptamer was also confirmed again by conducting two different experimental formats, the use of streptavidin-coated 96-well plates and the use of magnetic beads. The results clearly indicated that the structure of VDBA14 aptamer was changed upon the binding of the target, 25-HydroxyvitaminD, and so the indicator sequences (partially complementary to the aptamer sequence) tagged with an enzyme as a signaling molecule could be de-hybridized from the aptamer. Finally, the limit of detection for vitamin D based on AuNPs-based colorimetric assay using VDBA14 aptamer was found to be 1µM. All these results were taken together, the aptamer which was developed could play an exquisite role in the fields of early medical diagnosis of vitamin D deficiency with accurate, rapid and simple analytical method.
10.1016/j.bios.2016.08.011
Development of a ssDNA aptamer for detection of residual benzylpenicillin.
Lee A-Young,Ha Na-Reum,Jung In-Pil,Kim Sang-Heon,Kim A-Ru,Yoon Moon-Young
Analytical biochemistry
Antibiotics are useful for improving the living conditions of livestock. However, residual antibiotics induce several human diseases such as food-borne illness and infection of carbapenem-resistant Enterobacteriaceae (CRE). In this study, the identification of a benzylpenicillin-specific aptamer was selected by rGO-SELEX (reduced Graphene Oxide-Systematic Evolution of Ligands by EXponential enrichment). A random ssDNA library was incubated with rGO for adsorption and eluted with benzylpenicillin. As a result of the selection process, a DNA aptamer was found that specifically bound to benzylpenicillin with high binding affinity, K = 383.4 nM, and had a low limit of detection (LOD) of 9.2 nM. The characterization of the aptamer was performed through the fluorescence recovery signal from rGO surface. In addition, detection of benzylpenicillin was performed in pretreated milk samples, and its detection accuracy was shown to be 100± 10%. This represented that BBA1 was used for fluorescence aptasensor system in real sample. Furthermore, this benzylpenicillin binding aptamer showed high specificity against other antibiotics except for ampicillin. With these advantageous characteristics, we expect that this aptamer could be applied to an on-site detection system for residual benzylpenicillin.
10.1016/j.ab.2017.05.013
Development and characterization of DNA aptamers against florfenicol: Fabrication of a sensitive fluorescent aptasensor for specific detection of florfenicol in milk.
Sadeghi Atefeh Sarafan,Mohsenzadeh Mohammad,Abnous Khalil,Taghdisi Seyed Mohammad,Ramezani Mohammad
Talanta
Specific ssDNA aptamers for the antibiotic florfenicol (FF) were developed from an enriched nucleotide library using magnetic beads-based SELEX (Systematic Evolution of Ligands by EXponential enrichment) technique with high-binding affinity. After 12 rounds of selection, thirty-six sequences were obtained that were then divided into five major families, according to the primary sequence similarity. Binding affinity analyses of three fluorescently tagged aptamers belonging to different families demonstrated that the dissociation constants (K) were in the low nanomolar range (K = 52.78-211.4 nmol L). Furthermore, to verify the potential application of the aptamers, a fluorescent aptasensor was fabricated for detecting the FF residue in raw milk samples based on the energy transfer between graphene oxide as the acceptor and fluorescently tagged FF-specific aptamer as the donor. Under optimal conditions, the aptasensor displayed a wide linear range from 5 to 1200 nmol L and a detection limit of 5.75 nmol L with excellent selectivity in milk. The recovery rate in the milk was between 101% ± 0.14% and 110% ± 2.8%, indicating high accuracy. This fluorescent aptasensor possessed considerable potential for rapid analysis of FF in raw milk because of its simplicity of detection. Moreover, the interaction between the aptamer and FF was studied using molecular modeling.
10.1016/j.talanta.2018.01.083
In vitro selection of tacrolimus binding aptamer by systematic evolution of ligands by exponential enrichment method for the development of a fluorescent aptasensor for sensitive detection of tacrolimus.
Mansouri Atena,Abnous Khalil,Nabavinia Maryam Sadat,Ramezani Mohammad,Taghdisi Seyed Mohammad
Journal of pharmaceutical and biomedical analysis
Tacrolimus (TAC) is an immunosuppressant for preventing solid-organ transplant rejection. Because of its narrow therapeutic window, analytical methods which can detect TAC in serum samples with high accuracy and reliability are required. In this study, specific aptamers (Apt122 and Apt125) for TAC were isolated via systematic evolution of ligands by exponential enrichment method using magnetic beads to immobilize the target. After determination of binding constants of aptamers by flow cytometry analysis, Apt122 was selected and labeled with ATTO 647 N as a fluorophore to develop a fluorescent sensing platform for detection of TAC using graphene oxide (GO) as a fluorescence quencher. The designed aptasensor could detect TAC in phosphate buffer saline (10 mM PBS) and serum samples with detection limits as low as 1.4 and 2.5 nM, respectively.
10.1016/j.jpba.2019.112853
The sensitive detection of ODAM by using sandwich-type biosensors with a cognate pair of aptamers for the early diagnosis of periodontal disease.
Lee Bang Hyun,Kim Sang Hoon,Ko Youngkyung,Park Joo Cheol,Ji Suk,Gu Man Bock
Biosensors & bioelectronics
This research aims to develop biosensors which could diagnose periodontal diseases in early phases and predict the illness stage of patients, in order to give them adequate treatment timely. Human odontogenic ameloblast-associated protein (ODAM) is considered to be a potential biomarker for periodontal diseases, based on high correlation between the level of ODAM in gingival crevicular fluid (GCF) and the degree of periodontitis. Many aptamers, including a cognate pair of aptamers which can bind to the different sites of ODAM, were successfully screened in a very stringent condition employing saliva as a counter target through the graphene oxide-based systemic evolution of ligands by exponential enrichment (GO-SELEX). For the characterization of the aptamer candidates, GO-based fluorescence resonance energy transfer (GO-FRET) and surface plasmon resonance (SPR) assays were conducted. The sandwich-type binding of a cognate pair of aptamers to ODAM was additionally confirmed by employing circular dichroism (CD) and magnetic beads-based fluorescence imaging methods. The resulting cognate pair of aptamers, OD64 and OD35, were found to have their dissociation constant (Kd), 47.71 nM and 51.36 nM, respectively. The minimum detectable concentrations of a sandwich-type SPR biosensor were found to be 0.24 nM and 1.63 nM, respectively, for both buffered and saliva samples. Finally, using this cognate pair of aptamers, a sandwich-type lateral flow strip biosensor was successfully realized. This research shows the potential for implementation of a cognate pair of aptamers on point-of-care biosensors which enables simple, rapid, and non-invasive saliva-based diagnosis of periodontal-related diseases that can overcome current diagnostic methods and improve health care system.
10.1016/j.bios.2018.10.040
Screening and application of a broad-spectrum aptamer for acyclic guanosine analogues.
