Atomic Force Microscopy Recognition Imaging Research Articles

Recognition Imaging on Native Chromatin Extracted from Cancerous and Non-Cancerous Cell Lines

Atomic force microscopy (AFM) recognition imaging is a label-free technique which can identify specific molecules from compositionally complex samples while mapping the topography of the samples simultaneously in a liquid environment. It required a sensitive imaging mode. Typically, the so-called MAC mode is used, where the AFM tip is driven directly by a magnetic field which will reduce the movement of liquid surrounding the tip and increase signal to noise ratio. The AFM tips for MAC mode need to have a ferromagnetic coating like nickel in order to be driven by a magnetic field. Commercial MAC mode probes are typically silicon nitride or quartz. We developed a protocol to coat sharp commercial silicon probes with nickel, making them reliable MAC mode tips. We applied our new probes to samples prepared from the supernatant fraction of a nuclear extract of EPC2 (non-cancerous) and CPD (cancerous) esophageal cell lines. Salt fractionation is an efficient method for the extraction of intact chromatin fragments from cell nuclei, but the supernatant fraction was not studied in previous work. With anti-H3 antibody modified MAC mode tips, we identified histones H3 in samples of both cell lines, indicating that this fraction contains chromatin. We proved the specificity by blocking with a peptide mimicking the H3 binding site for our antibody. Further, we demonstrated the dependence of the recognition signals on the oscillation amplitude of the probe, indicating the robustness of our recognition signal. We also analyzed the nuclear extracts for the presence of SMC2, a structural maintenance protein for chromosomes. We applied AFM recognition imaging with anti-SMC2 antibody modified probes to the prepared chromatin samples and identified SMC2 proteins in samples of both cell lines. Western blots confirmed the presence of SMC2 proteins in the samples. Overall, AFM recognition imaging provides a robust way to analyze the structure and molecular components of nuclear extracts from human cells, providing insights into the link between chromatin structure and cell phenotypes.

Mapping molecular adhesion sites inside SMIL coated capillaries using atomic force microscopy recognition imaging

Capillary zone electrophoresis (CZE) is a powerful analytical technique for fast and efficient separation of different analytes ranging from small inorganic ions to large proteins. However electrophoretic resolution significantly depends on the coating of the inner capillary surface. High technical efforts like Successive Multiple Ionic Polymer Layer (SMIL) generation have been taken to develop stable coatings with switchable surface charges fulfilling the requirements needed for optimal separation. Although the performance can be easily proven in normalized test runs, characterization of the coating itself remains challenging. Atomic force microscopy (AFM) allows for topographical investigation of biological and analytical relevant surfaces with nanometer resolution and yields information about the surface roughness and homogeneity. Upgrading the scanning tip to a molecular biosensor by adhesive molecules (like partly inverted charged molecules) allows for performing topography and recognition imaging (TREC). As a result, simultaneously acquired sample topography and adhesion maps can be recorded. We optimized this technique for electrophoresis capillaries and investigated the charge distribution of differently composed and treated SMIL coatings. By using the positively charged protein avidin as a single molecule sensor, we compared these SMIL coatings with respect to negative charges, resulting in adhesion maps with nanometer resolution. The capability of TREC as a functional investigation technique at the nanoscale was successfully demonstrated.

Mapping single molecular binding kinetics of carbohydrate-binding module with crystalline cellulose by atomic force microscopy recognition imaging.

We studied the binding kinetics of family 3 carbohydrate-binding module (CBM3a) molecules to crystalline cellulose fibrils extracted from the poplar cell wall by atomic force microscopy (AFM) recognition imaging. The free CBM3a molecules of different concentrations were added to the buffer solution to bind to the crystalline cellulose sample immobilized on the AFM substrate. During in-situ AFM imaging, the CBM molecules were observed to bind to cellulose efficiently and regularly, especially in the first 60-120 min. A 1:1 single-molecule binding model was used to study the kinetics of the CBM3a-cellulose interaction. The saturation time when the concentration of occupied binding sites is 99% of the maximum bound CBM3a concentration at the end of reaction, t(0.99), was determined by fitting different concentrations of CBM3a against reaction time using the high resolution AFM images and the single-molecule kinetics equations. Based on the experimental data and kinetics calculations, the minimal effective initial CBM3a concentration was estimated to be 5.1 × 10(-7) M at 287 min reaction time. This study provides an in-depth understanding of the binding mechanism of CBM with crystalline cellulose at single molecule level.

