Development of Metal-Enhanced Fluorescence-Based Aptasensor for Thrombin Detection Using Silver Dendritic Nanostructures

Metal-enhanced fluorescence (MEF) phenomenon has shown a promising potential in the field of fluorescence-based biological sensing. In this study, we optimized the electroless metal deposition method to fabricate silver dendritic nanostructures as effective MEF active substrates. Then, an aptasensor was developed for thrombin detection using the established surfaces. For this purpose, thiolated 29-mer thrombin-binding aptamers (TBA₂₉ (12T) SH) as capturing aptamer were immobilized on the surface of silver dendritic nanostructures, then thrombin was sandwiched between the capturing aptamer and Cy5-labeled 15-mer thrombin aptamer (TBA_15-Cy5). Quantitative analysis was performed through fluorescence signal measurement. The established aptasensor presented satisfactory sensitivity and selectivity and exhibited a limit of detection (LOD) as low as 32 pM. This aptasensor was also able to detect thrombin in the human serum at picomolar levels. Furthermore, the ease and relatively low-cost of fabrication of this platform introduce it as a tool with great potential for the clinical diagnosis of diseases and also for improving sensitivity of a variety of technologies which exploit fluorescent dyes for analyte detection, at ultra-trace levels, in complex matrices.

     

A fluorescent aptasensor for sensitive analysis oxytetracycline based on silver nanoclusters

A fluorescent aptasensor for detection of oxytetracycline (OTC) was presented based on fluorescence quenching of DNA aptamer‐templated silver nanoclusters (AgNCs). The specific DNA scaffolds with two different nucleotides fragments were used: one was enriched with a cytosine sequence fragment (C12) that could produce DNA–AgNCs via a chemical reduction method, and another was the OTC aptamer fragment that could selectively bind to the OTC antibiotic. Thus, the as‐prepared AgNCs could exhibit quenched fluorescence after binding to the target OTC. The fluorescence ratio of the DNA–AgNCs was quenched in a linearly proportional manner to the concentration of the target in the range of 0.5 nM to 100 nM with a detection limit of 0.1 nM. This proposed nanobiosensor was demonstrated to be sensitive, selective, and simple, introducing a viable alternative for rapid determination of toxin OTC in honey and water samples. Copyright © 2016 John Wiley & Sons, Ltd.     

Time-resolved fluorescence biosensor for adenosine detection based on home-made europium complexes

In this protocol, the authors report a time-resolved fluorescence biosensor based on home-made europium complexes for highly sensitive detection of small molecules using adenosine as a model analyte. The fluorophore that used is europium complexes. Its signal can be measured in a time-resolved manner that eliminates most of the unspecific fluorescent background. The amino modified aptamer probe, which is designed to specifically recognize adenosine, is combined to the aldehyde-group modified glass slide by covalent bond. Europium complex-labeled a short ssDNA, designed to segment hybridize with aptamer probe is immobilized on the glass slide by hybridization reaction. In the presence of adenosine, the aptamer part is more inclined to bounds with adenosine and triggers structure-switching of the aptamer from aptamer/ssDNA duplex to aptamer/target complex. As a result, europium complexes-labeled ssDNA is forced to dissociate from the sensor interface, resulting in time-resolved fluorescence intensity decrease. The decrement intensity is proportional to the amount of adenosine. Under optimized assay conditions, a linear range (1.0×10(-8)M to 1.0×10(-7)M) is got with low detection limit of 5.61nM. The biosensor exhibits excellent selectivity and can provide a promising potential for aptamer-based adenosine detection.     

