RESUMO
Tumor-derived exosomes are vital for clinical dynamic and accurate tumor diagnosis, thus developing sensitive and multiple exosomes detection technology has attracted remarkable attention of scientists. Here, a novel herringbone microfluidic device with aptamer-functionalized barcodes integration for specific capture and multiple detection of tumor-derived exosomes is presented. The barcodes with core-shell constructions are obtained by partially replicating the periodically ordered hexagonal close-packaged colloidal crystal beads. As their inverse opal hydrogel shell possesses rich interconnected pores, the barcodes could provide abundant surface area for functionalization of DNA aptamers to realize specific recognition of target exosomes. Besides, the encoded structure colors of the barcodes can be maintained stably during the detection events as their hardish cores are with sufficient mechanical strength. It is demonstrated that by embedding these barcodes in herringbone groove microfluidic device with designed patterns, the specific capture efficiency and synergetic detection of multiple tumor-derived exosomes in peripheral blood can be significantly improved due to enhanced resistance of turbulent flow. These features make the aptamer-functionalized barcodes and herringbone microfluidics integrated platform promising for exosomes extraction and dynamic tumor diagnosis.
Assuntos
Aptâmeros de Nucleotídeos , Exossomos , Neoplasias , Aptâmeros de Nucleotídeos/análise , Exossomos/química , Humanos , MicrofluídicaRESUMO
Probing pro-metastatic biomarkers is of significant importance to evaluate the risk of tumor metastasis, but spatially selective imaging of such targets in extracellular microenvironment is particularly challenging. By introducing the bilinguality of PNA/peptide hybrid that can speak both peptide substrate and nucleobase-pairing languages to combine with aptamer technology, we designed a smart DNA nanodevice programmed to respond sequentially to dual pro-metastatic targets, MMP2/9 and ATP, in extracellular tumor microenvironment (TME). The DNA nanodevice is established based on the combination of an ATP-responsive aptamer sensor and a MMP2/9-hydrolyzable PNA/peptide copolymer with a cell membrane-anchoring aptamer module. Taking 4T1 xenograft as a highly aggressive tumor model, the robustness of the DNA nanodevice in spatioselective imaging of MMP2/9 and ATP in TME is demonstrated. We envision that this design will enable the simultaneous visualization of multiple pro-metastatic biomarkers, which allows to gain insights into their pathological roles in tumor metastasis.
Assuntos
Trifosfato de Adenosina/análise , Aptâmeros de Nucleotídeos/análise , Biomarcadores Tumorais/análise , DNA/química , Nanopartículas/química , Animais , Linhagem Celular Tumoral , Camundongos , Microambiente TumoralRESUMO
Analytical methods for molecular characterization of diagnostic or therapeutic targets have recently gained high interest. This review summarizes the combination of mass spectrometry and surface plasmon resonance (SPR) biosensor analysis for identification and affinity determination of protein interactions with antibodies and DNA-aptamers. The binding constant (KD) of a protein-antibody complex is first determined by immobilizing an antibody or DNA-aptamer on an SPR chip. A proteolytic peptide mixture is then applied to the chip, and following removal of unbound material by washing, the epitope(s) peptide(s) are eluted and identified by MALDI-MS. The SPR-MS combination was applied to a wide range of affinity pairs. Distinct epitope peptides were identified for the cardiac biomarker myoglobin (MG) both from monoclonal and polyclonal antibodies, and binding constants determined for equine and human MG provided molecular assessment of cross immunoreactivities. Mass spectrometric epitope identifications were obtained for linear, as well as for assembled ("conformational") antibody epitopes, e.g., for the polypeptide chemokine Interleukin-8. Immobilization using protein G substantially improved surface fixation and antibody stabilities for epitope identification and affinity determination. Moreover, epitopes were successfully determined for polyclonal antibodies from biological material, such as from patient antisera upon enzyme replacement therapy of lysosomal diseases. The SPR-MS combination was also successfully applied to identify linear and assembled epitopes for DNA-aptamer interaction complexes of the tumor diagnostic protein C-Met. In summary, the SPR-MS combination has been established as a powerful molecular tool for identification of protein interaction epitopes.
