Recent progress in aptamer-based microfluidics for the detection of circulating tumor cells and extracellular vesicles

Liquid biopsy is a technology that exhibits potential to detect cancer early,monitor therapies,and predict cancer prognosis due to its unique characteristics,including noninvasive sampling and real-time analysis.Circulating tumor cells(CTCs)and extracellular vesicles(EVs)are two important components of circulating targets,carrying substantial disease-related molecular information and playing a key role in liquid biopsy.Aptamers are single-stranded oligonucleotides with superior affinity and specificity,and they can bind to targets by folding into unique tertiary structures.Aptamer-based microfluidic platforms offer new ways to enhance the purity and capture efficiency of CTCs and EVs by combining the advantages of microfluidic chips as isolation platforms and aptamers as recognition tools.In this review,we first briefly introduce some new strategies for aptamer discovery based on traditional and aptamer-based microfluidic approaches.Then,we subsequently summarize the progress of aptamer-based microfluidics for CTC and EV detection.Finally,we offer an outlook on the future directional challenges of aptamer-based microfluidics for circulating targets in clinical applications.

Raloxifene-loaded and aptamer-bonded exosomes induce autophagic and apoptotic death in HeLa cells by enhancing the lysosomotropic effect

Background:Raloxifene,a selective estrogen receptor modulator,is also known to be a lysosomotropic agent.The bioavailability of raloxifene is around 2%due to extensive hepatic transport.Exosomes are nanosized vesicles that are naturally released from cells.Method:In this study,exosomes released from HeLa cervical cancer cells were loaded with raloxifene to increase its bioavailability,and an aptamer was attached to the exosome membrane for targeting only HeLa cells.Characterization of exosomes isolated from HeLa cells was performed by transmission electron microscopy,zeta sizer,and western blotting.In addition,the cytotoxic,apoptotic,autophagic,and lysosomotropic effects of the prepared Exo-Apt-Ral formulation on HeLa cervical cancer cells were investigated.Results:According to zeta analysis,the sizes of the empty exosome and Exo-Apt-Ral formulation were measured as 66±12 and 120±21 nm,respectively.There was a rise in the lysosomal permeability of HeLa cells after the Exo-Apt-Ral application.In addition,both apoptotic and autophagic death mechanisms were triggered in HeLa cells after the Exo-Apt-Ral application.Conclusion:This study showed that raloxifene functionalized by loading into aptamer-bound exosomes can be a new targeted drug carrier system for cervical cancer.

Recognition mechanism and sequence optimization of organophosphorus pesticides aptamers for better monitoring contaminations in food

Aptamers as a kind of biological recognition element have shown great potential in monitoring and the rapid quantification of organophosphorus pesticides(OPPs). However, molecules of OPPs are structurally similar and original aptamers selected by systematic evolution of ligands by exponential enrichment are usually long-chain bases, which hamper the further application under OPPs-aptamer recognition. The aim of the research was to develop a new strategy to design oligonucleotide sequences for binding OPPs by combination of experimental and molecular modeling methods. 3D models of aptamers binding OPPs were constructed, and binding energy and the most probable binding site for the OPPs were then determined by molecular docking, and the binding sites were further confirmed by the results of 2-AP replaced experiments. Based on the docking results, a new aptamer for detection 4 representative OPPs with only 29 bases was designed by reasonable truncation and mutation of the reported aptamer(named S4-29). The interaction between this new aptamer and OPPs were analyzed by molecular docking, microscale thermophoresis, circular dichroism and fluorometric analysis. The results revealed that the new aptamer exhibit more superior recognition performance to OPPs, which can be promote the monitoring ability of OPPs contaminations in food.

