The Development of Biosensors Using Gold Nanoparticles as Sensing Platform

基於納米金的生物傳感器方法研究

Student thesis: Doctoral Thesis

View graph of relations

Author(s)

Detail(s)

Awarding Institution
Supervisor(s)
Award date25 Jul 2017

Link(s)

Abstract

Metal nanoparticles have attracted scientists’ attention for decades because of their wide application in biomedical science and engineering. These metal nanoparticles can be synthesized in different sizes and shapes with various chemical functional groups, which allows them to conjugate with biomolecules such as antibodies, oligonucleotides, and drugs. Therefore, metal nanoparticles have several potential applications in biotechnology, such as drug delivery and diagnostic imaging.
Among these metal nanoparticles, gold nanoparticles (AuNPs) are very popular for applications in biomedical science and engineering. Because of their excellent biocompatibility, unique optical properties, good conductivity, and high surface-to-volume ratio, AuNPs have been widely used for biosensing, colorimetric detection, and electroanalysis. In this thesis, we introduce three amplification methods for the detection of oligonucleotides and botulinum neurotoxin (BoNT) by using AuNPs as the sensing platform.
First, AuNPs were used for the amplification detection of oligonucleotides. By using AuNPs as “nano sticky balls” to carry oligonucleotides, we could increase the detection limit of this method to 10 amol for single-strand DNA (ssDNA) (228 fM in 45 μL) and 75 amol for single-strand RNA (ssRNA) (1.67 pM in 45 μL). Moreover, this method was used for the detection of target oligonucleotides in blood serum and miRNA extracted from the metastatic epithelial cells of the human mammary gland (MDA-MB-231 cell line). This method was considerably more sensitive than other colorimetric detections such as AuNP aggregation or magnetophoretic assay and has many potential applications for disease diagnosis in low-resource settings.
Second, we developed an amplification method based on purified double-strand DNA (dsDNA). Here, the AuNPs were used for the purification of dsDNA. We developed a new modification method that allowed only dsDNA loading on AuNPs. Thus, we could obtain pure dsDNA for the amplification detection of oligonucleotides. With the assistance of polymerase, this method could detect target DNA concentrations of 2.5 pM and 0.01 U/μL of polymerase. This method might have potential applications in the detection of miRNA and polymerase activity in live cell research.
Finally, AuNPs were used as a color indicator for the colorimetric detection of active BoNT on the basis of their unique optical property, that is, a color change from red to purple after aggregation. In this scheme, because of the proteolytic activity of BoNT, the SNAPtide, a short peptide, would be cleaved. The cleaved peptide then caused the aggregation of AuNPs, resulting in the color change from red to purple. On the basis of this color change, our method could detect BoNT at a concentration of as low as 1 ng/mL (6.67 pM), and the limit of detection was 0.25 ng/mL (1.67 pM, 3σ/s) according to the spectral absorbance, which makes the proposed method, to the best of our knowledge, the most sensitive colorimetric method for BoNT detection. Therefore, this simple and sensitive method provides a broad and promising application for the onsite detection of BoNT. In summary, owing to these advantages of AuNPs, we believe that the use of AuNPs as a sensing platform will significantly improve the sensitivity of biosensors. Moreover, combined with the advanced technology of microfluidic chips and real sample preparation, we expect the proposed method to be helpful in the development of commercial products and real sample detection in the future.