Fluorescent metal nanoshell and CK19 detection on single cell image

In this article, we report the synthesis strategy and optical properties of a novel type of fluorescence metal nanoshell when it was used as imaging agent for fluorescence cell imaging. The metal nanoshells were made with 40 nm silica cores and 10 nm silver shells. Unlike typical fluorescence metal nanoshells which contain the organic dyes in the cores, novel metal nanoshells were composed of Cy5-labelled monoclonal anti-CK19 antibodies (mAbs) on the external surfaces of shells. Optical measurements to the single nanoparticles showed that in comparison with the metal free labelled mAbs, the mAb-Ag complexes displayed significantly enhanced emission intensity and dramatically shortened lifetime due to near-field interactions of fluorophores with metal. These metal nanoshells were found to be able to immunoreact with target cytokeratin 19 (CK19) molecules on the surfaces of LNCAP and HeLa cells. Fluorescence cell images were recorded on a time-resolved confocal microscope. The emissions from the metal nanoprobes could be clearly isolated from the cellular autofluorescence backgrounds on the cell images as either individuals or small clusters due to their stronger emission intensities and shorter lifetimes. These emission signals could also be precisely counted on single cell images. The count number may provide an approach for quantifying the target molecules in the cells.     

Graphene oxide based fluorescent aptasensor for adenosine deaminase detection using adenosine as the substrate

We present a novel fluorescent aptasensor for simple and accurate detection of adenosine deaminase (ADA) activity and inhibition on the basis of graphene oxide (GO) using adenosine (AD) as the substrate. This aptasensor consists of a dye-labeled single-stranded AD specific aptamer, GO and AD. The fluorescence intensity of the dye-labeled AD specific aptamer is quenched very efficiently by GO as a result of strong π-π stacking interaction and excellent electronic transference of GO. In the presence of AD, the fluorescence of the GO-based probe is recovered since the competitive binding of AD and GO with the dye-labeled aptamer prevents the adsorption of dye-labeled aptamer on GO. When ADA was introduced to this GO-based probe solution, the fluorescence of the probe was quenched owing to ADA can convert AD into inosine which has no affinity to the dye-labeled aptamer, thus allowing quantitative investigation of ADA activity. The as-proposed sensor is highly selective and sensitive for the assay of ADA activity with a detection limit of 0.0129U/mL in clean buffer, which is more than one order of magnitude lower than the previous reports. Meanwhile, a good linear relationship with the correlation coefficient of R=0.9922 was obtained by testing 5% human serum containing a series of concentrations of ADA. Additionally, the inhibition effect of erythro-9-(2-hydroxy-3-nonyl) adenine on ADA activity was investigated in this design. The GO-based fluorescence aptasensor not only provides a simple, cost-effective and sensitive platform for the detection of ADA and its inhibitor but also shows great potential in the diagnosis of ADA-relevant diseases and drug development.     

DNA-Ag nanoclusters as fluorescence probe for turn-on aptamer sensor of small molecules

As promising substitutes for organic dyes and quantum dots, few-atom fluorescent silver nanoclusters (Ag NCs) have recently gained much attention in a wide range from cellular imaging to chemical/biological detection applications owing to their ultrasmall size (<2 nm), excellent photostability, good biocompatibility and water solubility. Herein, we design an aptamer, guanine-rich (G-rich) DNA and Ag NCs nanocomplex to investigate its ability for the detection of small molecules. The design contains two DNA strands which are both chimeric conjugates of the DNA aptamer sequence fragment and G-rich sequence fragment. Using cocaine as a model molecule, the two DNA strands are in free state if there is no cocaine present, and the formed Ag NCs through the reduction of Ag(+) by NaBH(4) show weak fluorescence emission. In the presence of cocaine, however, the two aptamer fragments bind cocaine, which in turn puts the two G-rich sequence fragments in proximity and the fluorescent intensity of DNA-Ag NCs enhances greatly. As a result, DNA-Ag NCs are demonstrated as a novel, cost-effective and turn-on fluorescent probe for the analysis of cocaine, with a detection limit of 0.1 μM. Besides, successful detection of adenosine triphosphate (ATP) with detection limit of 0.2 μM demonstrates its potential to be a general method.     