Ren Le,Qi Shuo,Khan Imran Mahmood,Wu Shijia,Duan Nuo,Wang Zhouping
Analytical and bioanalytical chemistry
Acyclic guanosine analogues, a class of widely used antiviral drugs, can cause chronic toxicity and virus resistance. Therefore, it is essential to establish rapid and accurate methods to detect acyclic guanosine analogues. In this study, five acyclic guanosine analogues (acyclovir, famciclovir, ganciclovir, penciclovir, and valaciclovir) were used as positive targets to obtain broad-spectrum aptamers through Capture-SELEX technology. Real-time quantitative PCR (Q-PCR) was used to monitor the aptamer SELEX process. After the sixteen rounds of selection against mixed targets, sequences were obtained by high-throughput sequencing (HTS). Furthermore, a broad-spectrum aptamer, named CIV6, was found as the higher performance aptamer that was suitable for five acyclic guanosine analogues by graphene oxide (GO) polarization and fluorescence assay. Finally, the aptamer CIV6 was used to construct GO fluorescence assay to detect five acyclic guanosine analogues. The limits of detection (LOD) of acyclovir, famciclovir, ganciclovir, penciclovir, and valaciclovir were 0.48 ng·mL, 0.53 ng·mL, 0.50 ng·mL, 0.56 ng·mL, and 0.38 ng·mL, respectively.
10.1007/s00216-021-03446-w
Aptamer selection and aptasensor construction for bone density biomarkers.
Chinnappan Raja,Zaghloul Norhan Sameh,AlZabn Razan,Malkawi Abeer,Abdel Rahman Anas,Abu-Salah Khalid M,Zourob Mohammed
Talanta
Osteoporosis (OP) is a bone disease involved in dysregulation of one of the bone metabolism arms, formation, or desorption cause a porous bone. Osteocalcin (OC) and beta-crosslap (BC), are the well-known markers for OP, which are connected to bone formation and desorption, respectively. In addition to the OP biomarker, BC is also used as an estrogen replacement therapeutic monitoring. ELISA and other antibody-based detection methods are routinely used for measuring OC and BC. These methods have limitations that include thermostability, sensitivity, sacrificing animals, and cost of production. However, aptamer-based-assays are of interest to overcome these drawbacks and achieve the most specific and robust application. Herein, specific aptamers for OC and BC were selected by the systematic evolution of ligands by exponential enrichment (SELEX) method from the pool of ssDNA library with 60 random sequences. The binding affinity (K) of the selected aptamers were evaluated against the respective biomarkers. The high-affinity aptamers of OC and BC showed the K values of 59 and 55 nM respectively. A graphene oxide-based aptasensors were fabricated from the high-affinity aptamers, and the detection limits of OC and BC were found to be 0.4 pg/ml and 0.21 pg/ml, respectively. These aptasensors have been tested with OC and BC spiked buffer samples and validated using serum samples collected from osteoporotic rats.
10.1016/j.talanta.2020.121818
Isolation and Characterization of a ssDNA Aptamer against Major Soluble Antigen of .
Layman Brady,Mandella Brian,Carter Jessica,Breen Haley,Rinehart John,Cavinato Anna
Molecules (Basel, Switzerland)
Bacterial kidney disease (BKD) is a major health problem of salmonids, affecting both wild and cultured salmon. The disease is caused by (Rs), a fastidious, slow-growing and strongly Gram-positive diplobacillus that produces chronic, systemic infection characterized by granulomatous lesions in the kidney and other organs, often resulting in death. Fast detection of the pathogen is important to limit the spread of the disease, particularly in hatcheries or aquaculture facilities. Aptamers are increasingly replacing conventional antibodies as platforms for the development of rapid diagnostic tools. In this work, we describe the first instance of isolating and characterizing a ssDNA aptamer that binds with high affinity to p57 or major soluble antigen (MSA), the principal antigen found on the cell wall surface of Rs. Specifically, in this study a construct of the full-length protein containing a DNA binding domain (MSA-R2c) was utilized as target. Aptamers were isolated from a pool of random sequences using GO-SELEX (graphene oxide-systematic evolution of ligands by exponential enrichment) protocol. The selection generated multiple aptamers with conserved motifs in the random region. One aptamer with high frequency of occurrence in different clones was characterized and found to display a strong binding affinity to MSA-R2c with a of 3.0 ± 0.6 nM. The aptamer could be potentially utilized for the future development of a sensor for rapid and onsite detection of Rs in water or in infected salmonids, replacing time-consuming and costly lab analyses.
10.3390/molecules27061853
Insulin-binding aptamer-conjugated graphene oxide for insulin detection.
Pu Ying,Zhu Zhi,Han Da,Liu Huixia,Liu Jun,Liao Jie,Zhang Kejing,Tan Weihong
The Analyst
This paper describes a simple and sensitive aptamer/graphene oxide (GO) based assay for insulin detection. GO can protect DNA from nuclease cleavage, but aptamers can be detached from the GO surface by specific target binding. This exposes the aptamers to enzymatic cleavage and releases the target for a new cycle. Cycling of targets leads to significant signal amplification and low LOD.
10.1039/c1an15407a
Selection and identification of high-affinity aptamer of Kunitz trypsin inhibitor and their application in rapid and specific detection.
Food science & nutrition
Kunitz trypsin inhibitor (KTI), a harmful protein, seriously affects food hygiene and safety. Therefore, a sensitive, efficient, and rapid method for KTI detection is urgently needed. Aptamers are short and single-stranded (ss) DNA that recognize target molecules with high affinity. This work used graphene oxide-SELEX (GO-SELEX) to screen KTI aptamers. The positive and reverse screening was designed to ensure the high specificity and affinity of the selected aptamers. After 10 rounds of screening, multiple nucleic acid chains were obtained, and the chains were sequenced. Three aptamers with better affinity were obtained, and the values of the dissociation constant ( ) were calculated to be 52.6 nM, 22.7 nM, and 67.9 nM, respectively. Finally, a colorimetric aptamer biosensor based on gold nanoparticles (AuNPs) was constructed. The biosensor exhibited a broader linear range of 30-750 ng/ml, with a lower detection limit of 18 ng/ml, and the spiked recovery rate was between 98.2% and 103.3%. This experiment preliminary demonstrated the potential of the application of KTI aptamer in the real sample tests.
10.1002/fsn3.2729
Immobilization-free screening of aptamers assisted by graphene oxide.
Park Jee-Woong,Tatavarty Rameshwar,Kim Dae Woo,Jung Hee-Tae,Gu Man Bock
Chemical communications (Cambridge, England)
Graphene oxide (GO) has the ability to separate free short ssDNA in heterogeneous solution. This feature is applied as a label free platform for screening of aptamers that bind to their target with high affinity and specificity. Herein, we report an aptamer selection strategy for Nampt protein based on GO.
10.1039/c2cc16473f
Detection of Nonylphenol with a Gold-Nanoparticle-Based Small-Molecule Sensing System Using an ssDNA Aptamer.
Kim A-Ru,Kim Sang-Heon,Kim Dabin,Cho Seo Won,Son Ahjeong,Yoon Moon-Young
International journal of molecular sciences
Endocrine-disrupting chemicals (EDCs) threaten many kinds of life throughout the world. These compounds function the same as sexual hormones, inducing precocious puberty, gynecomastia, etc., in the human body. To prevent excess exposure to nonylphenol (NP), a simple and rapid detection system is needed. In this study, we develop a nonylphenol-specific aptamer from a random single-stranded DNA library and test a rapid sensor system based on the aptamer and gold nanoparticles (AuNPs). The aptamer was screened by a methodology involving reduced graphene oxide (rGO). As a result of screening and sequencing, a DNA aptamer was developed that recognizes the target with high binding affinity (K = 194.2 ± 65.9 nM) and specificity. The sensor system developed using the aptamer and gold nanoparticles is sensitive (LOD = 2.239 nM). Circular dichroism (CD) spectrometry results show that the free aptamer binds to the target molecule. The aptamer was characterized using gold nanoparticles to measure UV absorbance. Our results suggest that the sensor system developed using this aptamer is useful for field diagnosis of small molecules.