Assuntos
Anticorpos/análise , Aptâmeros de Nucleotídeos/análise , Técnicas Biossensoriais/métodos , Epitopos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Anticorpos/química , Anticorpos/imunologia , Afinidade de Anticorpos , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/imunologia , Epitopos/química , Epitopos/imunologia , Humanos , Ressonância de Plasmônio de Superfície/métodosRESUMO
Genetically encoded molecular tools are crucial for live cell RNA imaging, and few are available for endogenous RNA imaging. We develop a new genetically encoded sensor using conformation switching RNA induced fluorogenic proteins that enable multicolor and signal-amplified imaging of endogenous RNAs. The sensor system is designed with an RNA sensing module and a degron-fused fluorescent protein reporter. Target RNA induces conformation switching of the RNA sensing module to form RNA aptamers that stabilize the degron-fused protein for fluorogenic imaging. This sensor is demonstrated for high-contrast imaging of survivin mRNA abundance and dynamics in live cells. Moreover, the sensor system is extended to a multicolor palette by screening fluorogenic proteins of distinct colors, and engineered into a signal amplifier using the split fluorescent protein design. The sensor is further exploited for imaging lncRNA MALAT-1 and its translocation dynamics during mitosis. Our sensor system can afford a valuable platform for RNA imaging in biomedical research and clinical theranostics.
Assuntos
Aptâmeros de Nucleotídeos/análise , Proteínas de Fluorescência Verde/química , RNA Longo não Codificante/análise , RNA Mensageiro/análise , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Linhagem Celular Tumoral , Humanos , Hibridização de Ácido Nucleico , Imagem Óptica/métodos , Conformação Proteica , RNA Longo não Codificante/química , RNA Longo não Codificante/genética , RNA Mensageiro/química , RNA Mensageiro/genética , Survivina/genéticaRESUMO
The interaction of human epidermal growth factor receptor 3 (HER3) and heregulin (HRG) is involved in resistance to human epidermal growth factor receptor 2 (HER2)-targeted cancer treatment, such as therapies using anti-HER2 monoclonal antibody. Therefore, inhibition of the HER3/HRG interaction is potentially valuable therapeutic target for cancer treatment. In this study, we used in vitro selection, also known as systematic evolution of ligands by exponential enrichment (SELEX) against the extracellular domain of human HER3, and discovered a novel RNA aptamer. Pull-down and bio-layer interferometry assays showed that RNA aptamer discovered specifically bound to HER3 with a dissociation constant (KD) of 700 nM. Pull-down assays using chemiluminescence detection also revealed that the HER3-binding RNA aptamer inhibited interactions between HER3 and human HRG. These results indicated that the novel HER3-binding RNA aptamer has potential to be used as basic tool in a range of applications involving HER3/HRG interactions, including research, therapeutic, and diagnostic applications.
