Aptamer/thrombin/aptamer-AuNPs sandwich enhanced surface plasmon resonance sensor for the detection of subnanomolar thrombin.

A sensitive and selective aptamer/thrombin/aptamer-AuNPs sandwich enhanced surface plasmon resonance (SPR) sensor has been developed for real-time detection of subnanomolar thrombin. In this protocol, one thiol-modified thrombin aptamer (TBA29) was immobilized on gold nanoparticles (AuNPs) via Au-S bonding. The other biotinylated thrombin aptamer (TBA15) was grafted onto streptavidin pretreated SPR gold film through biotin-streptavidin recognition. The presence of thrombin would then induce the formation of a double aptamer sandwich structure on the SPR gold film and results in obvious enhancement of SPR signal, which was proportional to the concentration of thrombin. This proposed assay took advantage of sandwich binding of two affinity aptamers for increased specificity, AuNPs for signal enhancement, as well as SPR signal readout for real-time detection. The SPR signal had a good linear relationship with thrombin concentration in the range of 0.1-75nM, and the detection limit for thrombin was determined to be as low as 0.1nM. It was found that aptamer functionalized AuNPs enhanced the signal of SPR response and thus increased the limit of detection 4-fold and 5-fold compared to direct detection format without AuNPs. This sensor also showed good selectivity for thrombin without being affected by some other proteins, such as BSA and lysozyme. Furthermore, this proposed SPR sensing platform was successfully applied to thrombin analysis in diluted human serum samples.

Bai Y ，Feng F ，Zhao L ，Wang C ，Wang H ，Tian M ，Qin J ，Duan Y ，He X ... -  《-》

Electrochemiluminescence biosensor for the assay of small molecule and protein based on bifunctional aptamer and chemiluminescent functionalized gold nanoparticles.

An electrochemiluminescence (ECL) biosensor for simultaneous detection of adenosine and thrombin in one sample based on bifunctional aptamer and N-(aminobutyl)-N-(ethylisoluminol) functionalized gold nanoparticles (ABEI-AuNPs) was developed. A streptavidin coated gold nanoparticles modified electrode was utilized to immobilize biotinylated bifunctional aptamer (ATA), which consisted of adenosine and thrombin aptamer. The ATA performed as recognition element of capture probe. For adenosine detection, ABEI-AuNPs labeled hybridization probe with a partial complementary sequence of ATA reacted with ATA, leading to a strong ECL response of N-(aminobutyl)-N-(ethylisoluminol) enriched on ABEI-AuNPs. After recognition of adenosine, the hybridization probe was displaced by adenosine and ECL signal declined. The decrease of ECL signal was in proportion to the concentration of adenosine over the range of 5.0×10(-12)-5.0×10(-9) M with a detection limit of 2.2×10(-12) M. For thrombin detection, thrombin was assembled on ATA modified electrode via aptamer-target recognition, another aptamer of thrombin tagged with ABEI-AuNPs was bounded to another reactive site of thrombin, producing ECL signals. The ECL intensity was linearly with the concentration of thrombin from 5×10(-14)M to 5×10(-10) M with a detection limit of 1.2×10(-14) M. In the ECL biosensor, adenosine and thrombin can be detected when they coexisted in one sample and a multi-analytes assay was established. The sensitivity of the present biosensor is superior to most available aptasensors for adenosine and thrombin. The biosensor also showed good selectivity towards the targets. Being challenged in real plasma sample, the biosensor was confirmed to be a good prospect for multi-analytes assay of small molecules and proteins in biological samples.

Chai Y ，Tian D ，Cui H 《-》

Signal amplification for thrombin impedimetric aptasensor: sandwich protocol and use of gold-streptavidin nanoparticles.

In this work, we report a highly specific amplification strategy demonstrated for the ultrasensitive biosensing of thrombin with the use of gold-streptavidin nanoparticles (strep-AuNPs) and silver reduction enhancement. The biotinylated aptamer of thrombin was immobilized onto an avidin-graphite epoxy composite (AvGEC) electrode surface by affinity interaction between biotin and avidin; electrochemical impedance measurements were performed in a solution containing the redox marker ferrocyanide/ferricyanide. The change in interfacial charge transfer resistance (Rct) experimented by the redox marker, was recorded to confirm aptamer complex formation with target protein, thrombin (Thr), in a label-free first stage. A biotinylated second thrombin aptamer, with complementary recognition properties was then used in a sandwich approach. The addition of strep-AuNPs and silver enhancement treatment led to a further increment of Rct thus obtaining significant signal amplification. The AptThrBio1-Thr-AptThrBio2 sandwich formation was inspected by confocal microcopy after incubation with streptavidin quantum dots. In order to visualize the presence of gold nanoparticles, the same silver enhancement treatment was applied to electrodes already modified with the nanoparticle-sandwich conjugate, allowing direct observation by scanning electron microscopy (SEM). Results showed high sensitivity and selectivity for thrombin detection, with an improvement from ca. 4.7 pM in a simple assay to 0.3 pM in the amplified reported scheme.

Ocaña C ，del Valle M 《-》

Gold nanoparticles conjugates-amplified aptamer immunosensing screen-printed carbon electrode strips for thrombin detection.

Thrombin plays the role in cardiovascular diseases and regulates many processes in inflammation and could be a feature of many pathological conditions, including the thromboembolic disease, cancer and neurodegenerative diseases. An ultrasensitive and amplified electrochemical sandwich assay using screen-printed carbon electrode (SPCE) strips for thrombin detection was established in this study. The conductivity and sensing performance of the carbon electrodes were enhanced by using gold nanoparticles (AuNPs). The aptamer addressed on the strips was used as a primary probe to capture thrombin in the detected samples. An amplifier was invented for recognizing thrombin captured on the SPCE, which is the multiple molecules of anti-thrombin antibody (Ab) and horseradish peroxidase (HRP) co-modified AuNPs (AuNPs/Ab-HRP). Hydrogen peroxide was used as the substrate for HRP and then the response current (RC) could be detected. The optimization of these AuNPs conjugates-amplified aptamer immunosensing SPCE strips was conducted for thrombin detection. The detection sensitivity showed a linear relation between RC and thrombin concentration in the range of 10 pM-100 nM, and limit of detection (LOD) was 1.5 pM. The fabricated AuNPs/Ab-HRP-amplified aptamer immunosensing SPCE strips were further used to detect thrombin in human serum with a linear range of 100 pM-100 nM. This study provided the promising SPCE strips with highly sensitive and rapid detection for thrombin by the electrochemical aptasensor combined with AuNPs conjugates for amplifying the detection signal.

Yeh FY ，Liu TY ，Tseng IH ，Yang CW ，Lu LC ，Lin CS ... -  《-》

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

Wang J ，Munir A ，Li Z ，Zhou HS ... -  《-》