10.3390/ijms21010208
Selection and characterization, application of a DNA aptamer targeted to Streptococcus pyogenes in cooked chicken.
Huang Yukun,Wang Xin,Duan Nuo,Xia Yu,Wang Zhouping,Che Zhenming,Wang Lijun,Yang Xiao,Chen Xianggui
Analytical biochemistry
An aptamer against Streptococcus pyogenes was selected and identified, and a fluorescent method based on the reported aptamer was established to detect S. pyogenes in the cooked chicken. Through a twelve rounds of whole-bacterium SELEX (systematic evolution of ligands by exponential enrichment) selection in vitro, a set of aptamers binding to the whole cell of S. pyogenes were generated, harvesting a low-level dissociation constant (K) value of 44 ± 5 nmol L of aptamer S-12. Aptamer-based quantification of S. pyogenes in the cooked chicken sample was implemented in a fluorescence resonance energy transfer-based assay by using graphene oxide, resulting in a limit of detection of 70 cfu mL. The selected aptamer showed affinity and selectivity recognizing S. pyogenes; besides, more biosensors based on the selected aptamer as a molecular recognition element could be developed in the innovative determinations of S. pyogenes.
10.1016/j.ab.2018.04.015
Specific detection of avian influenza H5N2 whole virus particles on lateral flow strips using a pair of sandwich-type aptamers.
Kim Sang Hoon,Lee Junho,Lee Bang Hyun,Song Chang-Seon,Gu Man Bock
Biosensors & bioelectronics
We report a selection of a cognate pair of aptamers for whole avian influenza virus particles of H5N2 by using graphene-oxide based systemic evolution of ligands by exponential enrichment (GO-SELEX), and the application of a pair of sandwich-type binding aptamers on the lateral flow strips. The aptamers were characterized by GO-FRET assay, and Kd values of the selected aptamers were estimated to be from 6.913 × 10 to 1.27 × 10 EID/ml (EID/ml: 50% egg infective dose). Based on the evidence from confocal laser scanning microscope (CLSM), surface plasmon resonance (SPR), and circular dichroism (CD) spectrum analysis, the aptamers, JAPT and JHAPT, were found to be working as a cognate pair that binds to the target virus at the different sites simultaneously. This cognate pair of aptamers then was successfully applied on the lateral flow strips, clearly showing sandwich-type binding images with the presence of the certain numbers of H5N2 virus particles. On the newly developed lateral flow strips, the target virus was detectable down to 6 × 10 EID/ml in the buffer and 1.2 × 10 EID/ml in the duck's feces, respectively, by the naked eye. By using the ImageJ software, the LOD was found to be 1.27 × 10 EID/ml in the buffer and 2.09 × 10 EID/ml in the duck's feces, respectively. Interestingly, on the lateral flow strips, enhanced specificity towards the target virus (H5N2) appeared over other subtypes of H5Nx. To the best of our knowledge, this is the first report about the application of the cognate pair of aptamers for the detection of influenza virus on the lateral flow strips. This study shows the promising perspective of a cognate pair of aptamers for the on-site detection system which could be useful for rapid detection of avian influenza viruses for preventing the pandemic influenza viruses from spreading.
10.1016/j.bios.2019.03.061
Detection of adenosine triphosphate in HeLa cell using capillary electrophoresis-laser induced fluorescence detection based on aptamer and graphene oxide.
Fang Bi-Yun,Yao Ming-Hao,Wang Chun-Yuan,Wang Chao-Yang,Zhao Yuan-Di,Chen Fang
Colloids and surfaces. B, Biointerfaces
A method for ATP quantification based on dye-labeled aptamer/graphene oxide (aptamer/GO) using capillary electrophoresis-laser induced fluorescence (CE-LIF) detecting technique has been established. In this method, the carboxyfluorescein (FAM)-labelled ATP aptamers were adsorbed onto the surface of GO, leading to the fluorescence quenching of FAM; after the incubation with a limited amount of ATP, stronger affinity between ATP aptamer and ATP resulted in the desorption of aptamers and the fluorescence restoration of FAM. Then, aptamer-ATP complex and excess of aptamer/GO and GO were separated and quantified by CE-LIF detection. It was shown that a linear relation was existing in the CE-LIF peak intensity of aptamer-ATP and ATP concentration in range of 10-700 μM, the regression equation was F=1.50+0.0470C(ATP) (R(2)=0.990), and the limit of detection was 1.28 μM (3S/N, n=5), which was one order magnitude lower than that of detection in solution by fluorescence method. The approach with excellent specificity and reproducibility has been successfully applied to detecting concentration of ATP in HeLa cell.
10.1016/j.colsurfb.2015.12.043
Development of a fluorescence assay for highly sensitive detection of based on an aptamer-carbon dots/graphene oxide system.
RSC advances
An aptamer-based fluorescence assay for culture-independent detection of was developed. This assay was enabled by highly specific aptamers conjugated with photoluminescent carbon dots (CDs) as the fluorescent probe and graphene oxide (GO) as the quencher. Specially, high-throughput sequencing was achieved during systematic evolution of ligands exponential enrichment (SELEX) for accurate recognition of aptamers. This assay displayed high specificity towards and was resistant to interference by other ubiquitous bacteria including , , , , and . After the conditions were optimized, this assay achieved a wide detection range for varying from 10 CFU mL to 10 CFU mL. Notably, it approached an excellent detection limit as low as 9 CFU mL. Therefore, this fluorescence assay was considered successfully developed for highly sensitive detection of . This assay also detected the contamination of in tap water and commercial bottled water, thereby suggesting its potential application in real water samples.
10.1039/c8ra04819c
Development of aflatoxin B aptasensor based on wide-range fluorescence detection using graphene oxide quencher.
Joo Minyoung,Baek Seung Hoon,Cheon Seon Ah,Chun Hyang Sook,Choi Sung-Wook,Park Tae Jung
Colloids and surfaces. B, Biointerfaces
Aflatoxin B (AFB) is a carcinogenic substance produced by fungi of genus Aspergillus, especially Aspergillus flavus. Few nanograms of AFB that permeated through the skin is sufficient to cause liver cancer and stunted growth. In this study, a rapid aptamer-based assay for AFB was developed using the fluorescence quenching property of graphene oxide (GO) and a fluorescein amidite (FAM)-modified aptamer specific to AFB. The aptamer, modified with the fluorescence dye FAM on its 5'-end, was used as a probe. Once bound by AFB, a conformational change of the aptamer was caused that led to its interaction with the well-known fluorescence quencher GO, resulting in a decrease of the fluorescence intensity of the system. In the absence of AFB, the fluorescence intensity remained unchanged. The aptamer-based AFB assay process was conducted through 3 steps within 40min. The aptamer was incubated with AFB before the addition of GO. The amount of AFB present was measured by the change in fluorescence intensity. The detection system was evaluated with standard solutions of AFB of various concentrations. The results showed that the fluorescence intensity decreased linearly as the concentration of AFB gradually increased. Although the assay was specific to AFB, there was slight interference by other types of aflatoxin. When the assay was applied to a real sample, the limit of detection was 4.5 ppb, which was within the wide detection range of up to 300ppb with good linearity. Thus, this biosensor is considered to be competitive with the conventional detection methods in the field owing to its wide detection range and assay rapidity.
