Azhar Ilyas

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.

Synthesis of nano-textured biocompatible scaffolds from chicken eggshells

Cell adhesion, morphology and growth are influenced by surface topography at nano and micrometer scales. Nano-textured surfaces are prepared using photolithography, plasma etching and long polymer chemical etching which are cost prohibitive and require specialized equipment. This article demonstrates a simple approach to synthesize nano-textured scaffolds from chicken eggshells. Varieties of pattern are made on the eggshells like micro-needle forests and nanopores, giving very uniform nano-textures to the surfaces. The surfaces are characterized for chemical composition and crystal phase. The novel patterns are transferred to PDMS surfaces and the nano-textured PDMS surfaces are used to study the effect of texturing on human fibroblast cell growth and attachment. The effects of surface topographies, along with laminin coating on cell cultures, are also studied. We find an exciting phenomenon that the initial seeding density of the fibroblast cells affects the influence of the nano-texturing on cell growth. These nano-textured surfaces give 16 times more fibroblast growth when compared to flat PDMS surfaces. The novel nano-textured patterns also double the laminin adsorption on PDMS.

Pulsed plasma polymerization for controlling shrinkage and surface composition of nanopores

Solid-state nanopores have emerged as sensors for single molecules and these have been employed to examine the biophysical properties of an increasingly large variety of biomolecules. Herein we describe a novel and facile approach to precisely adjust the pore size, while simultaneously controlling the surface chemical composition of the solid-state nanopores. Specifically, nanopores fabricated using standard ion beam technology are shrunk to the requisite molecular dimensions via the deposition of highly conformal pulsed plasma generated thin polymeric films. The plasma treatment process provides accurate control of the pore size as the conformal film deposition depends linearly on the deposition time. Simultaneously, the pore and channel chemical compositions are controlled by appropriate selection of the gaseous monomer and plasma conditions employed in the deposition of the polymer films. The controlled pore shrinkage is characterized with high resolution AFM, and the film chemistry of the plasma generated polymers is analyzed with FTIR and XPS. The stability and practical utility of this new approach is demonstrated by successful single molecule sensing of double-stranded DNA. The process offers a viable new advance in the fabrication of tailored nanopores, in terms of both the pore size and surface composition, for usage in a wide range of emerging applications.

Parallel recognition of cancer cells using an addressable array of solid-state micropores

Early stage detection and precise quantification of circulating tumor cells (CTCs) in the peripheral blood of cancer patients are important for early diagnosis. Early diagnosis improves the effectiveness of the therapy and results in better prognosis. Several techniques have been used for CTC detection but are limited by their need for dye tagging, low throughput and lack of statistical reliability at single cell level. Solid-state micropores can characterize each cell in a sample providing interesting information about cellular populations. We report a multi-channel device which utilized solid-state micropores array assembly for simultaneous measurement of cell translocation. This increased the throughput of measurement and as the cells passed the micropores, tumor cells showed distinctive current blockade pulses, when compared to leukocytes. The ionic current across each micropore channel was continuously monitored and recorded. The measurement system not only increased throughput but also provided on-chip cross-relation. The whole blood was lysed to get rid of red blood cells, so the blood dilution was not needed. The approach facilitated faster processing of blood samples with tumor cell detection efficiency of about 70%. The design provided a simple and inexpensive method for rapid and reliable detection of tumor cells without any cell staining or surface functionalization. The device can also be used for high throughput electrophysiological analysis of other cell types.

Electrophysiological analysis of biopsy samples using elasticity as an inherent cell marker for cancer detection

Biopsy samples from patients are valuable for diagnosis. Herein, a label-free approach for the early diagnosis of cancer from biopsied samples is reported. The scheme relies on single solid-state micropores as the transducer component and cell elasticity as the indicator of cell health. The biomechanical discrimination of cancerous cells depends on purely intrinsic properties and is not limited by the availability of biomarkers. As a model, bladder cancer cells, which are very different from healthy cells, showed specific electrical signatures. The correlated detection of tumor cells was performed with an efficiency of 75%. The difference in cellular elasticity afforded one order of magnitude lower translocation time for tumor cells. The capability to recognize very few tumor cells solves the prime challenge in biopsy exams. A comparison of the motility and stiffness of cancer and normal urothelial cells showed distinctive quantifiable viscoelastic behavior.