Investigation on small molecule-aptamer dissociation equilibria based on antisense displacement probe

Food safety is a major issue to public health and have attracted global attention.Fast,sensitive,and reliable detection methods for food hazardous substances is highly desirable.Aptamers which can bind to the target molecules with high affinity and specificity represent an attractive tool for the recognition of food hazardous substances,which play an important role in the development and application of new food safety detection technology.But current assays for characterizing small molecule-aptamer binding are limited by either the mass sensitivity or the size differentiation ability.Herein,we proposed a comprehensive method for assessing the dissociation equilibria of small molecule-aptamer,which is immobilized-free under ambient conditions.The design employs the Le Chatelier’s principle and could be used to effectively measure small molecule-aptamer interactions.ATP binding aptamer and anti-aflatoxin B1 aptamer were used as the model system to determine their affinity,in which their dissociation equilibria measurements are in excellent close to their previous work.Due to the simplicity and sensitivity of this new method,we believe that it could be recommended as an effective tool for characterizing small molecule-aptamer interactions and promote the further application of small molecular aptamer in food safety.

Electrochemical Biosensor for Detection of Staphylococcus aureus Based on Nucleic Acid Aptamers

Staphylococcus aureus is a gram-staining positive cocci bacillus baterium and also one of the foodborne pathogens, which is a serious potential hazard to human health and food safety. We constructed an electrochemical biosensor for the detection of S. aureus based on nucleic acid aptamers to achieve highly specific detection of S. aureus. The detection of S. aureus was realized by using Aptamer (Apt) to capture S. aureus, which resulted in a change in the spatial conformation of Apt and a decrease in the electrochemical signal. Under the optimized experimental conditions, the detected electrochemical signals were positively correlated with the concentration of S. aureus with a linear range of 1 × 101 - 1 × 105 CFU/mL, a detection limit of 4.76 CFU/mL, and an experimental recovery of 97.43% - 99.37%. Therefore, we successfully constructed an electrochemical biosensor for the specific detection of S. aureus, which has the advantages of high specificity, sensitive detection and convenient operation.

SELEX技术及Aptamer研究的新进展

综述了近几年指数富集的配体系统进化(SELEX)技术的改良与寡核苷酸配基(aptamer)研究应用方面的新进展.Aptamer是指利用SELEX(systematicevolutionofligandsbyexponentialenrichment)技术,从随机寡核苷酸文库中筛选获得的能够与靶分子特异结合的短单链寡核苷酸配基,通常具有纳摩尔到皮摩尔的亲和力.高通量筛选的技术特点与aptamer精确识别、易体外合成与修饰等特性,使得aptamer在分析化学与生物医药研究方面具有广阔的应用前景.

基于RhD蛋白Aptamer筛选技术的随机ssDNA次级文库制备条件的优化

目的优化筛选RhD蛋白DNA-aptamer的ssDNA次级文库制备条件。方法对影响dsDNA扩增的主要参数(退火温度、循环数、模板用量、引物用量等)进行优化,在确定dsDNA扩增条件已优化的基础上,进而优化上、下游引物用量比例及扩增循环数,确定不对称PCR制备ssDNA的最佳条件。结果不对称PCR扩增的最佳条件为:上游引物终浓度为1 pmol/μL,上、下游引物浓度比例为20∶1,退火温度为59℃,循环数为30—35。结论制备条件优化后,经过不对称PCR扩增可获得浓度高、纯度好的ssDNA次级文库。

特异性鼠源肝癌细胞靶向的量子点标记Aptamer荧光生物探针

核酸适配体（Aptamer）是由几十个碱基组成的SSDNA，具有与靶物质特异性、高亲和结合的能力且配体范围非常广范，而量子点（Quantum dot，QDs）则具有独特的荧光性质和生物相容性，二者均在在细胞生物学领域具有重要的应用价值。相关文献表明，量子点标记核酸适配体的荧光生物探针可为癌症研究提供新的靶向研究技术。

Aptamer的原理、优越性与应用

从Aptamer能与靶分子特异结合的能力、抑制蛋白质活性的能力以及核酸本身易被修饰的性质等方面展开讨论,综述了Aptamer在科研、医药、工业方面的应用.

