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Functionalization of Polystyrene Microtiter Plates with Dendrons and Immunodetection of PSA by Force-Based Atomic Force Microscopy

Functionalization of Polystyrene Microtiter Plates with Dendrons and Immunodetection of PSA by Force-Based Atomic Force Microscopy
Roy, Dhruvajyoti
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Abstract Chapter 1. Towards Sensitive Biomarker Detection The development of sensitive biosensors and related techniques for the detection of biomarker has become a burgeoning field, which combines fundamental biological, chemical, and physical sciences with engineering sciences. Particularly, the impact of nanotechnology on sensing performance has led to extraordinary advances in molecular diagnostics. Recent developments in proteomics provide promising biomarker discovery related to diagnostic application and nanomaterials-based sensor has opened up possibilities for ultra-sensitive detection of those biomarkers. In this context, cancer related biomarkers have attended much importance for their early detection and particularly detection of prostate specific antigen (PSA) as a biomarker for prostate cancer has been extensively studied. In this section, various techniques have been highlighted for the detection of biomarker. The potential of nanomaterial-based biosensing has been addressed by recent technical developments. Applications of nanotools and sensitive read-out techniques for diagnostics have also been highlighted. The emergence of novel technologies has improved medical diagnostics but sensitive detection of biomarkers from complex biological samples is still challenging. The use of potential sensor platform in combination with suitable read-out system is of great interest to improve sensing performance. In addition, the molecularly controlled platforms have great impact to enhance the analytical performance of biosensors. In this respect, applications of mixed-self-assembled monolayer and the drawback of this approach have been depicted. Particular emphasis has been given on the application of dendron molecules as spacing template for the functionaliztion of spatially controlled nanoscale interfaces. The impact of lateral spacing for rational biomolecule immobilization has been exemplified. Chapter 2. Dendron-Modified Polystyrene Microtiter Plate: Influence of Spacing between Immobilized Amyloid Beta Proteins The design of proper surface and rational conjugation for controlled packing of biomolecules on polystyrene microtiter plates are of great interest in chemical, biological, and pharmaceutical sciences. In this chapter, a polystyrene microtiter plate was coated with a molecular layer of a cone-shaped dendron as a means of providing proper spacing between immobilized biomolecules. For the coating preparation, di-(ethylene glycol) vinyl ether was grafted onto the surface of the microtiter plate by a plasma process, followed by self-assembly of a second generation dendron (9-acid) or a third generation dendron (27-acid). Contact angle analysis revealed a pronounced increase in the hydrophilicity upon plasma grafting, while the hydrophilicity reverted/decreased after dendron immobilization. For the analysis by force-based atomic force microscopy (AFM), oligonucleotides were immobilized onto the AFM tip and the plate. The DNA-DNA interaction was observed at all spots examined, which implied that coating of the dendrons was uniform over the entire surface. The effectiveness for biomolecular assays of the spacing on dendron-modified microtiter plates was examined by carrying out an enzyme-linked immunosorbent assay (ELISA), where enhanced detection of different fragments of amyloid beta protein (Aβ) was observed when compared with other conventional plates, such as untreated polystyrene or maleic anhydride activated plates. The positive influence of the mesospacing between biomolecules on the microtiter plates for this assay was confirmed. Chapter 3. “Seeing and Counting” Individual PSAs Captured on a Microarrayed Spot with Force-Based Atomic Force Microscopy The substantial interest in the detection of proteins at ultra-low level is quite challenging, because numerous important biological markers for cancer, infectious diseases, or biochemical processes are present at very low levels during the early stages of the disease development. In this chapter, we demonstrate that the force-based AFM can be employed as a force spectroscopy tool to investigate the commercial immunoassay substrates. The mapping capability of atomic force microscopy (AFM) enabled us to see captured prostate specific antigens (PSAs) on a spot microarrayed with the corresponding antibody and count the number of the antigens in a submicron area. To enhance the reliability and the reproducibility of the approach, a third generation dendron was employed for the surface treatment. The specific force between the captured PSA and the detection antibody (5A6) was measured after cross-linking, and the mean unbinding force was 56 ± 2 pN. At 100 fM there were 12 captured antigens in 4.32 × 104 nm2, and the number was dependent upon the concentration. A larger hydrodynamic distance (8 ± 2 nm) of the immunocomplex resulted in a cluster of pixels corresponding to the single complex in a map recorded over a selected area with a positional interval of 3 nm, and this feature helped to discriminate between pixels of the specific interaction and the nonspecific ones. The results indicate that the approach can be applicable to the quantitative analysis of the antigen in a sample and imply that it can be extended to a sample of very low copy numbers as long as the size of the microrrayed spot is reduced.
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