Colorimetric Aptasensor Using Unmodified Gold Nanoparticles for Homogeneous Multiplex Detection

Colorimetric aptasensors using unmodified gold nanoparticles (AuNPs) have attracted much attention because of their low cost, simplicity, and practicality, and they have been developed for various targets in the past several years. However, previous research has focused on developing single-target assays. Here, we report the development of a homogeneous multiplex aptasensor by using more than one class of aptamers to stabilize AuNPs. Using sulfadimethoxine (SDM), kanamycin (KAN) and adenosine (ADE) as example targets, a KAN aptamer (750 nM), an SDM aptamer (250 nM) and an ADE aptamer (500 nM) were mixed at a 1∶1∶1 volume ratio and adsorbed directly onto the surface of unmodified AuNPs by electrostatic interaction. Upon the addition of any of the three targets, the conformation of the corresponding aptamer changed from a random coil structure to a rigid folded structure, which could not adsorb and stabilize AuNPs. The AuNPs aggregated in a specific reaction buffer (20 mM Tris-HCl containing 20 mM NaCl and 5 mM KCl), which led to a color change from red to purple/blue. These results demonstrate that the multiplex colorimetric aptasensor detected three targets simultaneously while maintaining the same sensitivity as a single-target aptasensor for each individual target. The multiplex aptasensor could be extended to other aptamers for various molecular detection events. Due to its simple design, easy operation, fast response, cost effectiveness and lack of need for sophisticated instrumentation, the proposed strategy provides a powerful tool to examine large numbers of samples to screen for a small number of potentially positive samples containing more than one analyte, which can be further validated using sophisticated instruments.     

4-(dimethylamino)butyric acid@PtNPs as enhancer for solid-state electrochemiluminescence aptasensor based on target-induced strand displacement

A solid-state electrochemiluminescence (ECL) aptasensor based on target-induced aptamer displacement for highly sensitive detection of thrombin was developed successfully using 4-(dimethylamino)butyric acid (DMBA)@PtNPs labeling as enhancer. Such a special aptasensor included three main parts: ECL substrate, ECL intensity amplification and target-induced aptamer displacement. The ECL substrate was made by modifying the complex of Pt nanoparticles (PtNPs) and tris(2,2-bipyridyl) ruthenium (II) (Ru(bpy)(3)(2+)) (Ru-PtNPs) onto nafion@multi-walled carbon nanotubes (nafion@MWCNTs) modified electrode surface. A complementary thrombin aptamer labeled by DMBA@PtNPs (Aptamer II) acted as the ECL intensity amplification. The thrombin aptamer (TBA) was applied to hybridize with the labeled complementary thrombin aptamer, yielding a duplex complex of TBA-Aptamer II on the electrode surface. The introduction of thrombin triggered the displacement of Aptamer II from the self-assembled duplex into the solution and the association of inert protein thrombin on the electrode surface, decreasing the amount of DMBA@PtNPs and increasing the electron transfer resistance of the aptasensor and thus resulting large decrease in ECL signal. With the synergistic amplification of DMBA and PtNPs to Ru(bpy)(3)(2+) ECL, the aptasensor showed an enlarged ECL intensity change before and after the detection of thrombin. As a result, the change of ECL intensity has a direct relationship with the logarithm of thrombin concentration in the range of 0.001-30 nM. The detection limit of the proposed aptasensor is 0.4 pM. Thus, the approach is expected to open new opportunities for protein diagnostics in clinical as well as bioanalysis in general.     