Detecting Protein Aggregates on Untreated Human Tissue Samples by Atomic Force Microscopy Recognition Imaging

We apply topography and recognition (TREC) imaging to the analysis of whole, untreated human tissue for what we believe to be the first time. Pseudoexfoliation syndrome (PEX), a well-known cause of irreversible blindness worldwide, is characterized by abnormal protein aggregation on the anterior lens capsule of the eye. However, the development of effective therapies has been hampered by a lack of detailed knowledge of the protein constituents in these pathological deposits and their distribution. Using both TREC and immunofluorescence, one of the proteins implicated in the PEX pathology—the apolipoprotein clusterin—was detected, and differences in its distribution pattern on the surface of untreated human lens capsule tissue in both PEX and normal control samples were investigated. Our study shows the potential of TREC imaging for the analysis of whole, untreated human tissue samples.

Simple, Clickable Protocol for Atomic Force Microscopy Tip Modification and Its Application for Trace Ricin Detection by Recognition Imaging

A simple two-step protocol for modification of atomic force microscopy (AFM) tip and substrate by using a "click reaction" has been developed. The modified tip and substrate would be applied to detect trace amounts of ricin by using atomic force microscopy. A key feature of the approach is the use of a PEG (polyethylene glycol) derivative functionalized with one thiol and one azide ending group. One end of the PEG was attached to the gold-coated AFM tip by a strong Au-thiol bond. The azide group hanging at the other end of the immobilized PEG was used for the attachment of an antiricin antibody modified with an alkyne group using a "click reaction". The latter reaction is highly efficient, compatible with the presence of many functional groups and could proceed under mild reaction conditions. In a separate step, ricin was immobilized on the gold substrate surface that was modified by active esters. For this process, a novel bifunctional reagent was employed containing an active ester and a thioctic acid moiety. By these modification processes, AFM recognition imaging was used to detect the toxin molecules and the results show fg/mL detection sensitivity, surpassing the existing detection techniques. With measurement of the unbinding force between the antiricin antibody and ricin, which was statistically determined to be 64.89 +/- 1.67 pN, the single molecular specificity of this sensing technique is realized.

Characterization of an antibody scFv that recognizes fibrillar insulin and β-amyloid using atomic force microscopy

Fibrillar amyloid is the hallmark feature of many protein aggregation diseases, such as Alzheimer's and Parkinson's diseases. A monoclonal single-chain variable fragment (scFv) targeting insulin fibrils was isolated using phage display technology and an atomic force microscopy (AFM) mica substrate. Specific targeting of the scFv to insulin fibrils but not monomers or other small oligomeric forms, under similar conditions, was demonstrated both by enzyme-linked immunosorbent assays and AFM recognition imaging. The scFv also recognizes β-amyloid fibrils, a hallmark feature of Alzheimer's disease. The results suggest that the isolated scFv possibly targets a shared fibrillar motif—probably the cross-β-sheet characteristic of amyloid fibrils. The techniques outlined here provide additional tools to further study the process of fibril formation. The scFvs isolated can have potential use as diagnostic or therapeutic reagents for protein aggregation diseases.

Two-component atomic force microscopy recognition imaging of complex samples

Biological complexes are typically multisubunit in nature and the processes in which they participate often involve protein compositional changes in themselves and/or their target substrates. Being able to identify more than one type of protein in complex samples and to track compositional changes during processes would thus be very useful. Toward this goal, we describe here a single-molecule technique that can simultaneously identify two types of proteins in compositionally complex samples. It is an adaptation of the recently developed atomic force microscopy (AFM) recognition imaging technique but involves the tethering of two different types of antibodies to the AFM tip and sequential blocking with appropriate antigenic peptides to distinguish the recognition from each antibody. The approach is shown to be capable of simultaneously identifying in a single AFM image two specific components, BRG1 and β-actin, of the human Swi–Snf ATP-dependent nucleosome remodeling complex and two types of histones, H2A and H3, in chromatin samples.

Nanobiotechnology

Nanobiotechnology