Aptamer-gold Nanoparticle Research Articles

Theophylline detection in serum using a self-assembling RNA aptamer-based gold nanoparticle sensor.

Recently, DNA aptamer-gold nanoparticle (AuNP) conjugates have emerged as novel biosensing tools. Although RNA aptamers are more advantageous than DNA aptamers, their vulnerable nature during the construction of these conjugates restricts the development of RNA aptasensors. In this study, we developed an RNA aptamer-based AuNP sensor for the detection of theophylline in serum, combining the high binding affinity and selectivity of a theophylline RNA aptamer and the fluorescence quenching ability of AuNPs. In order to prevent nuclease degradation during the experimental process, the single strand of the theophylline RNA aptamer (33-mer) was split at the end loop region into two shorter halves, which were able to reassemble to form the theophylline-binding pocket. One fragment was linked to a DNA sequence that included a 15 thymine (T15) spacer and a polyadenine (polyA, A12) tail. The chimeric RNA/DNA oligonucleotide was attached to AuNPs within a few minutes via adsorption of the polyA tail. The other fragment was labeled with a fluorophore (Cy3). The two individual fragments self-assembled in the presence of theophylline. Upon ligand binding, the fragments came into close proximity, resulting in fluorescence quenching. This sensor exhibited a low detection limit of 0.05 µM, with a linear dynamic range from 0.1 to 10 µM in serum. Moreover, the sensor did not recognize theophylline-related compounds (e.g., caffeine and theobromine), demonstrating its high selectivity. This strategy offers new possibilities for the application of RNA aptasensors in clinical settings.

Colorimetric detection with aptamer-gold nanoparticle conjugates coupled to an android-based color analysis application for use in the field

The feasibility of using aptamer-gold nanoparticle conjugates (Apt-AuNPs) to design colorimetric assays for in the field detection of small molecules was investigated. An assay to detect cocaine was designed using two clones of a known cocaine-binding aptamer. The assay was based on the AuNPs difference in affinity for single-stranded DNA (non-binding) and double stranded DNA (target bound). In the first assay, a commonly used design was followed, in which the aptamer and target were incubated to allow binding followed by exposure to the AuNPs. Interactions between the non-bound analytes and the AuNPs surface resulted in a number of false positives. The assay was redesigned by incubating the AuNPs and the aptamer prior to target addition to passivate the AuNPs surface. The adsorbed aptamer was able to bind the target while preventing non-specific interactions. The assay was validated with a number of masking and cutting agents and other controlled substances showing minimal false positives. Studies to improve the assay performance in the field were performed, showing that assay activity could be preserved for up to 2 months. To facilitate the assay analysis, an android application for automatic colorimetric characterization was developed. The application was validated by challenging the assay with cocaine standards of different concentrations, and comparing the results to a conventional plate reader, showing outstanding agreement. Finally, the rapid identification of cocaine in mixtures mimicking street samples was demonstrated. This work established that Apt-AuNPs can be used to design robust assays to be used in the field.

Assembly of a Dual Aptamer Gold Nanoparticle Conjugate Ensemble in the Specific Detection of Thrombin when Coupled with Dynamic Light Scattering Spectroscopy

We demonstrate an extremely facile, rapid, specific and selective method for detecting proteins using aptamerconjugated gold nanoparticles coupled with dynamic light scattering (DLS) at ambient conditions. Addition of proteins to aptamer-conjugated gold nanoparticles (GNPs) induced the formation of protein-aptamer-gold nanoparticle conjugate complexes. The average hydrodynamic diameter of the nanoconjugate complexes as measured by DLS, increased with the corresponding increase in protein concentration. This correlation formed the analytical basis of the assay. The nanoparticles and nanoconjugate complexes were characterized by transmission electron microscopy, ultra-violet visible spectroscopy and DLS. Various parameters affecting the assay were optimized. Using thrombin as the model analyte, we demonstrated the detection of as low as 1.41 nM (0.05 μg/mL) of the protein. A linear dynamic range of up to 300 nM (11 μg/mL) was realized. The presence of other interfering proteins such as BSA showed no effect on the assay response.The presence of other interfering proteins such as bovine serum albumin (BSA) showed no significant effect on the assay response.