Assuntos
Aptâmeros de Nucleotídeos/análise , Receptores ErbB/antagonistas & inibidores , Neuregulina-1/antagonistas & inibidores , Neuregulina-1/metabolismo , Receptor ErbB-3/antagonistas & inibidores , Receptor ErbB-3/metabolismo , Técnica de Seleção de Aptâmeros , Aptâmeros de Nucleotídeos/isolamento & purificação , Sequência de Bases , Receptores ErbB/metabolismo , Humanos , Cinética , Luminescência , Ligação Proteica/efeitos dos fármacos , Receptor ErbB-3/químicaRESUMO
This study was to find the aptamers with high affinity and specificity binding to acute promyelocytic leukemia (APL) NB4 cell line. These aptamers targeted NB4 cells were selected from a random single-stranded DNA (ssDNA) library of systematic evolution of ligands by exponential enrichment (CELL-SELEX). The binding rate of FITC-ssDNA library and NB4 cells was monitored using flow cytometry and fluorescence microscope. After cloned and sequenced, the structure, specificity, and affinity of these candidate aptamers were further analyzed. After a total of 19 rounds of selection, the ssDNA library was enriched and the BR (19.9%) of the 16th round was 12 times of the first round (1.6%). Three enriched aptamers were obtained from 21 positive clones of the 16th round, and the predicted secondary structures of these aptamers were mainly stem-loop. The aptamer CX9 had the highest affinity, and the equilibrium dissociation constant (Kd) was 16.2 nM. The fluorescence intensity of CX9 binding to NB4 cells was stronger than HL60 and K562 cells under fluorescence microscopy. The study indicates that aptamer CX9 exhibits high affinity and specificity with NB4 cells and lay a foundation for the rapid diagnostic method to detect APL with fluorescence-labeled aptamer.
Assuntos
Aptâmeros de Nucleotídeos/análise , DNA de Cadeia Simples/metabolismo , Aptâmeros de Nucleotídeos/química , Linhagem Celular Tumoral , Humanos , Cinética , Conformação de Ácido Nucleico , Técnica de Seleção de AptâmerosRESUMO
Biochemical properties and flexibility of nitrogenous bases allow DNA to fold into higher-order structures. Among different DNA secondary structure, G-quadruplexes (tetrapelexes-G4) - which are formed in guanine rich sequences - have gained more attention because of their biological significance, therapeutic intervention, and application in molecular device and biosensor. G4-quadruplex studies categorize into three main fields, in vivo, in vitro, and in silico. The in vitro field includes G4 synthetic oligonucleotides. This review focuses on the G-quadruplex synthetic aptamers structure features and considers the applicability of G4-aptamers for cancer biomarkers detection. Various biosensing methods will be reviewed based on G-quadruplex aptamers for cancer detection.
Assuntos
Aptâmeros de Nucleotídeos/análise , Biomarcadores Tumorais/análise , DNA de Neoplasias/química , Neoplasias/diagnóstico , Quadruplex G , Humanos , Neoplasias/químicaRESUMO
Abnormal glycan structures are valuable biomarkers for disease states; the development of glycan-specific binders is thereby significantly important. However, the structural homology and weak immunogenicity of glycans pose major hurdles in the evolution of antibodies, while the poor availability of complex glycans also has extremely hindered the selection of anti-glycan aptamers. Herein, we present a new approach to efficiently screen aptamers toward specific glycans with a complex structure, using a glycosylated peptide as a scaffold. In this method, using peptide-imprinted magnetic nanoparticles (MNPs) as a versatile platform, a glycopeptide tryptically digested from a native glycoprotein was selectively entrapped for positive selection, while a nonglycosylated analogue with an identical peptide sequence was synthesized for negative selection. Alternating positive and negative selection steps were carried out to guide the directed evolution of glycan-binding aptamers. As proof of the principle, the biantennary digalactosylated disialylated N-glycan A2G2S2, against which there have been no antibodies and lectins so far, was employed as the target. With the glycoprotein transferrin as a source of target glycan, two satisfied anti-A2G2S2 aptamers were selected within seven rounds. Since A2G2S2 is upregulated in cancerous liver cells, carboxyfluorescein (FAM)-labeled aptamers were prepared as fluorescent imaging reagents, and successful differentiation of cancerous liver cells over normal liver cells was achieved, which demonstrated the application feasibility of the selected aptamers. This approach obviated a tedious glycan preparation process and allowed favorable expose of the intrinsic flexible conformation of natural glycans. Therefore, it holds great promise for developing glycan-specific aptamers for challenging applications such as cancer targeting.