10.1016/j.colsurfb.2017.03.010
Aptamer-graphene oxide for highly sensitive dual electrochemical detection of Plasmodium lactate dehydrogenase.
Jain Priyamvada,Das Smita,Chakma Babina,Goswami Pranab
Analytical biochemistry
A 90 mer ssDNA aptamer (P38) enriched against Plasmodium falciparum lactate dehydrogenase (PfLDH) through SELEX process was immobilized over glassy carbon electrode (GCE) using graphene oxide (GO) as an immobilization matrix, and the modified electrode was investigated for detection of PfLDH. The GO was synthesized from powdered pencil graphite and characterized by XRD based on the increased interlayer distance between graphitic layers from 0.345 nm for graphite to 0.829 nm for GO. The immobilization of P38 on GO was confirmed by I/I intensity ratio in Raman spectra where, the ratio were 0.67, 0.915, and 1.35 for graphite, GO and P38-GO, respectively. The formation of the P38 layer over GO-GCE was evident from an increase in the surface height in AFM analysis of the electrode from ∼3.5 nm for GO-GCE to ∼27 nm for P38-GO-GCE. The developed aptasensor when challenged with the target, a detection of as low as 0.5 fM of PfLDH was demonstrated. The specificity of the aptasensor was confirmed through a voltametric measurement at 0.65 V of the reduced co-factor generated from the PfLDH catalysis. Studies on interference from some common proteins, storage stability, repeatability and analysis of real samples demonstrated the practical application potential of the aptasensor.
10.1016/j.ab.2016.09.013
Multiple GO-SELEX for efficient screening of flexible aptamers.
Nguyen Van-Thuan,Kwon Young Seop,Kim Jae Hoon,Gu Man Bock
Chemical communications (Cambridge, England)
We describe a simple, high-speed, high-throughput aptamer screening for a group of small molecules using graphene oxide (simple Multi-GO-SELEX) without immobilizing targets. The affinities of ten different ssDNA aptamers successfully obtained for three pesticides were in the range of 10-100 nM. Besides a specific aptamer for each target, we found a couple of flexible multi-target aptamers, which can bind with 2 or 3 different molecules. These flexible aptamers developed for binding with a mixture of targets are not only significant for the rapid screening of a group of small molecules but also offer great promise for aptamer-based biosensor applications.
10.1039/c4cc03953j
Aptamer selection for fishing of palladium ion using graphene oxide-adsorbed nanoparticles.
Cho Yea Seul,Lee Eun Jeong,Lee Gwan-Ho,Hah Sang Soo
Bioorganic & medicinal chemistry letters
A new aptamer selection method using graphene oxide (GO)-adsorbed nanoparticles (GO-adsorbed NPs) was employed for specific fishing of palladium ion. High affinity ssDNA aptamers were isolated through 13 rounds of selection and the capacity of the selected DNA aptamers for palladium ion uptake was measured, clarifying that DNA01 exhibits the highest affinity to palladium ion with a dissociation constant (Kd) of 4.60±1.17 μM. In addition, binding ability of DNA01 to palladium ion was verified against other metal ions, such as Li(+), Cs(+), Mg(2+), and Pt(2+). Results of the present study suggest that future modification of DNA01 may improve palladium ion-binding ability, leading to economic recovery of palladium from water solution.
10.1016/j.bmcl.2015.10.056
Selection of DNA aptamers against Mycobacterium tuberculosis Ag85A, and its application in a graphene oxide-based fluorometric assay.
Ansari Najmeh,Ghazvini Kiarash,Ramezani Mohammad,Shahdordizadeh Mahin,Yazdian-Robati Rezvan,Abnous Khalil,Taghdisi Seyed Mohammad
Mikrochimica acta
The Mycobacterium Ag85 complex is the major secretory protein of M. tuberculosis. It is a potential marker for early diagnosis of tuberculosis (TB). The authors have identified specific aptamers for Ag85A (FbpA) via protein SELEX using magnetic beads. After twelve rounds of selection, two aptamers (Apt8 and Apt22) were chosen from different groups, and their binding constants were determined by flow cytometry. Apt22 (labeled with Atto 647N) binds to FbpA with high affinity (K = 63 nM) and specificity. A rapid, sensitive, and low-cost fluorescent assay was designed based on the use of Apt22 and graphene oxide, with a limit of detection of 1.5 nM and an analytical range from 5 to 200 nM of FbpA. Graphical abstract Schematic illustration of graphene oxide-based aptasensor for fluorometric determination of FbpA.
10.1007/s00604-017-2550-3
Screening and identification of DNA aptamers against T-2 toxin assisted by graphene oxide.
Chen Xiujuan,Huang Yukun,Duan Nuo,Wu Shijia,Xia Yu,Ma Xiaoyuan,Zhu Changqing,Jiang Yuan,Wang Zhouping
Journal of agricultural and food chemistry
A high-affinity ssDNA aptamer that specifically binds to T-2 toxin was generated by the systemic evolution of ligands by exponential enrichment (SELEX) procedure assisted by graphene oxide (GO). After 10 rounds of selection against T-2 toxin, a highly enriched ssDNA pool was sequenced and the representative aptamers were subjected to binding assays to evaluate their affinity and specificity. Circular dichroism spectroscopy was also used to study the inherent interaction of T-2 toxin and the preferred aptamer Seq.16, which demonstrated a low dissociation constant (Kd) of 20.8 ± 3.1 nM and excellent selectivity for T-2 toxin. Using the selected aptamer Seq.16 as the recognition element, an aptamer-based fluorescent bioassay was developed for the measurement of T-2 in beer samples with a linear range from 0.5 to 37.5 μM (R(2) = 0.988) and a limit of detection (LOD) of 0.4 μM. The results indicate that GO-SELEX technology is appropriate for the screening of aptamers against small-molecule toxins, offering a promising application for aptamer-based biosensors.
10.1021/jf5032058
Graphene oxide and Lambda exonuclease assisted screening of L-carnitine aptamers and the site-directed mutagenesis design of C-rich structure aptamer.
Xing Ligang,Zhao Yanmei,Gong Mingzhu,Liu Xia,Zhang Yuhui,Li Dan,He Zefeng,Yan Ping,Yang Jidong
Biochemical and biophysical research communications
In this study, Graphene Oxide (GO) was used to screen the binding with the aptamers of L-carnitine chiral enantiomers. The ssDNA library was prepared by the method of Lambda exonuclease. In addition, a simple casing device was designed to improve the purification and recovery efficiency of the small ssDNA fragments in the process of screening. Finally, more than 160,000 aptamer sequences were obtained by high-throughput sequencing. We determined the strongest affinity aptamer sequence, CA04, by the Resonance Rayleigh scattering (RRS) technology. We also analyzed the key binding sites (in the 16th position case) of the truncated aptamer sequence CAD10. Interestingly, we found that aptamer CA10 and CA06 were both C-rich bases through sequence alignment and analysis, and the aptamer CA10 was confirmed that the CA10 and CA06 were formed under acidic conditions (pH 4.5) by CD spectrum and ESI-MS analysis. The interaction between gold nanoparticles (AuNPs) and functionalized aptamer CA10 was analyzed. We used Site-directed mutagenesis design and QGRS Mapper to optimize aptamer CA10, where an optimal aptamer CA10-03 were obtained after affinity analysis. It is also proved to be an effective method to obtain stronger affinity aptamer. Meanwhile, Native-PAGE and UV spectrum analysis were performed on the mutation sequences, and the interaction with ThT was analyzed. Finally, it is hoped that my study can provide help for later identification and detection of L-carnitine.