H22肿瘤细胞aptamer的筛选与结构分析

目的:获得与H22肿瘤细胞特异性结合的aptamer。方法:利用SELEX技术,以小鼠DC细胞为反筛选细胞,以H22肿瘤细胞为靶细胞,从体外合成的80bp随机单链DNA文库中筛选出能与H 22肿瘤细胞特异结合的aptamer。采用荧光标记引物法检测aptamer与H 22肿瘤细胞的亲和力。所获得的aptamer采用MACAWv2.05软件进行aptamer序列的一级结构同源序列比较,用DNASIS v2.5软件计算分析序列最低二级结构能量值,获得其二级结构模拟图。结果:本实验设计的文库序列:5′-CGTCGCTGCACATTCCG-N46-CGCACAGCTGGGA GTAC-3′具有较高的扩增效率,适合于aptamer与以细胞为靶物质的筛选。经过11轮循环筛选,随机单链DNA文库与靶细胞结合的荧光强度从1%上升到59%,结合曲线进入平台期,表明结合已基本处于稳定状态,因此可以判断aptamer与靶细胞的结合基本已经处于饱和状态。对所获得的32个aptam er进行测序,然后进行一级结构和二级结构分析。一级结构分析获得5个保守序列:AGGGA、AGAAGG、GTGAXAA、ATAGT、CAAGG,其余10个aptamer无同源序列。二级结构分析表明,aptamer形成的茎环、凸环结构可能是与H 22肿瘤细胞特异性结合的结构基础。亲和力检测结果表明第24号aptamer具有最强的亲和力。结论:利用随机单链寡核苷酸文库成功获得与H22肿瘤细胞特异结合的aptamer,其一级和二级结构与亲和力密切相关。

Aptamer-Based Extraction of Ergot Alkaloids from Ergot Contaminated Rye Feed

Ergot alkaloids are mycotoxins which can be found in food based on cereal-crops, due to a contamination of plants by fungi of the genus Claviceps. The ingestion of ergot contaminated cereal crops can lead to a severe poisoning known as ergotism. For food and feed safety purposes, the extraction of ergot alkaloids from ergot contaminated flour was investigated. For the specific recognition of ergot alkaloids, DNA aptamer ligands specially selected for ergot alkaloids were grafted onto silica gel in order to construct a specific solid phase extraction system. The aptamer-functionalized silica gels were used to extract ergot alkaloids from a contaminated rye feed sample. The presence of ergot alkaloids eluted from the aptamer-functionalized silica gels was analyzed using LC-QTOF-MS. By using this simple system, it was possible to specifically extract ergosine, ergokryptine and ergocornine from an ergot contaminated rye feed sample. This aptamer-based extraction tool shows the applicability of aptamers for the specific extraction of toxins or natural compounds from turbid matrices in a one-step procedure.

Screening of aptamers and their potential application in targeted diagnosis and therapy of liver cancer

Aptamers are a class of single oligonucleotide molecules(DNA or RNA)that are screened from random DNA or RNA oligonucleotide chain libraries by the systemic evolution of ligands by exponential enrichment technology.The selected aptamers are capable of specifically binding to different targeting molecules,which is achieved by the three-dimensional structure of aptamers.Aptamers are similar in function to monoclonal antibodies,and therefore,they are also referred to as"chemical antibodies".Due to their high affinity and specificity and low immunogenicity,aptamers are topics of intense interest in today's biological targeting research especially in tumor research.They not only have high potential for clinical advances in tumor targeting detection but also are highly promising as targeted tumor drug carriers for use in tumor therapy.Various experimental studies have shown that aptamer-based diagnostic and therapeutic methods for liver cancer have great potential for application.This paper summarizes the structure,characteristics,and screening methods of aptamers and reviews the recent research progress on nucleic acid aptamers in the targeted diagnosis and treatment of liver cancer.