Sequence-dependent fluorescence of cyanine dyes on microarrays

Cy3 and Cy5 are among the most commonly used oligonucleotide labeling molecules. Studies of nucleic acid structure and dynamics use these dyes, and they are ubiquitous in microarray experiments. They are sensitive to their environment and have higher quantum yield when bound to DNA. The fluorescent intensity of terminal cyanine dyes is also known to be significantly dependent on the base sequence of the oligonucleotide. We have developed a very precise and high-throughput method to evaluate the sequence dependence of oligonucleotide labeling dyes using microarrays and have applied the method to Cy3 and Cy5. We used light-directed in-situ synthesis of terminally-labeled microarrays to determine the fluorescence intensity of each dye on all 1024 possible 5'-labeled 5-mers. Their intensity is sensitive to all five bases. Their fluorescence is higher with 5' guanines, and adenines in subsequent positions. Cytosine suppresses fluorescence. Intensity falls by half over the range of all 5-mers for Cy3, and two-thirds for Cy5. Labeling with 5'-biotin-streptavidin-Cy3/-Cy5 gives a completely different sequence dependence and greatly reduces fluorescence compared with direct terminal labeling.     

Label-free and sensitive faradic impedance aptasensor for the determination of lysozyme based on target-induced aptamer displacement

A label-free and sensitive faradic impedance spectroscopy (FIS) aptasensor based on target-induced aptamer displacement was developed for the determination of lysozyme as a model system. The aptasensor was fabricated by self-assembling the partial complementary single strand DNA (pcDNA)–lysozyme binding aptamer (LBA) duplex on the surface of a gold electrode. To measure lysozyme, the change in interfacial electron transfer resistance of the aptasensor using a redox couple of [Fe(CN)₆]^3−/4− as the probe was monitored. The introduction of target lysozyme induced the displacement of the LBA from the pcDNA–LBA duplex on the electrode into the solution, decreasing the electron transfer resistance of the aptasensor. The decrease in the FIS signal is linear with the concentration of lysozyme in the range from 0.2 nM to 4.0 nM, with a detection limit of 0.07 nM. The fabricated aptasensor shows a high sensitivity, good selectivity and satisfactory regeneration. This work demonstrates that a high sensitivity of the fabricated aptasensor can be obtained using a relatively short pcDNA. This work also demonstrates that the target-induced aptamer displacement strategy is promising in the design of an electrochemical aptasensor for the determination of lysozyme with good selectivity and high sensitivity.     

A binary Cy3 aptamer probe composed of folded modules

Aptamers are short single-stranded DNA or RNA sequences that are selected in vitro based on their high affinity to a target molecule. Dye-binding aptamers are promising tools for real-time detection of not only DNA or RNA sequences but also proteins of interest both in vitro and in vivo. In this study, we aimed to isolate an RNA aptamer to Cy3, a widely used, membrane-permeant, and nontoxic fluorescent cyanine dye. Extensive selection of affinity RNA molecules to Cy3 yielded a unique sequence aptamer named Cy3_apt. The selected Cy3_apt was 83 nucleotides long and successfully shortened to 49 nucleotides long with increased affinity to Cy3 by multiple base changes. The shortest Cy3_apt is composed of two separate hairpin modules that are required for the affinity to Cy3 as monitored by the surface plasmon resonance (SPR) assay. Also, the fluorescence of Cy3 increased on binding to Cy3_apt. The two modules of Cy3_apt, when detached from each other, functioned as a binary aptamer probe. We demonstrate that the binary Cy3_apt probe is applicable to the detection of target oligonucleotides or RNA-RNA interaction by tagging with target sequences. This binary probe consists of two folded modules, referred to as a folded binary probe.     

A fluorescent aptasensor for analysis of adenosine triphosphate based on aptamer–magnetic nanoparticles and its single‐stranded complementary DNA labeled carbon dots