Masking Nanoparticle Surfaces for Sensitive and Selective Colorimetric Detection of Proteins

We have developed a convenient and efficient colorimetric detection system for protein targets using aptamer-gold nanoparticle conjugates. We take advantage of the correlation between the catalytic properties and the exposed surface area of the nanoparticles, which is inversely proportional to the amount of the aptamer-bound protein targets. As the concentration of the protein target increases, the nanoparticle surface area becomes more masked, thus increasing the reduction time of 4-nitrophenol for the color change. We also reduce the detection time by either redesigning the aptamer sequences or regulating their density. This detection system is highly selective, discriminating the target protein even at a concentration 1000 times higher than the limit of detection (LOD). Importantly, to the best of our knowledge, the LOD with the unaided eye in this work is the lowest for a colorimetric detection system using lysozyme as a model protein (16 nM). Lysozyme in chicken egg whites is directly analyzed using our detection system, whose results are in excellent agreement with the enzyme-linked immunosorbent assay (ELISA) analysis.

Colorimetric Sensing of Tetracyclines in Milk Based on the Assembly of Cationic Conjugated Polymer-Aggregated Gold Nanoparticles

We have developed an unlabeled sensing strategy for tetracycline (TET) detection in aqueous solutions and milk by using an aptamer–gold nanoparticle (AuNP)-based colorimetric method. Aptamers were found to be absorbed on the Au surface which stabilizes the AuNPs against conditions of high poly(diallyldimethylammonium) strength. The introduction of TET had reduced aptamers to form a TET–aptamer complex, so that the following cationic polymers could aggregate AuNPs and cause a remarkable change in color. Through this phenomenon, TET can be detected qualitatively and quantitatively by means of absorbance. This unlabeled strategy saves both the time and cost for detection, which could selectively detect TET at concentrations as low as 1 μM and 45.8 nM for the naked eyes and colorimetric detector, respectively. In addition, this biosensor exhibited high selectivity over other antibiotics with an excellent recovery in the detection of milk. Because of the advantages of this sensor, we hope this will be the basis for an assay for the sensitive, colorimetric detection of a wide range of molecular analytes.

Colorimetric detection with aptamer–gold nanoparticle conjugates: effect of aptamer length on response

A riboflavin binding aptamer (RBA) was used in combination with gold nanoparticles (AuNPs) to detect riboflavin in vitro. The RBA–AuNP conjugates (RBA–AuNPs) responded colorimetrically to the presence of riboflavin and this response could be followed by the naked eye. This system was used as a model to study how modifications on the aptamer sequence affect the RBA–AuNPs’ stability and their response to their target. To mimic primers and other sequence modifications typically used in aptamer work, the RBA was extended by adding extra bases to its 5′ end. These extra bases were designed to avoid interactions with the RBA binding site. The response of these RBA–AuNPs was evaluated and compared. Dynamic light scattering and UV-aggregation kinetics studies showed that the length of the aptamer significantly affected the RBA–AuNPs’ stability and, as a consequence, the magnitude of the detection response to riboflavin. The addition of thymine nucleotides instead of random tails to the RBA showed that the effects observed were not specific to the sequence used. This study shows that modifications of the aptamer sequence provide a means to improve the stability of aptamer–AuNPs conjugates and their sensing response.

A Conjugated Aptamer-Gold Nanoparticle Fluorescent Probe for Highly Sensitive Detection of rHuEPO-α

We present here a novel conjugated aptamer-gold nanoparticle (Apt-AuNPs) fluorescent probe and its application for specific detection of recombinant human erythropoietin-α (rHuEPO-α). In this nanobiosensor, 12 nm AuNPs function as both a nano-scaffold and a nano-quencher (fluorescent energy acceptor), on the surface of which the complementary sequences are linked (as cODN-AuNPs) and pre-hybridized with carboxymethylfluorescein (FAM)-labeled anti-rHuEPO-α aptamers. Upon target protein binding, the aptamers can be released from the AuNP surface and the fluorescence signal is restored. Key variables such as the length of linker, the hybridization site and length have been designed and optimized. Full performance evaluation including sensitivity, linear range and interference substances are also described. This nanobiosensor provides a promising approach for a simple and direct quantification of rHuEPO-α concentrations as low as 0.92 nM within a few hours.