Development of a Label-Free Colorimetric Aptasensor with Rationally Utilized Aptamer for Rapid Detection of Okadaic Acid

Okadaic acid(OA)is a typical marine toxin with strong toxicity causing diarrheic shellfish poisoning(DSP).Aptamers show great advantages in toxin detection and attract increasing attentions in the field of food analysis.In this study,a label-free col-orimetric aptasensor was constructed for visual and rapid detection of OA in shellfish.To exploit the binding capability of the anti-OA aptamer,the inherent molecular recognition mechanism of aptamer and OA was studied,based on molecular docking,fluorescent assay,and biolayer interferometry.Consistent results showed that the stem-loop near the 3’terminal of the aptamer exhibit dominate binding capacity.Based on the revealed recognition information,the aptamer was thus rationally utilized and combined with AuNPs and cationic polymer polydiallyl dimethyl ammonium chloride(PDDA)for the development of the label-free colorimetric aptasensor,in which the 3’terminal was thoroughly exposed to OA.The aptasensor provided robust performance with a linear detection range of 100-1200 nmol L-1,a limit of detection of 41.30 nmol L-1,recovery rates of 91.6%-106.2%,as well as a high selectivity towards OA in shellfish samples.The whole detection process can be completed within 1 h.To our best knowledge,this is the first time that the anti-OA aptamer was thoroughly studied,and a label-free colorimetric aptasensor was rationally designed in this way.This study not only provides a rapid detection method for highly sensitive and specific detection of OA,but also serves as a reference for the design of efficient aptasensors in the future.

Aptamer-based Electrochemical Biosensors for Highly Selective and Quantitative Detection of Adenosine

A new adenosine biosensor based on aptamer probe is introduced in this article. An amino-labeled aptamer probe was immobilized on the gold electrode modified with an o-phenylenediamine electropolymerized film. When adenosine is bound specifically to the aptamer probe, the interface of the biosensor is changed, resulting in the decrement of the peak current. The response current is proportional to the amount of adenosine in sample. The used electrode can be easily regenerated in hot water. The proposed biosensor represents a linear response to adenosine over a concentration range of 1.0x 10^-7-l.0x10^-4 mol/L with a detection limit of 1.0xl0^-8 mol/L. The presented biosensor exhibits a nice specificity towards adenosine. It offers a promising approach for adenosine assay due to its excellent electrochemical properties that are believed to be very attractive for electrochemical studies and electroanalytical applications.

Aptameric SERS sensor for Hg^(2+) analysis using silver nanoparticles

Aptamer-silver nanoparticles （AgNPs） based surface-enhanced Raman scattering （SERS） sensor has been developed for Hg^2＋ detection by employing the structure-switching aptamer in the presence of spermine. This simple method shows excellent sensitivity and selectivity owing to the sensitive SERS detection technique and high specificity of aptamer for binding Hg^2＋.

Highly sensitive chemiluminescence technology for protein detection using aptamer-based rolling circle amplification platform

A robust, selective and highly sensitive chemiluminescent （CL） platform for protein assay was presented in this paper. This novel CL approach utilized rolling circle amplification （RCA） as a signal enhancement technique and the 96-well plate as the immobilization and separation carrier. Typically, the antibody immobilized on the surface of 96-well plate was sandwiched with the protein target and the aptamer-primer sequence. This aptamer-primer sequence was then employed as the primer of RCA. Based on this design, a number of the biotinylated probes and streptavidin-horseradish peroxidase （SA-HRP） were captured on the plate, and the CL signal was amplified. In summary, our results demonstrated a robust biosensor with a detection limit of 10 fM that is easy to be established and utilized, and devoid of light source. Therefore, this new technique .will broaden the perspective for future development of DNA-based biosensors for the detection of other protein biomarkers related to clinical diseases, by taking advantages of high sensitivity and selectivity.