A new fluorimetric aptasensor was designed for the determination of adenosine triphosphate (ATP) based on magnetic nanoparticles (MNPs) and carbon dots (CDs). In this analytical strategy, an ATP aptamer was conjugated on MNPs and a complementary strand of the aptamer (CS) was labeled with CDs. The aptamer and its CS were hybridized to form a double helical structure. The hybridized aptamers could be used for the specific recognition of ATP in a biological complex matrix using a strong magnetic field to remove the interfering effect. In the absence of ATP, no CDs–CS could be released into the solution and this resulted in a weak fluorescence signal. In the presence of ATP, the target binds to its aptamer and causes the dissociation of the double helical structure and liberation of the CS, such that a strong fluorescence signal was generated. The increased fluorescence signal was proportional to ATP concentration. The limit of detection was estimated to be 1.0 pmol L^–1 with a dynamic range of 3.0 pmol L^–1 to 5.0 nmol L^–1. The specific aptasensor was applied to detect ATP in human serum samples with satisfactory results. Moreover, molecular dynamic simulation (MDS) studies were used to analyze interactions of the ATP molecule with the aptamer.     

A universal fluorescent aptasensor based on AccuBlue dye for the detection of pathogenic bacteria

We report a universal fluorescent aptasensor based on the AccuBlue dye, which is impermeant to cell membranes, for the detection of pathogenic bacteria. The sensor consists of AccuBlue, an aptamer strand, and its complementary strand (cDNA) that partially hybridizes to the aptamer strand. We have fabricated two models by changing the sequence of the reaction between the elements in the system. One is the “signal on” model in which the aptamer is first bound to the target, followed by the addition of cDNA and AccuBlue, at which time the cDNA hybridizes with the free unreacted aptamer and forms a double-stranded DNA (dsDNA) duplex. Such hybridization causes AccuBlue to insert into the dsDNA and exhibit significantly increased fluorescence intensity because of the specific intercalation of the AccuBlue into dsDNA rather than single-stranded DNA (ssDNA). The other model, “signal off,” involves hybridization of the aptamer with cDNA first, resulting in high fluorescence intensity on the addition of AccuBlue. When the target is added, the aptamer binds the target, causing the cDNA to detach from the dsDNA duplex and resulting in low fluorescence as a result of the liberation of AccuBlue. Because this design is based purely on DNA hybridization, and AccuBlue is impermeant to cell membranes, it could potentially be adapted to a wide variety of analytes.     

适配子纤维蛋白靶向性验证及其抗凝活性测定

目的:验证适配子G81的纤维蛋白靶向性,评估适配子对凝血系统的影响。方法:以复钙法制备鼠源、人源体外纤维蛋白,将不同浓度Cy5.5标记的适配子溶液与之孵育,置于激光共聚焦显微镜下以固定的参数成像,用ImageJ软件进行相对荧光强度分析;将适配子G81溶液加入血浆中,通过倍比稀释法得到含浓度梯度适配子的血浆,采用SYSMEX CS-5100全自动血凝仪检测PT、APTT、TT,评估适配子G81对凝血功能的影响。结果:激光共聚焦显微镜显示适配子能与纤维蛋白结合,随着加入适配子量的增加其相对荧光强度逐渐增强,表明适配子可与纤维蛋白结合,统计分析提示荧光强度与适配子存在量效关系;人源、鼠源纤维蛋白结合的荧光强度无统计学差异(P0.05)。在抗凝活性检测中,血浆中适配子G81浓度达到200 pmoL/mL时,各浓度统计分析结果均显示P0.05,表明适配子对PT、APTT、TT的测量均没有统计学差异上的影响。结论:适配子G81具有纤维蛋白靶向性,且当加入的适配子剂量低于200 pmol/mL时对内、外源性凝血功能、凝血酶时间均无明显影响。     

Determination of urinary adenosine using resonance light scattering of gold nanoparticles modified structure-switching aptamer

A novel sensitive method has been developed for the detection of adenosine (AD) in human urine by using enhanced resonance light scattering (RLS). This method is based on the specific recognition and signal amplification of adenosine aptamer (Apt) coupled with gold nanoparticles (GNPs) via G-quartet-induced nanoparticle assembly, which was fabricated by triggering a structure switching of the 3′ terminus G-rich sequence and aptamer duplex. RLS signal linearly correlated with the concentration of adenosine over the range of 6-115 nM. The limit of detection (LOD) for adenosine is 1.8 nM with relative standard deviations (RSD) of 2.90-4.80% (n = 6). The present method has been successfully applied to determination of adenosine in real human urine, and the obtained results were in good agreement with those obtained by the HPLC method. Our investigation shows that the combination of the excellent selectivity of aptamer with the high sensitivity of the RLS technique could provide a promising potential for aptamer-based small molecule detection, and be beneficial in extending the application of RLS.     