Assuntos
Aptâmeros de Nucleotídeos/análise , Peptídeos/química , Polissacarídeos/análise , Linhagem Celular , Glicosilação , Células Hep G2 , Humanos , Nanopartículas de Magnetita/química , Microscopia ConfocalRESUMO
Electrochemical aptamer-based (E-AB) sensors, exploiting binding-induced changes in biomolecular conformation, are rapid, specific, and selective and perform well even in a complex matrix, such as directly in whole blood and even in vivo. However, like all sensors employing biomolecular recognitions, E-AB sensors suffer from an inherent limitation of single-site binding, i.e., its fixed dose-response curve. To circumvent this, we employ here distal-site mutation and allosteric inhibition to rationally tune the dynamic range of E-AB sensors, achieving sets of sensors with a significantly varied target affinity (â¼3 orders of magnitude). Using their combination, we recreate several approaches to narrow (down to 5-fold) or extend (up to 2000-fold) the dynamic range of biological receptors. The thermodynamic consequences of aptamer-surface interactions are estimated via the free-energy difference in solution-phase and surface-bound biosensors employing the same aptamer as a recognition element, revealing that an allostery strategy provides a more predictable and efficient means to finely control the target affinity and dynamic range. Such an ability to rationally modulate the affinity of biomolecule receptors would open the door to applications including cancer therapy, bioelectronics, and many other fields employing biomolecule recognition.
Assuntos
Aptâmeros de Nucleotídeos/análise , Técnicas Biossensoriais , Técnicas Eletroquímicas , Termodinâmica , Regulação Alostérica/efeitos dos fármacos , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/farmacologia , Mutação , Propriedades de SuperfícieRESUMO
Exosomes, small extracellular vesicles, are released by various cell types. They are found in bodily fluids, including blood, urine, serum, and saliva, and play essential roles in intercellular communication. Exosomes contain various biomarkers, such as nucleic acids and proteins, that reflect the status of their parent cells. Since they influence tumorigenesis and metastasis in cancer patients, exosomes are excellent noninvasive potential indicators for early cancer detection. Aptamers with specific binding properties have distinct advantages over antibodies, making them effective versatile bioreceptors for the detection of exosome biomarkers. Here, we review various aptamer-based exosome detection approaches based on signaling methods, such as fluorescence, colorimetry, and chemiluminescence, focusing on electrochemical strategies that are easier, cost-effective, and more sensitive than others. Further, we discuss the clinical applications of electrochemical exosome analysis strategies as well as future research directions in this field.
Assuntos
Aptâmeros de Nucleotídeos/análise , Técnicas Eletroquímicas , Exossomos/química , HumanosRESUMO
Aptamers are chemically synthetic single-stranded DNA or RNA molecules selected by molecular evolution. They have been widely used as attractive tools in biosensing and bioimaging because they can bind to a large variety of targets with high sensitivity and high affinity and specificity. As recognition elements, aptamers contribute in particular to cancer diagnostics by recognizing different cancer biomarkers, while they can also facilitate ultrasensitive detection by further employing signal amplification elements. Optical techniques have been widely used for direct and real-time monitoring of cancer-related biomolecules and bioprocesses due to the high sensitivity, quick response, and simple operation, which has greatly benefited cancer diagnostics. In this review, we highlight recent advances in optical platform-based sensing strategies for cancer diagnostics aided by aptamers. Limitations and current challenges are also discussed.