PLGA Micro- and Nanoparticles Loaded Into Gelatin Scaffold for Controlled Drug Release

Curcumin and bovine serum albumin (BSA) were used as model drugs and loaded into microand nanoparticles of biodegradable poly(lactic-co-glycolic acid) (PLGA). The PLGA was incorporated into hydrophilic and biocompatible gelatin scaffolds to design a controlled drug release system. The gelatin scaffolds were cross-linked using glutaraldehyde. The controlled delivery of drugs from biologically active PLGA microand nanoparticles was measured and these showed consistent release for 30 days. Curcuminand BSA-loaded PLGA micro/nanoparticles-based gelatin scaffolds define a novel approach to embed multiple drug molecules to overcome multidrug resistance as well as depict a new type of biocompatible and biodegradable implant. Such scaffold constructs can be used for breast implants after lumpectomy to not only overcome cosmetic issues, but also to provide sustained drug release during healing process. In one type of construct, only BSA-loaded microparticles were mixed with gelatin, while in the other type of construct, both BSAand curcumin-loaded PLGA microparticles were embedded. BSAand curcumin-loaded nanoparticles were also embedded into gelatin constructs to see the effects of particle size on drug release. After 30 days, cumulative BSA release from PLGA microand nanoparticles embedded in gelatin scaffold were measured to be 69.87% and 86.11%, respectively. The cumulative release of curcumin was measured to be 53.11% and 60.42% from curcumin-loaded PLGA microand nanoparticles, respectively. A statistically significant difference was seen in cumulative drug release from these scaffolds (p value <; 0.05).

Differentiating Metastatic and Non-metastatic Tumor Cells from Their Translocation Profile through Solid-State Micropores

Cancer treatment, care, and outcomes are much more effective if started at early stages of the disease. The presence of malignant cancer cells in human samples such as blood or biopsied tissue can be used to reduce overtreatment and underdiagnosis as well as for prognosis monitoring. Reliable quantification of metastatic tumor cells (MTCs) and non-metastatic tumor cells (NMTCs) from human samples can help in cancer staging as well. We report a simple, fast, and reliable approach to identify and quantify metastatic and non-metastatic cancer cells from whole biological samples in a point-of-care manner. The metastatic (MDA MB-231) and non-metastatic (MCF7) breast cancer cells were pushed through a solid-state micropore made in a 200 nm thin SiO2 membrane while measuring current across the micropore. The cells generated very distinctive translocation profiles. The translocation differences stemmed from their peculiar mechanophysical properties. The detection efficiency of the device for each type of tumor cells was ∼75%. MTCs showed faster translocation (36%) and 34% less pore blockage than NMTCs. The micropore approach is simple, exact, and quantitative for metastatic cell detection in a lab-on-a chip setting, without the need for any preprocessing of the sample.

Salt-Leaching Synthesis of Porous PLGA Nanoparticles

Poly(lactic-co-glycolic acid) (PLGA) nanoparticles are widely used for controlled delivery of bioactive agents in therapeutic applications. These nanoparticles show bioavailability, better encapsulation, controlled release, biocompatibility, and in vivo biodegradability. This paper reports a novel approach to synthesize porous PLGA nanoparticles and their use as controlled release vehicles. Bovine serum albumin (BSA) loaded PLGA nanoparticles (porous and nonporous) were synthesized using water-in-oil-in-water double emulsion method. Specifically, PLGA nanoparticles were prepared using chloroform and polyvinyl alcohol, and freeze drying was employed for the phase separation to obtain the nanoparticles. The porous nanoparticles were prepared through the salt-leaching process where sodium bicarbonate was used as an extractable porogen. In vitro drug release behavior of porous and nonporous nanoparticles was monitored over a period of 30 days. A much more enhanced BSA release was observed in case of porous polymeric nanoparticles when compared to nonporous nanoparticles. The characterization was done using laser scattering, zeta potential analysis, and scanning electron microscopy. The drug loading efficiencies for BSA in porous and nonporous PLGA nanoparticles were 65.50% and 77.59%, respectively. Over a period of 30 days, the cumulative BSA released from PLGA porous and nonporous nanoparticles were measured to be 87.41% and 59.91%, respectively. The synthesis of porous nanoparticles with this novel, rapid, and inexpensive method opens a new horizon of using a wide range of cheap and easily-accessible water-soluble salts that can be extracted through leaching process to introduce porous morphology on the nanoparticle surfaces. The porous nanoparticles can have useful applications in controlled drug delivery systems.