10.1016/j.bbrc.2021.01.044
Graphene oxide-mediated fluorescence turn-on GO-FAM-FRET aptasensor for detection of sterigmatocystin.
Analytical methods : advancing methods and applications
Sterigmatocystin (STC) is a toxic fungal secondary metabolite recognized by the FAO and WHO as a genotoxic and carcinogenic substance. STC contaminates several foods and feed commodities, posing a health risk to humans. The present study proposes to develop a graphene oxide-mediated aptasensor platform for the one-step detection of STC. In this study, DNA aptamers were generated against STC by using a target immobilization-free graphene oxide (GO)-SELEX protocol. The champion aptamers were subjected to maturation using a genetic algorithm to improve binding affinity. Further, MSA-C6 and STC interactions were characterized by MD simulation, bio-layer interferometry ( 27.9 nM) and flow cytometry. GO was immobilized on a polypropylene surface and functionalized with FAM labelled MSA-C6 to develop a simple one-step fluorescence turn-on aptasensor. The linear detection range of the aptasensor was found to be 80-720 ppb with LOD 23.56 ± 4.93 ppb and LOQ 132.43 ± 3.25 ppb. Insignificant interference of salts and detergents as well as negligible cross-reactivity with other structurally similar mycotoxins were observed. Recovery studies in simulated contaminated samples indicated appreciable recoveries (71-89%) using aptasensing assay. The results of the study indicate the successful development of a simple one-step detection platform for STC, useful for the measurement and monitoring of samples for the presence of STC. It also reports a high-affinity aptamer, which can be exploited in other sensing platforms.
10.1039/d2ay01405j
On-chip selection of adenosine aptamer using graphene oxide-coated magnetic nanoparticles.
Biomicrofluidics
Systematic evolution of ligands by exponential enrichment (SELEX) is a method that is generally used for developing aptamers, which have arisen the promising alternatives for antibodies. However, conventional SELEX methods have limitations, such as a limited selection of target molecules, time-consuming and complex fabrication processes, and labor-intensive processes, which result in low selection yields. Here, we used (i) graphene oxide (GO)-coated magnetic nanoparticles in the selection process for separation and label-free detection and (ii) a multilayered microfluidic device manufactured using a three-dimensionally printed mold that is equipped with automated control valves to achieve precise fluid flows. The developed on-chip aptamer selection device and GO-coated magnetic nanoparticles were used to screen aptamer candidates for adenosine in eight cycles of the selection process within approximately 2 h for each cycle. Based on results from isothermal titration calorimetry, an aptamer with a dissociation constant of 18.6 ± 1.5 M was selected. Therefore, the on-chip platform based on GO-coated magnetic nanoparticles provides a novel label-free screening technology for biosensors and micro/nanobiotechnology for achieving high-quality aptamers.
10.1063/5.0095419
Graphene oxide assisted light-up aptamer selection against Thioflavin T for label-free detection of microRNA.
Islam Md Mamunul,Ghielmetti Victoria Michele,Allen Peter B
Scientific reports
We selected an aptamer against a fluorogenic dye called Thioflavin T (ThT). Aptamers are single-stranded DNA that can bind a specific target. We selected the ThT aptamer using graphene oxide assisted SELEX and a low-cost Open qPCR instrument. We optimized, minimized, and characterized the best aptamer candidate against ThT. The aptamer, ThT dye, and the enzymatic strand displacement amplification (SDA) were used in a label-free approach to detect the micro RNA miR-215 in saliva and serum. The aptamer confers higher specificity than intercalating dyes but without expensive covalently modified DNA probes. This isothermal, low-cost, simple method can detect both DNA and RNA. The target, miR-215, was detected with a limit of detection of 2.6 nM.
10.1038/s41598-021-83640-z
Pattern recognition of enrichment levels of SELEX-based candidate aptamers for human C-reactive protein.
Yu Xinliang,Yu Ruqin,Yang Xiaohai
Biomedizinische Technik. Biomedical engineering
Selecting aptamers for human C-reactive protein (CRP) would be of critical importance in predicting the risk for cardiovascular disease. The enrichment level of DNA aptamers is an important parameter for selecting candidate aptamers for further affinity and specificity determination. This paper is the first report on pattern recognition used for CRP aptamer enrichment levels in the systematic evolution of ligands by exponential enrichment (SELEX) process, by applying structure-activity relationship models. After generating 10 rounds of graphene oxide (GO)-SELEX and 1670 molecular descriptors, eight molecular descriptors were selected and five latent variables were then obtained with principal component analysis (PCA), to develop a support vector classification (SVC) model. The SVC model (C=8.1728 and γ=0.2333) optimized by the particle swarm optimization (PSO) algorithm possesses an accuracy of 88.15% for the training set. Prediction results of enrichment levels for the sequences with the frequencies of 6 and 5 are reasonable and acceptable, with accuracies of 70.59% and 76.37%, respectively.
10.1515/bmt-2015-0230
Aptamer based fluorometric sulfamethazine assay based on the use of graphene oxide quantum dots.
He Yanhua,Zhang Bingyan,Fan Zhefeng
Mikrochimica acta
The authors have developed a homogeneous "off-on" fluorometric method for the determination of the antibiotic sulfamethazine (SMZ). Aptamer against SMZ was labeled with graphene oxide quantum dots upon which the Graphene oxide quenched the blue fluorescence of the GOQDs. On addition of SMZ, the aptamers will bind SMZ and this will cause the release of GOQDs. As a result, fluorescence will be regenerated. Fluorescence, best measured at excitation/emission wavelengths of 365/455 nm, increases linearly in the 8 pg·mL to 60 ng·mL SMZ concentration range, with a 5 pg·mL detection limit. The method is reliable and was successfully applied to the determination of SMZ in spiked milk samples, with recoveries ranging from 89 to 96% depending on analyte concentration. Graphical abstract Graphene oxide quantum dots (GOQDs) were covalently bound to the aptamer (apt) against sulfamethazine (SMZ) and adsorbed on the surface of graphene oxide (GO). This results in quenching of the fluorescence of GOQDs. On addition of SMZ, fluorescence is restored due to the release of GOQD@apt from GO.
10.1007/s00604-018-2695-8
Selection of DNA aptamers to Streptococcus pneumonia and fabrication of graphene oxide based fluorescent assay.
Bayraç Abdullah Tahir,Donmez Sultan Ilayda
Analytical biochemistry
Pneumococci are one of the leading causes of infections throughout the world causing problems mainly in children, elderly, and immune-deficient patients. In recent years antibiotic resistant Streptococcus pneumoniae strains become widespread. Therefore simple, rapid, and specific detection methods are needed for public health. In this study, DNA aptamer probes against S. pneumoniae were selected using bacterial Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and these probes were integrated in to a graphene oxide (GO) based fluorescent assay. Among the tested aptamers three candidates Lyd-1, Lyd-2 and Lyd-3 showed K values of 844.7 ± 123.6, 1984.8 ± 347.5, and 661.8 ± 111.3 nM, respectively. These candidates showed binding affinity to S. pneumoniae and no specific binding to the bacteria used in negative selection. The binding of aptamers were showed by fluorescence spectroscopy and flow cytometry. GO based label-free fluorescent assay developed using Lyd-3 aptamer had a unique detection limit of 15 cfu mL. Thus we believe that the selected aptamers and fabricated GO based assay has potential to be used in the detection of S. pneumoniae. Selected aptamers selectively bind to S. pneumonia with anti-pneumococcal potential and holds great potential to be used as molecular probes for identifying and targeting.