Assuntos
Aptâmeros de Nucleotídeos/análise , Técnicas Biossensoriais/métodos , Neoplasias/diagnóstico , Óptica e Fotônica/métodos , Técnica de Seleção de Aptâmeros/métodos , Biomarcadores Tumorais/análise , HumanosRESUMO
Human mucin-1 (MUC1) has attracted considerable attention owing to its overexpression in diverse malignancies. Here, for the rapid and efficient detection of MUC1, we present a SERS-colorimetric dual-mode aptasensor, by integrating SERS probes with magnetic separation, which has several distinctive advantages. Using such a dual-mode aptasensor, the colorimetric functionality is distinguishable by the naked eye, providing a fast and straightforward screening ability for the detection of MUC1. Moreover, SERS-based detection greatly improves the detection sensitivity, reaching a limit of detection of 0.1 U/mL. In addition, the combination of SERS and colorimetric method holds the advantages of these two techniques and thereby increases the reliability and efficiency of MUC1 detection. On the one hand, the magnetic nanobeads functionalized with MUC1-specific aptamer were utilized as an efficient capturing substrate for separating MUC1 from biological complex medium. On the other hand, the gold-silver core-shell nanoparticles modified with Raman reporters and the complementary sequences of MUC1 were used as the signal indicator, which could simultaneously report the SERS signal and colorimetric change. This strategy can achieve a good detection range and realize MUC1 analysis in real patients' samples. Thus, we anticipate that this kind of aptasensor would provide promising potential applications in the diagnosis and prognosis of cancers. Graphical abstract.
Assuntos
Aptâmeros de Nucleotídeos/análise , Biomarcadores Tumorais/análise , Colorimetria/métodos , Mucina-1/análise , Neoplasias/diagnóstico , Análise Espectral Raman/métodos , Humanos , Neoplasias/patologia , PrognósticoRESUMO
Colorimetric platform using the aggregation of gold nanoparticles (AuNPs) is a pretty simple method for biosensing, but advanced instruments such as specterophotometer is still needed to achieve accurately quantitative readout. Aggregated AuNPs exhibit excellent photothermal properties under near-infrared laser irradiation, which is significantly different from non-aggregated AuNPs. Herein, given the different photothermal effect, we translated the AuNPs-based colorimetric assay into a photothermal assay for the quantitative detection of adenosine using a thermometer as readout. Short single-stranded DNA (ssDNA, adenosine aptamer) was adsorbed on the surface of AuNPs and hence prevented the aggregation of AuNPs under high ionic concentration. The presence of adenosine caused the structural change of ssDNA and the AuNPs became aggregated. The enhanced temperature under NIR-laser irradiation has a linear response to the concentration of adenosine in the range of 2.0-50.0 µM. The detection limit was 1.7 µM. This proposed method is portable, easy and applicable to the quantitative assay of other targets by simply replacing of the sequence of ssDNA.
Assuntos
Adenosina/análise , Aptâmeros de Nucleotídeos/análise , Ouro/química , Nanopartículas Metálicas/química , Temperatura , Adsorção , Técnicas Biossensoriais , Colorimetria , Tamanho da Partícula , Processos Fotoquímicos , Propriedades de Superfície , TermômetrosAssuntos
Simulação por Computador , Microscopia Crioeletrônica/tendências , Terapia Genética/tendências , Genoma/fisiologia , Microbiota/fisiologia , Análise de Sequência de RNA/tendências , Análise de Célula Única/tendências , Aptâmeros de Nucleotídeos/análise , Progressão da Doença , Elementos Facilitadores Genéticos/genética , Genoma/genética , Humanos , Microbiota/genética , Neoplasias/genética , Neoplasias/patologiaRESUMO
Precise detection and effective treatment are crucial to prolong cancer patients' lives. Surface-enhanced Raman scattering (SERS) imaging coupled with photothermal therapy has been considered a precise and effective strategy for cancer theranostics. Nevertheless, Raman reporters employed in the literature usually possessed multiple shift peaks in the fingerprint region, which are overlapped with background signals from endogenous biological molecules. Herein, we fabricated a new kind of bioorthogonal Raman reporter and aptamer functionalized SERS nanotags. The SERS nanotags demonstrated a strong Raman signal at 2205 cm-1 in the biologically Raman-silent region and recognized MCF-7 breast cancer cells for Raman imaging with high specificity. Laser irradiation induced serious toxicity of MCF-7 cells due to the excellent photothermal capability of the SERS nanotags. After intravenous administration of the SERS nanotags, tumor Raman spectral detection and mapping in living mice were successfully achieved. Further in vivo antitumor experiments manifested that the aptamer-modified SERS nanotags significantly restrained tumor growth after laser irradiation with 99% inhibition rate and good biocompatibility. These results clearly revealed that the SERS nanotags could serve as a novel and precise theranostic platform for in vivo cancer diagnosis and photothermal therapy.