The novel amyloid-beta peptide aptamer inhibits intracellular amyloid-beta peptide toxicity

Amyloid β peptide binding alcohol dehydrogenase （ABAD） decoy peptide （DP） can competitively antagonize binding of amyloid β peptide to ABAD and inhibit the cytotoxic effects of amyloid β peptide. Based on peptide aptamers, the present study inserted ABAD-DP into the disulfide bond of human thioredoxin （TRX） using molecular cloning technique to construct a fusion gene that can express the TRX1-ABAD-DP-TRX2 aptamer. Moreover, adeno-associated virus was used to allow its stable expression. Immunofluorescent staining revealed the co-expression of the transduced fusion gene TRX1-ABAD-DP-TRX2 and amyloid β peptide in NIH-3T3 cells, indicating that the TRXl-ABAD-DP-TRX2 aptamer can bind amyloid β peptide within cells. In addition, cell morphology and MTT results suggested that TRX1-ABAD-DP-TRX2 attenuated amyloid β peptide-induced SH-SY5Y cell injury and improved cell viability. These findings confirmed the possibility of constructing TRX-based peptide aptamer using ABAD-DP. Moreover, TRXl-ABAD-DP-TRX2 inhibited the cytotoxic effect of amyloid β peptide.

Exosome-mediated aptamer S58 reduces fibrosis in a rat glaucoma filtration surgery model

AIM:To confirm whether exosome-mediated delivery of aptamer S58(Exo-S58) has a better antifibrotic effect than naked S58 in human conjunctival fibroblasts(HCon Fs) and a rat glaucoma filtration surgery(GFS) model.METHODS:To enhance the effective reaction time of aptamer S58 in vivo, we loaded aptamer S58 into exosomes derived from HEK293 T cells by PEI transfection to determine the effect of Exo-S58 in HCon Fs and a rat GFS model.RESULTS:Exo-S58 can significantly reduce cell proliferation, migration and fibrosis in TGF-β2-induced HCon Fs. In an in vivo experiment, Exo-S58 treatment prolonged filtering bleb retention and reduced fibrosis compared with naked S58 treatment in GFS rats.CONCLUSION:The exosomes are safe and valid carriers to deliver aptamers. Furthermore, Exo-S58 exhibited superior antifibrotic effect than naked S58 both in HCon Fs cells and rat GFS models.

Selection of DNA aptamer that specific binding human carcinoembryonic antigen in vitro

Objective：To select the specific aptamer of carcinoembryonic antigen （CEA）, one of the most attractive molecule for cancer target therapy and imaging. Methods： Seven rounds in vitro selection were performed against the purified CEA protein. Ligand-mediated target purification and Co-immunoprecipitation were adopted to verify the specific binding of the aptamer to the purified and native protein separately. Results：The CEA-specific aptamer which can bind both the purified and native protein with the high specificity was obtained. Conclusion：This is the first time the CEA specific apatmer was produced. The results in this study provides the preliminary evidence for further investigation and application of CEA-aptamer in the future.

In vitro Selection of DNA Aptamers and Fluorescence-Based Recognition for Rapid Detection Listeria monocytogenes

Aptamers are specific nucleic acid sequences that can bind to a wide range of nucleic acid and non-nucleic acid targets with high affinity and specificity. Nucleic acid aptamers are selected in vitro from single stranded DNA or RNA ligands containing random sequences of up to a few hundred nucleotides. Systematic evolution of ligands by exponential enrichment （SELEX） was used to select and PCR amplify DNA sequences （aptamers） capable of binding to and detecting Listeria monocytogenes, one of the major food-borne pathogens. A simplified affinity separation approach was employed, in which L. monocytogenes in exponential （log） phase of growth was used as the separation target. A fluorescently-labeled aptamer assay scheme was devised for detecting L. monoeytogenes. This report described a novel approach to the detection of L. monocytogenes using DNA aptamers. Aptamers were developed by nine rounds of SELEX. A high affinity aptamer was successfully selected from the initial random DNA pool, and its secondary structure was also investigated. One of aptamers named e01 with the highest affinity was further tested in aptamer-peroxidase and aptamer-fluorescence staining protocols. This study has proved the principle that the whole-cell SELEX could be a promising technique to design aptamer-based molecular probes for dectection of pathogenic microorganisms without tedious isolation and purification of complex markers or targets.