Samir M. Iqbal

Surface-immobilized aptamers for cancer cell isolation and microscopic cytology

Exposing rare but highly malignant tumor cells that migrate from the primary tumor mass into adjacent tissue(s) or circulate in the bloodstream is critical for early detection and effective intervention(s). Here, we report on an aptamer-based strategy directed against epidermal growth factor receptor (EGFR), the most common oncogene in glioblastoma (GBM), to detect these deadly tumor cells. GBMs are characterized by diffuse infiltration into normal brain regions, and the inability to detect GBM cells renders the disease surgically incurable with a median survival of just 14.2 months. To test the sensitivity and specificity of our platform, anti-EGFR RNA aptamers were immobilized on chemically modified glass surfaces. Cells tested included primary human GBM cells expressing high levels of the wild-type EGFR, as well as genetically engineered murine glioma cells overexpressing the most common EGFR mutant (EGFRvIII lacking exons 2-7) in Ink4a/Arf-deficient astrocytes. We found that surfaces functionalized with anti-EGFR aptamers could capture both the human and murine GBM cells with high sensitivity and specificity. Our findings show how novel aptamer substrates could be used to determine whether surgical resection margins are free of tumor cells, or more widely for detecting tumor cells circulating in peripheral blood to improve early detection and/or monitoring residual disease after treatment.

Direct current electrical characterization of ds-DNA in nanogap junctions

Measurements of DNA conductivity, hybridization, and melting using electronic means can have wide applications in molecular electronics and biological sensors. We have fabricated nanogap break-junctions by electromigration through thin gold-on-titanium films. 18-mer thiolated ds-DNA molecules were covalently attached between the electrodes and dc electrical measurements were done. The conductance was measured through the molecule before and after a temperature ramp from 300 to 400 K. A dramatic decrease in conductance was observed, analogous to an electrical fuse, possibly attributed to complete or partial denaturing of the ds-DNA molecules bridging the nanogaps. We also show evidence that the dc resistance of dry DNA strands of the same length decreases with increasing guanine-cytosine content in the sequence with values ranging from 10 M Ω to 2 G Ω. These findings can have important consequences in DNA-based molecular electronics and direct label-free detection of DNA hybridization.

Nanotextured substrates with immobilized aptamers for cancer cell isolation and cytology

The detection of a small number of circulating tumor cells (CTCs) is important, especially in the early stages of cancer. Small numbers of CTCs are hard to detect, because very few approaches are sensitive enough to differentiate these from the pool of other cells. Improving the affinity of a selective, surface‐functionalized molecule is important given the scarcity of CTCs in vivo. There are several proteins and aptamers that provide such high affinity; however, using surface nanotexturing increases this affinity even further.

Fabrication and characterization of solid-state nanopores using a field emission scanning electron microscope

The fabrication of solid-state nanopores using the electron beam of a transmission electron microscope (TEM) has been reported in the past. Here, we report a similar method to fabricate solid-state nanopores using the electron source of a conventional field-emission scanning electron microscope (FESEM) instead. Micromachining was used to create initial pore diameters between 50nm and 200nm, and controlled pore shrinking to sub 10nm diameters was performed subsequently during in situ processing in the FESEM. Noticeably, different shrinking behavior was observed when using irradiation from the electron source of the FESEM than the TEM. Unlike previous reports of TEM mediated pore shrinkage, the mechanism of pore shrinkage when using the FESEM could be a result of surface defects generated by radiolysis and subsequent motion of silicon atoms to the pore periphery.

Capture, isolation and release of cancer cells with aptamer-functionalized glass bead array

Early detection and isolation of circulating tumor cells (CTC) can enable better prognosis for cancer patients. A Hele-Shaw device with aptamer functionalized glass beads is designed, modeled, and fabricated to efficiently isolate cancer cells from a cellular mixture. The glass beads are functionalized with anti-epidermal growth factor receptor (EGFR) aptamer and sit in ordered array of pits in polydimethylsiloxane (PDMS) channel. A PDMS encapsulation is then used to cover the channel and to flow through cell solution. The beads capture cancer cells from flowing solution depicting high selectivity. The cell-bound glass beads are then re-suspended from the device surface followed by the release of 92% cells from glass beads using combination of soft shaking and anti-sense RNA. This approach ensures that the cells remain in native state and undisturbed during capture, isolation and elution for post-analysis. The use of highly selective anti-EGFR aptamer with the glass beads in an array and subsequent release of cells with antisense molecules provide multiple levels of binding and release opportunities that can help in defining new classes of CTC enumeration devices.