Assuntos
Aptâmeros de Nucleotídeos/uso terapêutico , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/terapia , Ouro/uso terapêutico , Nanotubos , Células 3T3-L1 , Animais , Aptâmeros de Nucleotídeos/análise , Feminino , Ouro/análise , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanotubos/análise , Nanotubos/ultraestrutura , Terapia Fototérmica/métodos , Análise Espectral Raman/métodos , Nanomedicina Teranóstica/métodosRESUMO
Mycotoxins are toxic compounds produced by the metabolism of certain fungi that threaten the food and agricultural industry. Over hundreds of mycotoxins, one of the most common toxins, zearalenone (ZEN), has toxic effects on human and animal health due to its mutagenicity, treatogenicity, carcinogenicity, nephrotoxicity, immunotoxicity, and genotoxicity. In this work, attenuated internal reflection spectroscopic ellipsometry (AIR-SE) combined with the signal amplification via surface plasmon resonance conditions that were proved to be a highly sensitive analytical tool in bio-sensing was developed for the sensitive and selective ZEN detection in cereal products such as corn, wheat, rice, and oat. Combined with the oligonucleotide aptamer for ZEN recognition, our proposed method showed good performance with yielding 0.08 ng/mL LOD and 0.01-1000 ng/mL detection range. A mini-review was also introduced in, to compare various methods for ZEN detection.
Assuntos
Grão Comestível/química , Contaminação de Alimentos/análise , Análise Espectral/métodos , Zearalenona/análise , Aptâmeros de Nucleotídeos/análise , Oryza/química , Análise Espectral/instrumentação , Triticum/química , Zea mays/químicaRESUMO
Theranostics cover emerging technologies for cell biomarking for disease diagnosis and targeted introduction of drug ingredients to specific malignant sites. Theranostics development has become a significant biomedical research endeavor for effective diagnosis and treatment of diseases, especially cancer. An efficient biomarking and targeted delivery strategy for theranostic applications requires effective molecular coupling of binding ligands with high affinities to specific receptors on the cancer cell surface. Bioaffinity offers a unique mechanism to bind specific target and receptor molecules from a range of non-targets. The binding efficacy depends on the specificity of the affinity ligand toward the target molecule even at low concentrations. Aptamers are fragments of genetic materials, peptides, or oligonucleotides which possess enhanced specificity in targeting desired cell surface receptor molecules. Aptamer-target binding results from several inter-molecular interactions including hydrogen bond formation, aromatic stacking of flat moieties, hydrophobic interaction, electrostatic, and van der Waals interactions. Advancements in Systematic Evolution of Ligands by Exponential Enrichment (SELEX) assay has created the opportunity to artificially generate aptamers that specifically bind to desired cancer and tumor surface receptors with high affinities. This article discusses the potential application of molecular dynamics (MD) simulation to advance aptamer-mediated receptor targeting in targeted cancer therapy. MD simulation offers real-time analysis of the molecular drivers of the aptamer-receptor binding and generate optimal receptor binding conditions for theranostic applications. The article also provides an overview of different cancer types with focus on receptor biomarking and targeted treatment approaches, conventional molecular probes, and aptamers that have been explored for cancer cells targeting.
