Visualization Detection Methods For Proteins Based On Silver Nanoparticles

Aptamers are single-stranded oligonucletides generated from an in vitro process known as SELEX. Based on their high binding affinity and specificity towards target molecules, aptamers have been utilized in applications ranging from biosening to diagnostics. Meanwhile, nanomaterials possessing unique optical, electronic and catalytic properties can interact with biomolecules to yield improved signal amplication and target recognition. By combining both technologies, aptamer conjugated nanoparticles offer great promise for applications in bioanalysis.With the rapid development of nanoscience and nanotechnology, nanoparticles of various shapes, sizes, and compositions have been successfully used in bioanalysis such as bioassay and bioimaging due to their unique optical properties, high surface-to-volume ratio as well as other size-dependent qualities.We developed a simple and rapid colorimetric assay for protein detection based on the reduction of dye molecules catalyzed by silver nanoparticles (AgNPs). Aptamer-modified magnetic particles and aptamer-functionalized AgNPs were employed as capture and detection probes, respectively. Introduction of thrombin as target protein could form a sandwich-type complex involving catalytically active AgNPs, whose catalytic activity was monitored on the catalytic reduction of rhodamine B (RhB) by sodium borohydride (NaBH4). The amount of immobilized AgNPs on the complex increased along with the increase of the thrombin concentration, thus the detection of thrombin was achieved via recording the decrease in absorbance corresponding to RhB. This method has adopted several advantages from the key factors involved, i.e., the sandwich binding of affinity aptamers contributed to the increased specificity; magnetic particles could result in rapid capture and separation processes; the conjugation of AgNPs would lead to a clear visual detection. It allows for the detection limit of thrombin down to picomolar level by the naked eye, with remarkable selectivity over other proteins. Moreover, it is possible to apply this method to the other targets with two binding sites as well.Microfluidic platform offers a unique alternative approach for the integration of bioassays. It possesses advantages over traditional methods, such as short analysis time, portable, high throughout and minimal sample consumption. We demonstrated a sensitive and facile protocol for protein detection in the microfluidic device. A PDMS-glass chip consists of seven parallel channels was designed, in which biotin modified aptamer 15 was absorbed uniformly. The combination of aptamer 29-functionalized AgNPs and thrombin was captured and enriched in a sandwich form on the surface of chip. The visual analysis was achieved through the inherent optical properties of AgNPs. The results indicated that the color of channels displayed deeper along with the increase of thrombin concentration. The grayscale was linear to the concentration of thrombin in the range from 0.02 nM to 20 nM. This suggested method made full use of advantages in microfluidic systems and showed high sensitivity and excellent specificity. The integration of nanomaterials and microfluidic devices have the potential to develop separation and detection assays for proteins and started a new scope of operation of biomarkers diagnoses.