Microreactor array device.

We report a device to fill an array of small chemical reaction chambers (microreactors) with reagent and then seal them using pressurized viscous liquid acting through a flexible membrane. The device enables multiple, independent chemical reactions involving free floating intermediate molecules without interference from neighboring reactions or external environments. The device is validated by protein expressed in situ directly from DNA in a microarray of ~10,000 spots with no diffusion during three hours incubation. Using the device to probe for an autoantibody cancer biomarker in blood serum sample gave five times higher signal to background ratio compared to standard protein microarray expressed on a flat microscope slide. Physical design principles to effectively fill the array of microreactors with reagent and experimental results of alternate methods for sealing the microreactors are presented.

Measurement of small molecule binding kinetics on a protein microarray by plasmonic-based electrochemical impedance imaging.

We report on a quantitative study of small molecule binding kinetics on protein microarrays with plasmonic-based electrochemical impedance microscopy (P-EIM). P-EIM measures electrical impedance optically with high spatial resolution by converting a surface charge change to a surface plasmon resonance (SPR) image intensity change, and the signal is not scaled to the mass of the analyte. Using P-EIM, we measured binding kinetics and affinity between small molecule drugs (imatinib and SB202190) and their target proteins (kinases Abl1 and p38-α). The measured affinity values are consistent with reported values measured by an indirect competitive binding assay. We also found that SB202190 has weak bindings to ABL1 with KD > 10 µM, which is not reported in the literature. Furthermore, we found that P-EIM is less prone to nonspecific binding, a long-standing issue in SPR. Our results show that P-EIM is a novel method for high-throughput measurement of small molecule binding kinetics and affinity, which is critical to the understanding of small molecules in biological systems and discovery of small molecule drugs.

NanoProbeArrays for the analysis of ultra-low-volume protein samples using piezoelectric liquid dispensing technology.

Antibody microarrays are gaining popularity as a high-throughput technology to investigate the proteome. However, protein extracts from most body fluid or biopsy samples are available in very small volumes and are often unsuitable for large-scale antibody microarray studies. To demonstrate the potential for protein analysis with as little as a few nanoliters of sample, we have developed a new technology called NanoProbeArrays based on piezoelectric liquid dispensing for non-contact printing and probing of antibody arrays. Instead of flooding the protein sample on the antibody microarray surface, as in conventional microarray screening, a piezoelectric inkjet printer is used to dispense nanoliters of fluorescently labeled proteins over the antibody spots on the array. The ability of NanoProbeArrays to precisely identify and reliably distinguish between test proteins from different sources, without any loss of sensitivity and specificity as compared with conventional antibody microarrays, is illustrated here. The utility of NanoProbeArrays for biomarker identification in a complex biological sample was tested by detecting the cytokine interleukin-4 in serum. The significant reduction in volume of sample during NanoProbeArray analysis, as compared with conventional antibody microarrays, offers new opportunities for basic and applied proteomic research.

Single cells and intracellular processes studied by a plasmonic-based electrochemical impedance microscopy.

Electrochemical impedance spectroscopy is a crucial tool for the detection and study of various biological substances, from DNA and proteins to viruses and bacteria. It does not require any labelling species, and methods based on it have been developed to study cellular processes (such as cell spreading, adhesion, invasion, toxicology and mobility). However, data have so far lacked spatial information, which is essential for investigating heterogeneous processes and imaging high-throughput microarrays. Here, we report an electrochemical impedance microscope based on surface plasmon resonance that resolves local impedance with submicrometre spatial resolution. We have used an electrochemical impedance microscope to monitor the dynamics of cellular processes (apoptosis and electroporation of individual cells) with millisecond time resolution. The high spatial and temporal resolution makes it possible to study individual cells, but also resolve subcellular structures and processes without labels, and with excellent detection sensitivity (~2 pS). We also describe a model that simulates cellular and electrochemical impedance microscope images based on local dielectric constant and conductivity.

Efficacy of TRAIL treatment against HPV16 infected cervical cancer cells undergoing senescence following siRNA knockdown of E6/E7 genes.

In this study we investigated E6 and E7 oncogenes from the Human Papilloma Virus as targets for siRNA knockdown in order to boost the efficacy of the anti-cancer drug 'tumor necrosis factor-related apoptosis inducing ligand' (TRAIL). SiHa cells were treated with TRAIL following transfection with E6/E7 siRNA and the expression of death receptors DR4 and DR5, cell viability, apoptosis, senescence and cell cycle analysis were undertaken using flow cytometry, MTT viability assay and cellular ß-galactosidase activity assays. E6/E7 siRNA resulted in significant upregulation of death receptors DR4 and DR5 but did not result in an enhanced sensitivity to TRAIL. Our results indicate that E6/E7-siRNA induces senescence rather than apoptosis in SiHa cells. The occurrence of senescence in drug resistant cervical cancer cells such as the SiHa cell line by E6/E7 siRNA, among other factors, may prevent TRAIL induced activation of extrinsic and intrinsic pathways that lead to apoptotic cell death. Our findings are significant for combinatorial strategies for cancer therapy since the induction of senescence can preclude apoptosis rendering cells to be recalcitrant to TRAIL treatment.

NanoMonitor: a miniature electronic biosensor for glycan biomarker detection.

AIM: The goal of our research is to develop an ultrasensitive diagnostic platform called 'NanoMonitor' to enable rapid label-free analysis of a highly promising class of biomarkers called glycans (oligosaccharide chains attached to proteins) with high sensitivity and selectivity. The glycosylation of fetuin - a serum protein - and extracts from a human pancreatic cancer line was analyzed to demonstrate the capabilities of the NanoMonitor. MATERIAL & METHODS: The NanoMonitor device consists of a silicon chip with an array of gold electrodes forming multiple sensor sites and works on the principle of electrochemical impedance spectroscopy. Each sensor site is overlaid with a nanoporous alumina membrane that forms a high density of nanowells on top of each electrode. Lectins (proteins that bind to and recognize specific glycan structures) are conjugated to the surface of the electrode. When specific glycans from a test sample bind to lectins at the base of each nanowell, a perturbation of electrical double-layer occurs, which results in a change in the impedance. Using the lectins Sambucs nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA), subtle variations to the glycan chains of fetuin were investigated. Protein extracts from BXPC-3, a cultured human pancreatic cancer cell line were also analyzed for binding to SNA and MAA lectins. The performance of the NanoMonitor was compared to a conventional laboratory technique: lectin-based enzyme linked immunosorbent assay (ELISA). RESULTS & DISCUSSION: The NanoMonitor was used to identify glycoform variants of fetuin and global differences in glycosylation of protein extracts from cultured human pancreatic cancerous versus normal cells. While results from NanoMonitor correlate very well with results from lectin-based ELISA, the NanoMonitor is rapid, completely label free, requires just 10 microl of sample, is approximately five orders of magnitude more sensitive and highly selective over a broad dynamic range of glycoprotein concentrations. CONCLUSION: Based on its performance metrics, the NanoMonitor has excellent potential for development as a point-of-care handheld electronic biosensor device for routine detection of glycan biomarkers from clinical samples.