10.1016/j.ab.2018.06.024
Development of fluorescent aptasensor for detection of acephate by utilizing graphene oxide platform.
Talanta
A fluorescent graphene oxide based aptasensing platform was developed for the detection of acephate. The aptamers specific to the acephate were screened through GO-SELEX (Graphene Oxide - Systematic Evolution of Ligands by EXponential enrichment) method for six rounds. The screened aptamers were analyzed for their binding affinity and specificity by using fluorescence-based assay. The aptamer AAPT3 that demonstrated highest affinity (K = 9 ± 1 nM) and an excellent selectivity, was employed in the development of fluorescent aptasensor. Under optimal conditions, the aptasensor showed low limit of detection (4 ng mL) and a wide dynamic linear range (5-80 ng mL). The aptasensor was also validated against water samples spiked with acephate, which showed fluorescence recovery from 94 to 107% and coefficient of variation 1-5%. These results indicate that the developed aptasensor can be used for sensitive, selective, and accurate detection of acephate in various samples.
10.1016/j.talanta.2022.123843
Selection and Application of ssDNA Aptamers against Clenbuterol Hydrochloride Based on ssDNA Library Immobilized SELEX.
Duan Nuo,Gong Wenhui,Wu Shijia,Wang Zhouping
Journal of agricultural and food chemistry
Clenbuterol hydrochloride (CLB) is often abused as additive feed for livestock to decrease adipose tissue deposition and to increase growth rate. It raises a potential risk to human health through the consumption of animal product. In this study, aptamers with higher affinity and specificity were screened through 16 selection rounds based on the ssDNA library immobilized systematic evolution of ligands by exponential enrichment (SELEX) technique. After cloning and sequencing, five aptamer candidates were picked out for affinity and specificity assays based on a graphene oxide (GO) adsorption method. The results showed that the aptamer CLB-2 binds specifically against CLB with a dissociation constant, K, value of 76.61 ± 12.70 nM. In addition, an aptamer-based fluorescence bioassay was established for CLB analysis. The correlation between the CLB concentration and fluorescent signal was found to be linear within the range of 0.10 to 50 ng/mL with a limit of detection of 0.07 ng/mL. It has been further applied for the determination of CLB in pork samples, showing its great potential for sensitive analysis in food safety control.
10.1021/acs.jafc.6b04951
Development of a ssDNA aptamer system with reduced graphene oxide (rGO) to detect nonylphenol ethoxylate in domestic detergent.
Kim A-Ru,Ha Na-Reum,Jung In-Pil,Kim Sang-Heon,Yoon Moon-Young
Journal of molecular recognition : JMR
Endocrine-disrupting chemicals are a major public health problem throughout the world. In the human body, these compounds functionalize the same as sexual hormones, inducing precocious puberty, gynecomastia, etc. To help prevent this occurrence, a simple detection system is needed. In this study, a nonylphenol ethoxylate (NPE)-specific aptamer was selected by reduced graphene oxide-systematic evolution of ligands by exponential enrichment. A random ssDNA library was incubated with rGO for adsorption, followed by elution with the target molecule. As a result of screening, a DNA aptamer was found that specifically bounds to the target with high binding affinity (K = 100.9 ± 13.2 nM) and had a low limit of detection (LOD = 696 pM). Furthermore, this NPE-binding aptamer bounds selectively to the target. Characterization of the aptamer was confirmed by measuring the fluorescence signal recovery from rGO. In addition, detection of NPE was performed with several water samples, and the detection accuracy was 100 ± 10%. From these results, we expect that this aptamer could be applied to an on-site detection system for NPE in industrial sites or domestic fields.
10.1002/jmr.2764
An ssDNA library immobilized SELEX technique for selection of an aptamer against ractopamine.
Duan Nuo,Gong Wenhui,Wu Shijia,Wang Zhouping
Analytica chimica acta
An improved SELEX technique was developed for selecting aptamers against ractopamine (RAC) by immobilizing ssDNA library on the magnetic beads. After sixteen selection rounds, a highly enriched ssDNA pool was sequenced and nine families were grouped according to their homology and secondary structures analysis. One representative aptamer candidate from each family was picked out for binding affinity identification by graphene oxide (GO) adsorption platform. The aptamer RAC-6 was demonstrated as the optimal aptamer with high specificity and dissociation constant (K) value of 54.22 ± 8.02 nM. To prove the potential application of aptamer RAC-6 in the quantitative determination of RAC, a fluorescent bioassay with aptamer RAC-6 was developed. The linear range for RAC was from 0.10 ng/mL to 100 ng/mL and the limit of detection was as low as 0.04 ng/mL. Furthermore, the method was validated for the analysis of RAC spiked real samples, and the recoveries were between 82.57% and 104.65%.
10.1016/j.aca.2017.01.008
Rapid identification and quantitation of the viable cells of Lactobacillus casei in fermented dairy products using an aptamer-based strategy powered by a novel cell-SELEX protocol.
Song Shixi,Wang Xingyu,Xu Ke,Ning Lufang,Yang Xingbin
Journal of dairy science
An aptamer-based strategy was developed for qualitative and quantitative analysis of viable Lactobacillus casei in dairy products. Three highly specific aptamers for L. casei were obtained using systematic evolution of ligands by exponential enrichment protocol using the whole bacterium cell as the target (cell-SELEX) facilitated by polyethyleneglycol and chitosan modified graphene oxide and complementary ring-mediated rolling circle amplification. Two aptamers, one for separating and enriching the L. casei cells and the other for generating fluorescence signals, were employed to develop an aptamer-based strategy, which was demonstrated for the selective detection of L. casei in commercial dairy drinks, with a dynamic range of 10 to 10 cfu/mL. Viable and nonviable L. casei cells could be discriminated based on the significant difference in fluorescence intensity. This established strategy is of high selectivity and sensitivity, and can be used for rapid analysis of viable L. casei in quality control and food surveillance areas.
10.3168/jds.2019-16693
Selection of specific aptamer against enrofloxacin and fabrication of graphene oxide based label-free fluorescent assay.
Dolati Somayeh,Ramezani Mohammad,Nabavinia Maryam Sadat,Soheili Vahid,Abnous Khalil,Taghdisi Seyed Mohammad
Analytical biochemistry
Specific ssDNA aptamers for the antibiotic enrofloxacin (ENR) were isolated from an enriched nucleotide library by SELEX (Systematic Evolution of Ligands by EXponential enrichment) method with high binding affinity. After seven rounds, five aptamers were selected and identified. Apt58 with highest affinity and sensitivity (K = 14.19 nM) was employed to develop a label-free fluorescent biosensing approach based on aptamer, graphene oxide (GO) and native fluorescence of ENR for determination of ENR residue in raw milk samples. Under optimized experimental conditions, the linear range was from 5 nM to 250 nM and LOD was calculated to be 3.7 nM, and the recovery rate was between 94.1% and 108.5%. The integration of aptamer and GO in this bioassay provides a promising way for rapid, sensitive and cost-effective detection of ENR in real samples like raw milk.
