The Detection Of β-amyloid Peptide Based On Quantum Dots And Immunoassay

Quantum dots(QDs) were made of semiconductor materials, also known as semiconductor nanocrystals, is quasi zero dimensional nanomaterials, and the three dimensions are less than 100 nm. The appearance like a tiny point, mainly spherical or have spherical shape, consisting of IIB ~ ⅥA or IIIA ~ VA element and the particles’ diameter are also nanoscale. Due to the quantum dots with quantum effects, size effect,quantum confinement effect, macroscopic quantum tunneling effect and surface effect.When it compared with conventional organic fluorescent reagent, it has exhibits excellent fluorescent properties, including wide excitation spectrum and narrow optical emission spectroscopy, good light stability, large Stokes shift, good biocompatible, so it is a kind ideal fluorescent probe.Therefore, in recent years, the application of QDs in the science of life and related disciplines caused more and more person’s attention,especially QDs in biological molecular fluorescent labeling. Most of the application of QDs in this area has focused on the fluorescent probes’ identification and imaging,such as the interaction between biological macromolecules, cells and tissues of single color and multi-color labeling, biological tissue and in vivo optical imaging, and its application in biological samples such as protein, DNA testing.Alzheimer’s disease(AD) is a progressive neurodegenerative pathology causing dementia in humans. The clinical manifestations of AD included a decline in cognitive ability, alterations in behavior, irreversible memory loss, and language impairment.According to the 2015 world Alzheimer’s disease report shows that today over 46 million people live with dementia worldwide, more than the population of Spain. This number is estimated to increase to 131.5 million by 2050. Obviouly, there is a growingawareness of dementia, and the high prevalence and morbidity of the disease in the elderly, the treat time is long and the cost is expensive, which has a tremendous burden on the patient’s family and society. It is no doubt that Alzheimer’s disease is one of the biggest global public health and social care challenges facing people today and in the future. Methods for early diagnosis are critical for the development of effective treatments to combat this debilitating disease. Studies have confirmed that deposition of extracellular amyloid plaque is the main cause of AD, and the major component of amyloid plaque is the 42-residue-long peptide(Aβ(1-42)), which has great tendency to misfold and form toxic aggregates. With regard to the close relationship between AD and Aβ(1-42), Aβ(1-42) is considered as a promising biomarker for AD. In recent years,some attempts have been made to detect Aβ species in both aqueous solutions and cell derived samples. These methods, however, have some limitations. Some methods need expensive instruments, which limit their applications in routine testing. And the sensitivity of some methods is not very high. Therefore, it is highly desirable to develop a sensitive method for Aβ(1-42) without using expensive instruments.Based on the superior properties of QDs, the main works of this thesis are summarized as follows:(1) A new sandwich immunoassay method for the detection of Aβ(1-42) based on quantum dot(QDs) nanolabels and magnetic separation. With beads as the substrate,first of all, the biotinylated C-terminal antibody bonding on the streptavidin modified magnetic beads by biotin- avidin binding interaction, and then bind Aβ(1-42) and biotinylated N-terminal antibody specifically,finally the biotinylated N-terminal antibody bound with streptavidin modified QDs, respectively. In the presence of Aβ(1-42), QDs linked to magnetic beads(MB) via the formation of immune-sandwich complex and can be removed by a magnetic field. And as a result, fluorescence intensity from QDs in the supernatant decreased. Under the optimized conditions, there is a linear relationship between the fluorescence intensity of supernatant solution and the concentration of Aβ(1-42) from 0.50 to 8.0 nmol L-1 with a limit of detection 0.2nmol L-1(3δ). This immunoassay was applied to detect Aβ(1-42) in human cerebrospinal fluid(CSF) successfully.(2) Based on the first job, in order to reduce the reaction time, make the operation more simple, while improve the detection sensitivity,this work established a high sensitivity sandwich immunassay for detecting Aβ(1-42) with QDs as thefluorescent signal label. First of all, the the undecorated C-terminal antibody(C- Ab)which has special combination with one end of Aβ(1-42) will be binded on the 96-well plates. And then combing with Aβ(1-42) and biotinylated N-terminal antibodies(N-Ab) which has special combination with the other end of Aβ(1-42); Finally adding QDs. Removing the supernatant and determining fluorescence intensity, we find that the fluorescent intensity at 525 nm of the supernatant solution showed a good linear relationship with Aβ(1-42), and the concentration of Aβ(1-42) from 5 to 100 pmol L-1.Therefore, another sandwich immunassay was developed, therefore, applying this method to determine the content of Aβ(1-42), the limit of detection is 1.7 pmol L-1(3δ).This method can better to detect Aβ(1-42) in human cerebrospinal fluid(CSF).