Enzyme-free fluorescence aptasensor for amplification detection of human thrombin via target-catalyzed hairpin assembly

Aptamers have many advantages, such as simple synthesis, good stability, high binding affinity and wide applicability, making them suitable candidates for protein detection. Since the disease-related protein may be present in very small amounts in biological samples, the development of amplification paths for aptasensors is essential. In this paper, we develop a simple and enzyme-free amplified aptasensor for protein detection via target-catalyzed hairpin assembly. This aptasensor contains two DNA hairpins termed as H1 and H2. H1, which is modified at its 5' and 3' ends with a fluorophore and a quencher respectively, consists of the aptamer sequence of human thrombin. Meanwhile, H2 is partially complementary to H1. These two hairpins H1 and H2 interact slowly with each other. Upon the addition of target protein, it can facilitate the opening of the hairpin structure of H1 and thus accelerate the hybridization between H1 and H2, resulting in the significant fluorescence enhancement of the system. By monitoring the change in fluorescence intensity, we could detect the target protein with high sensitivity. The detection limit of this aptasensor is 20 pM, which is more than two orders of magnitude lower than that of reported unamplified aptasensors. Furthermore, this amplified aptasensor shows high selectivity toward its target protein. Thus, the proposed aptasensor could be used as a simple, sensitive and selective platform for target protein detection.     

Effect of silver nanoparticles on the parameters of chlorophyll fluorescence and P700 reaction in the green alga Chlamydomonas reinhardtii

Acute toxicity of silver nanoparticle (AgNP) for photosynthesis in Chlamydomonas reinhardtii was studied using an M-PEA2 fluorimeter. Analysis of the fluorescence induction curves in the presence of AgNP at low concentrations revealed inhibited electron transport on the PS2 photosystem and increased content of Q_B-nonreducing centers. No direct effect of AgNP on the reactions of P₇₀₀ oxidation in PS1 was found, while the energization of the photosynthetic membranes was affected. Investigation of the parameters of the prompt and delayed fluorescence is proposed as a method for early detection of AgNP in the environment.     

Determination of glucose and uric acid with bienzyme colorimetry on microfluidic paper-based analysis devices

An aptamer is an artificial functional oligonucleic acid, which can interact with its target molecule with high affinity and specificity. Enzyme linked aptamer assay (ELAA) is developed to detect cocaine using aptamer fragment/cocaine configuration based on the affinity interaction between aptamer fragments with cocaine. The aptasensor was constructed by cleaving anticocaine aptamer into two fragments: one was assembled on a gold electrode surface, while the other was modified with biotin at 3'-end, which could be further labelled with streptavidin-horseradish peroxidase (SA-HRP). Upon binding with cocaine, the HRP-labelled aptamer fragment/cocaine complex formed on the electrode would increase the reduction current of hydroquinone (HQ) in the presence of H(2)O(2). The sensitivity and the specificity of the proposed electrochemical aptasensor were investigated by differential pulse voltammetry (DPV). The results indicated that the DPV signal change could be used to sensitively detect cocaine with the dynamic range from 0.1 μM to 50 μM and the detection limit down to 20 nM (S/N=3). The proposed aptasensor has the advantages of high sensitivity and low background current. Furthermore, a new configuration for ELAA requiring only a single aptamer sequence is constructed, which can be generalized for detecting different kinds of targets by cleaving the aptamers into two suitable segments.