10.1016/j.ab.2018.03.021
Targeted inhibition of methicillin-resistant biofilm formation by a graphene oxide-loaded aptamer/berberine bifunctional complex.
Drug delivery
Biofilm formation is known to promote drug resistance in methicillin-resistant (MRSA), which is closely related to persistent infections in hospital settings. In this study, a DNA aptamer specific to penicillin-binding protein 2a (PBP2a) with a dissociation constant () of 82.97 ± 8.86 nM was obtained after 14 cycles of systematic evolution of ligands by exponential enrichment (SELEX). Next, a bifunctional complex containing the aptamer intercalated by berberine into the double-strand region was prepared and adsorbed onto the surface of graphene oxide GO) by π-stacking interactions. The GO-loaded aptamer/berberine bifunctional complex showed significantly higher inhibition of MRSA biofilm formation than the control. Furthermore, this study shows that the complex possesses anti-biofilm activity, which can be attributed to the ability of the aptamer to reduce cell-surface attachment by blocking the function of PBP2a and berberine to attenuate the level of the accessory gene regulator () system, which plays an important role in mediating MRSA biofilm formation. Therefore, the simultaneous delivery of berberine and PBP2a-targted aptamer using GO may have potential for the treatment of chronic infections caused by MRSA biofilms. It also provides a new avenue for multitarget treatment of bacterial biofilms.
10.1080/10717544.2022.2079768
Magnetic Separation-Based Multiple SELEX for Effectively Selecting Aptamers against Saxitoxin, Domoic Acid, and Tetrodotoxin.
Gu Huajie,Duan Nuo,Xia Yu,Hun Xu,Wang Haitao,Wang Zhouping
Journal of agricultural and food chemistry
In this study, a novel magnetic separation-based multiple systematic evolution of ligands by exponential enrichment (SELEX) was applied to select aptamers simultaneously against three kinds of marine biotoxins, including domoic acid (DA), saxitoxin (STX), and tetrodotoxin (TTX). Magnetic reduced graphene oxide (MRGO) was prepared to adsorb unbound ssDNAs and simplify the separation step. In the multiple SELEX, after the initial twelve rounds of selection against mixed targets and the subsequent four respective rounds of selection against each single target, the three resulting ssDNA pools were cloned, sequenced, and analyzed. Several aptamer candidates were selected and subjected to the binding affinity and specificity test. Finally, DA-06 ( K = 62.07 ± 19.97 nM), TTX-07 ( K = 44.12 ± 15.38 nM), and STX-41 ( K = 61.44 ± 23.18 nM) showed high affinity and good specificity for DA, TTX, and STX, respectively. They were also applied to detect and quantify DA, TTX, and STX successfully. The other two multitarget aptamers, DA-01 and TTX-27, were also obtained, which can bind with either DA or TTX. These aptamers provide alternative recognition molecules to antibodies for biosensor applications.
10.1021/acs.jafc.8b02771
In Vivo Monitoring of Intracellular Metabolite in a Microalgal Cell Using an Aptamer/Graphene Oxide Nanosheet Complex.
Jin Cho Rok,Kim Jee Young,Kim Da Hee,Jeon Min Seo,Choi Yoon-E
ACS applied bio materials
Real-time sensing and imaging of intracellular metabolites in living cells are crucial tools for the characterization of complex biological processes, including the dynamic fluctuation of metabolites. Therefore, additional efforts are required to develop detection strategies for the visualization and quantification of specific target metabolites, particularly in microalgae. In this study, we developed a strategy to monitor a specific microalgal metabolite in living cells using an aptamer/graphene oxide nanosheet (GOnS) complex. As a proof-of-concept, β-carotene, an antioxidant pigment that accumulates in most microalgal species, was chosen as a target metabolite. To achieve this, a β-carotene-specific aptamer was selected through graphene oxide-assisted systematic evolution of ligands by exponential enrichment (GO-SELEX) and characterized thereafter. The aptamer could sensitively sense the changes in the concentration of β-carotene (, the target metabolite) and more specifically bind to β-carotene than to nontargets. The selected aptamer was labeled with a fluorophore (fluorescein; FAM) and allowed to form an aptamer/GOnS complex that protected the aptamer from nucleic cleavages. The aptamer/GOnS complex was delivered into the cells via electroporation, thus enabling the sensitive monitoring of β-carotene in the cell by quantifying the aptamer fluorescence intensity. The results suggest that our biocompatible strategy could be employed to visualize and semiquantify intracellular microalgae metabolites , which holds a great potential in diverse fields such as metabolite analysis and mutant screening.
10.1021/acsabm.1c00322
Development of a Novel ssDNA Sequence for a Glycated Human Serum Albumin and Construction of a Simple Aptasensor System Based on Reduced Graphene Oxide (rGO).
Biosensors
Diabetes is one of the top 10 global causes of death. About one in 11 global adults have diabetes. As the disease progresses, the mortality rate increases, and complications can develop. Thus, early detection and effective management of diabetes are especially important. Herein, we present a novel glycated human serum albumin (GHSA) aptamer, i.e., GABAS-01, which has high affinity and specificity. The aptamer was selected by reduced graphene oxide-based systematic evolution of ligands by exponential enrichement (rGO-based SELEX) against GHSA. After five rounds of selection through gradually harsher conditions, GABAS-01 with high affinity and specificity for the target was obtained. GABAS-01 was labeled by FAM at the 5'-end and characterized by measuring the recovery of a fluorescence signal that is the result of fluorescence quenching effect of rGO. As a result, GABAS-01 had low-nanomolar Kd values of 1.748 ± 0.227 nM and showed a low limit of detection of 16.40 μg/mL against GHSA. This result shows the potential application of GABAS-01 as an effective on-site detection probe of GHSA. In addition, these properties of GABAS-01 are expected to contribute to detection of GHSA in diagnostic fields.
10.3390/bios10100141
Non-immobilized GO-SELEX of aptamers for label-free detection of thiamethoxam in vegetables.
Kong Qianqian,Yue Fengling,Liu Mengyue,Huang Jingcheng,Yang Fengzhen,Liu Junjie,Li Jiansen,Li Falan,Sun Xia,Guo Yemin,Zhu Yelong
Analytica chimica acta
Due to the massive use of thiamethoxam (TMX) pesticide and the accumulated potential hazards exposure, the detection of TMX is of great significance to food and ecological safety. In this study, aptamers with affinity for TMX were obtained through graphene oxide assisted systematic evolution of ligands by exponential enrichment (GO-SELEX). After 9 rounds of positive and counter selection, 5 candidate sequences were obtained, among which seq.20 had the highest affinity for TMX, and its dissociation constant (K) was 210.47 ± 79.37 nM. Then, the aptamer was further truncated based on structural analysis. The truncated aptamers (seq.20-1, seq.20-2) exhibited higher affinity (K = 118.34 ± 13.85 nM, K = 123.35 ± 29.80 nM), which seq.20-2 had only 37 bases. Furthermore, circular dichroism spectroscopy showed that TMX induced the conformation of aptamer from B-form structure to hairpin structure, and then formed a stable TMX-ssDNA complex. Finally, the truncated aptamer (seq.20-2) and the original aptamer (seq.20) were used as recognition elements to construct colorimetric aptasensors based on gold nanoparticles for the detection of TMX. It was found that the sensitivity of the former (LOD = 1.67 ± 0.12 nM, S/N = 3) was better than that of the latter (LOD = 3.33 ± 0.23 nM, S/N = 3). Feasibility of truncated aptamer as recognition element in the detection of TMX in vegetable samples was preliminarily verified.