Assuntos
Aptâmeros de Nucleotídeos/análise , Biomarcadores Tumorais/análise , Simulação de Dinâmica Molecular , Sondas Moleculares/química , Neoplasias/diagnóstico , Animais , HumanosRESUMO
A disposable package of biosensors was developed along with the corresponding guidelines for early detection of the acute lymphoblastic leukemia cancer. This proposed cancer-screening package included a DNA sensor and an aptasensor as two main types of biosensors. The biosensors were used simultaneously. This combination of sensors can detect not only the presence of mutant genes but also the biomarkers of cancer. At current work, the combination of sensors were used to detect the presence of BCR-ABL1 as a mutant gene and CEA as a biomarkers of cancer, such a capability makes the package liable for early and certain detection of acute lymphoblastic leukemia. To construct both the DNA sensor and the aptasensor, a nanocomposite consisting of electrosynthesis carbon quantum dots and biosynthesized gold nanoparticles was applied. The construction of these biosensors was characterized using four different electrochemical methods including DPV (Differential Pulse Voltammetry), EIS (Electrochemical Impedance Spectroscopy), CV (Cyclic Voltammetry) and chronoamperometry. The peak current of a catechol solution that was used as an electroactive probe on the biosensor was linearly related to the logarithm of the concentrations of the target DNA and the target antigen in the range of 10 pM to 100⯵M and 1â¯pgâ¯mL-1 to 0.001â¯gâ¯mL-1 with the detection limits of 1.5 pM and 0.26â¯pgâ¯mL-1 respectively, which are quite good results.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Sondas de DNA/análise , DNA de Neoplasias/análise , Detecção Precoce de Câncer , Técnicas Eletroquímicas , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Aptâmeros de Nucleotídeos/análise , Eletrodos , Humanos , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
The detection of alpha-fetoprotein (AFP) is of great importance for hepatocellular carcinoma (HCC) diagnosis, but it needs to be further improved because of poor sensitivity and complicated operating steps. In this paper, a simple and sensitive homogeneous apatasensor for AFP has been developed based on Förster resonance energy transfer (FRET) where the AFP aptamer labeled luminescent CdTe quantum dots (QDs) as a donor and anti-AFP antibody functional gold nanoparticles (AuNPs) as an acceptor. In the presence of AFP, the bio-affinity between aptamer, target, and antibody made the QDs and AuNPs close enough, thus the fluorescence of CdTe QDs quenched though the FRET between QD and AuNP. The fluorescent aptasensor for AFP showed a concentration-dependent decrease of fluorescence intensity in the low nanomolar range and a detecting linear range of 0.5-45â¯ngâ¯mL-1, with a detection limit of 400â¯pgâ¯mL-1. Moreover, this homogeneous aptasensor is simple and reliable, and obtained satisfying results for the detection of AFP in human serum samples. With more and more aptamers for biomarkers have been selected gradually, this approach could be easily extended to detection of a wide range of biomarkers. The proposed aptasensor has great potential for carcinoma screening in point-of-care testing and even in field use.
Assuntos
Aptâmeros de Nucleotídeos/química , Biomarcadores Tumorais/sangue , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Ouro/química , Nanopartículas Metálicas/química , Pontos Quânticos/química , Adulto , Aptâmeros de Nucleotídeos/análise , Técnicas Biossensoriais , Corantes Fluorescentes/análise , Humanos , Espectrometria de Fluorescência , alfa-Fetoproteínas/análiseRESUMO
To explore thermofluorimetric analysis (TFA) in detail, we compared two related aptamers. The first, LINN2, is a DNA aptamer previously selected against EGFR recombinant protein. In this work we selected a second aptamer, KM4, against EGFR-overexpressing A549 cells. The two aptamers were derived from the same pool and bind the same target but behave differently in TFA. Our results suggest four overall conclusions about TFA of aptamers: 1. Some aptamers show reduced fluorescence upon target binding suggesting that target-bound aptamer is not always fluorescent. 2. Many aptamers do not obey the intuitive assumptions that aptamer-target interactions stabilize a folded conformation. 3. TFA may be most appropriate for aptamers with significant double-stranded structure. 4. Kinetic effects may be significant and the order of operations in preparing samples should be carefully optimized.