Quantitative detection of adenosine in urine using silver enhancement of aptamer–gold nanoparticle aggregation and progressive dilution

We implement the progressive dilution strategy to bring assays based on gold nanoparticles to a quantitative level. This is demonstrated by the detection of adenosine in urine by combining progressive dilution with the silver enhancement of aptamer-gold nanoparticle aggregation, giving good accuracy, high selectivity, and an unlimited dynamic range above LOD.

A surface energy transfer nanoruler for measuring binding site distances on live cell surfaces.

Measuring distances at molecular length scales in living systems is a significant challenge. Methods like Förster resonance energy transfer (FRET) have limitations due to short detection distances and strict orientations. Recently, surface energy transfer (SET) has been used in bulk solutions; however, it cannot be applied to living systems. Here, we have developed an SET nanoruler, using aptamer-gold nanoparticle conjugates with different diameters, to monitor the distance between binding sites of a receptor on living cells. The nanoruler can measure separation distances well beyond the detection limit of FRET. Thus, for the first time, we have developed an effective SET nanoruler for live cells with long distance, easy construction, fast detection, and low background. This is also the first time that the distance between the aptamer and antibody binding sites in the membrane protein PTK7 was measured accurately. The SET nanoruler represents the next leap forward to monitor structural components within living cell membranes.

Ultrasensitive electrochemical aptasensor for thrombin based on the amplification of aptamer–AuNPs–HRP conjugates

Successful development of an ultrasensitive and highly specific electrochemical aptasensor for thrombin based on amplification of aptamer–gold nanoparticles–horseradish peroxidase (aptamer–AuNPs–HRP) conjugates was reported. In this electrochemical protocol, aptamer1 (Apt1) was immobilized on core/shell Fe 3O 4/Au magnetic nanoparticles (AuMNPs) and served as capture probe. Aptamer2 (Apt2) was dual labeled with AuNPs and HRP and used as detection probe. In the presence of thrombin, the sandwich format of AuMNPs–Apt1/thrombin/Apt2–AuNPs–HRP was fabricated. Remarkable signal amplification was realized by taking the advantage of AuNPs and catalytic reactions of HRP. Other proteins, such as human serum albumin, lysozyme, fibrinogen, and IgG did not show significant interference with the assay for thrombin. Linear response to thrombin concentration in the range of 0.1–60 pM and lower detection limit down to 30 fM ( S/ N = 3) was obtained with the proposed method. This electrochemical aptasensor is simple, rapid (the whole detection period for a thrombin sample is less than 35 min), sensitive and highly specific, it shows promising potential in protein detection and disease diagnosis.

Aptamer–Au NPs conjugates-enhanced SPR sensing for the ultrasensitive sandwich immunoassay

The goal of this work is to explore the amplification effect of aptamer–gold nanoparticles (Au NPs) conjugates for ultrasensitive detection of large biomolecules by surface plasmon resonance (SPR). A novel sandwich immunoassay is designed to demonstrate the amplification effect of aptamer–Au NPs conjugates by using human immunoglobulin E (IgE) as model analyte. Human IgE, captured by immobilized goat anti-human IgE on SPR gold film, is sensitively detected by SPR spectroscopy with a lowest detection limit of 1 ng/ml after anti-human IgE aptamer–Au NPs conjugates is used as amplification reagent. Meanwhile, the non-specific adsorption of aptamer–Au NPs conjugates on goat anti-human IgE is confirmed by SPR spectroscopy and then it is minimized by treating aptamer–Au NPs conjugates with 6-mercaptohexan-1-ol (MCH). These results confirm that aptamer–Au NPs conjugates is a powerful sandwich element and an excellent amplification reagent for SPR-based sandwich immunoassay.