10.1016/j.aca.2022.339677
Selection of DNA aptamers for tramadol through the systematic evolution of ligands by exponential enrichment method for fabrication of a sensitive fluorescent aptasensor based on graphene oxide.
Hedayati Narges,Taghdisi Seyed Mohammad,Yazdian-Robati Rezvan,Mansouri Atena,Abnous Khalil,Ahmad Mohajeri Seyed
Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy
Tramadol hydrochloride (TH), as an atypical opioid and a 4-phenyl-piperidine analogue of codeine, is mainly used for treating moderate to severe pains. Due to its extensive application, the consequent need for its analysis in various samples is essential. The current study focuses on the introduction of a rapid fluorescent assay using graphene oxide (GO) and aptamer for determination of tramadol in serum samples. Specific ssDNA aptamers for TH were developed by SELEX (Systematic Evolution of Ligands by EXponential Enrichment) technique using GO as a fluorescence quencher. After 10 rounds, two aptamers (Apt19 and Apt39) were selected from various families. Then, the binding constants of aptamers were measured using fluorometric assay and finally Apt39 (labeled with ATTO 647N) was chosen for development of a fluorescent aptasensor because this aptamer bound to TH with high affinity (K = 178.4 nM) and specificity. The current analytical system showed detection limits of 1.04 nM and 2.56 nM in serum sample and phosphate buffer saline (10 mM PBS), respectively.
10.1016/j.saa.2021.119840
Selection and truncation of aptamers for ultrasensitive detection of sulfamethazine using a fluorescent biosensor based on graphene oxide.
Kou Qiming,Wu Ping,Sun Qi,Li Chenxi,Zhang Lei,Shi Haixing,Wu Juan,Wang Yarong,Yan Xueling,Le Tao
Analytical and bioanalytical chemistry
We developed a fluorescent aptamer/graphene oxide (GO)-based biosensor to detect sulfamethazine (SMZ) residues in animal-derived foods. The SMZ-bound aptamers were identified and screened with an improved GO-SELEX technique using non-immobilizing ssDNA library. After seven rounds of selection, six SMZ aptamers were sequenced and analyzed for secondary structure, and their affinity and specificity were assessed by binding assays. The truncated aptamer (SMZ1S: 5'-CGTTAGACG-3') with a unique stem-loop structure showed the highest affinity (K = 24.6 nM) to SMZ and was used to develop a GO-based fluorescent aptasensor. The binding mechanism between SMZ1S and SMZ was further analyzed by molecular docking. Under optimal conditions, the fluorescent aptasensor showed low detection limits (0.35 ng/mL) and a wide dynamic linear range (from 2 to 100 ng/mL). The aptasensor was also validated against real samples spiked with SMZ, which showed a fluorescence recovery from 93.9 to 108.8% and a coefficient of variation of < 12.7%. Taken together, these results suggest that this novel aptasensor can be used to sensitively, selectively, and accurately detect SMZ residues in foods. Schematic illustration of fluorescent aptasensor based on aptamer/graphene oxide complex detection of of SMZ.
10.1007/s00216-020-03044-2
Graphene oxide-assisted non-immobilized SELEX of okdaic acid aptamer and the analytical application of aptasensor.
Gu Huajie,Duan Nuo,Wu Shijia,Hao Liling,Xia Yu,Ma Xiaoyuan,Wang Zhouping
Scientific reports
Okadaic acid (OA) is a low-molecular-weight marine toxin from shellfish that causes abdominal pain, vomiting and diarrhea, i.e., diarrheic shellfish poisoning. In this study, a ssDNA aptamer that specifically binds to OA with high affinity was obtained via Systematic Evolution of Ligands by Exponential Enrichment (SELEX) assisted by graphene oxide (GO). This aptamer was then applied to fabricate a novel direct competitive enzyme-linked aptamer assay (ELAA). At the optimized conditions, this ELAA method showed a low detection limit (LOD of 0.01 ng/mL), wide linear range (from 0.025 to 10 ng/mL), good recovery rate (92.86-103.34% in OA-spiked clam samples) and repeatability (RSD of 2.28-4.53%). The proposed method can be used to detect OA in seafood products with high sensitivity and can potentially be adapted for the determination of other small molecular analytes.
10.1038/srep21665
Graphene oxide-assisted non-immobilized SELEX of chiral drug ephedrine aptamers and the analytical binding mechanism.
Xing Ligang,Zhang Yuhui,Yang Jidong
Biochemical and biophysical research communications
Here, we describe a study of screen characterization of aptamers targeting the chiral drug ephedrine using the non-immobilized graphene oxide (GO) SELEX. The improved method of long and short chains was here used to prepare the ssDNA library. The Resonance Rayleigh Scattering (RRS) method was first used to monitor the screening process. Through high-throughput sequencing, the genetic sequence data of 90,487 aptamers were obtained. Through the analysis of the parameters of free energy value and secondary structure prediction model of high repeatability sequence, the 10 candidate sequences were identified. Finally, a best-fit aptamer named EP08 was identified by combining the dissociation experiment. The binding affinity and binding mechanism of the aptamer and target were analyzed using an isothermal titration colorimetry (ITC) experiment and circular dichromatic (CD) experiment. The binding affinity (Kd) of the EP08 aptamer to ephedrine is approximately 2.86 ± 0.24 μM. This novel DNA aptamer will help in the future development of a new method for the identification and detection of chiral drug ephedrine.
10.1016/j.bbrc.2019.04.067
Selection of highly specific aptamers to Vibrio parahaemolyticus using cell-SELEX powered by functionalized graphene oxide and rolling circle amplification.
Song Shixi,Wang Xingyu,Xu Ke,Li Qiao,Ning Lufang,Yang Xingbin
Analytica chimica acta
Cell-SELEX is a powerful tool to screen aptamers binding to living cellular organisms such as bacteria, fungi and even oncocytes. Here, we developed an advanced cell-SELEX strategy featuring functionalized graphene oxide (GO) and isothermal rolling circle amplification (RCA) to select aptamers against a prevailing foodborne pathogen, Vibrio parahaemolyticus. Polyethyleneglycol (PEG) and chitosan (CTS) were grafted onto the sheet-like GO molecules to synthesize a PC-GO material. TEM and FT-IR characterization demonstrated that the PC-GO composites were near-nanometric scale and tethered with PEG and CTS moieties, a property that significantly improved its solubility in biological buffer solutions used in cell-SELEX process. PC-GO could bind with ssDNAs with lower affinities to target cells, therefore the selection efficiency is greatly enhanced. The cell-binding aptamer candidates (CACs) were amplified by 10 fold using complementary ring mediated (CRM-RCA), a created amplification method that generate single-stranded products, which could be directly used in the next round selection. As fueled by PC-GO and CRM-RCA, four highly specific aptamers with lowest K value of 10.3 ± 2.5 nM were obtained. Flow cytometry analysis showed that all the four aptamers exhibited more than 75% binding affinity to V. parahaemolyticus than to other foodborne bacteria (less than 18%). Simple procedure, high efficiency, and free from expensive thermal cycler (required by PCR amplification) will enable the established strategy to find its applications in aptamer selecting against fungi, stem and cancerous cells as well.
10.1016/j.aca.2018.11.047