Velocity Effect on Aptamer-Based Circulating Tumor Cell Isolation in Microfluidic Devices

The isolation and detection of rare circulating tumor cells (CTCs) has been one of the focuses of intense research recently. In a microfluidic device, a number of factors can influence the enrichment capability of surface-bound probe molecules. This article analyzes the important factor of flow velocity in a microfluidic channel. The competition of surface-grafted anti-EGFR aptamers to bind the overexpressed EGFR on cell membranes against the drag force from the fluid flow is an important efficiency determining factor. The flow rate variations are applied both in experiments and in simulation models to study their effects on CTC capture efficiency. A mixture of mononuclear cells and human Glioblastoma cells is used to isolate cancer cells from the cellular flow. The results show interdependence between the adhesion probability, isolation efficiency, and flow rate. This work can help in designing flow-through lab-on-chip devices that use surface-bound probe affinities against overexpressed biomarkers for cell isolation. This work demonstrates that microfluidic based approaches have strong potential applications in CTC detection and isolation.

Electrical fingerprinting, 3D profiling and detection of tumor cells with solid-state micropores

Solid-state micropores can provide direct information of ex vivo or in vitro cell populations. Micropores are used to detect and discriminate cancer cells based on the translocation behavior through micropores. The approach provides rapid detection of cell types based on their size and mechano-physical properties like elasticity, viscosity and stiffness. Use of a single micropore device enables detection of tumor cells from whole blood efficiently, at 70% CTC detection efficiency. The CTCs show characteristic electrical signals which easily distinguish these from other cell types. The approach provides a gentle and inexpensive instrument that can be used for specific blood analysis in a lab-on-a-chip setting. The device does not require any preprocessing of the blood sample, particles/beads attachment, surface functionalization or fluorescent tags and provides quantitative and objective detection of cancer cells.

Shrinking of Solid-state Nanopores by Direct Thermal Heating

Solid-state nanopores have emerged as useful single-molecule sensors for DNA and proteins. A novel and simple technique for solid-state nanopore fabrication is reported here. The process involves direct thermal heating of 100 to 300 nm nanopores, made by focused ion beam (FIB) milling in free-standing membranes. Direct heating results in shrinking of the silicon dioxide nanopores. The free-standing silicon dioxide membrane is softened and adatoms diffuse to a lower surface free energy. The model predicts the dynamics of the shrinking process as validated by experiments. The method described herein, can process many samples at one time. The inbuilt stress in the oxide film is also reduced due to annealing. The surface composition of the pore walls remains the same during the shrinking process. The linear shrinkage rate gives a reproducible way to control the diameter of a pore with nanometer precision.

Electrical detection of cancer biomarker using aptamers with nanogap break-junctions

Epidermal growth factor receptor (EGFR) is a cell surface protein overexpressed in cancerous cells. It is known to be the most common oncogene. EGFR concentration also increases in the serum of cancer patients. The detection of small changes in the concentration of EGFR can be critical for early diagnosis, resulting in better treatment and improved survival rate of cancer patients. This article reports an RNA aptamer based approach to selectively capture EGFR protein and an electrical scheme for its detection. Pairs of gold electrodes with nanometer separation were made through confluence of focused ion beam scratching and electromigration. The aptamer was hybridized to a single stranded DNA molecule, which in turn was immobilized on the SiO(2) surface between the gold nanoelectrodes. The selectivity of the aptamer was demonstrated by using control chips with mutated non-selective aptamer and with no aptamer. Surface functionalization was characterized by optical detection and two orders of magnitude increase in direct current (DC) was measured when selective capture of EGFR occurred. This represents an electronic biosensor for the detection of proteins of interest for medical applications.

Cell detachment: Post-isolation challenges

Rare cells already have become established indicators for disease diagnosis, to help track prognosis, and in developing personalized therapy. Numerous techniques have been developed to effectively and specifically detect and sort rare cells and cell isolation techniques have gained much attention among researchers in the last few decades. Recent developments in nanotechnologies and microfluidics have been used with great promise towards these goals. The research emphasis has also shifted from simple detection with microfluidic devices to comprehensive isolation, collection and subsequent analysis with integrated and automated systems. The first challenge in post-isolation analysis is cell detachment from substrates, while keeping cells viable and unperturbed. In this review, various methods used for cell detachments are discussed. For effective cell sorting, the detachment is identified as critical criteria for selecting substrates and methods.