Threshold-Responsive Colorimetric Sensing System for the Continuous Monitoring of Gases.

Colorimetric sensors are widely used because of their inherent advantages including accuracy, rapid response, ease-of-use, and low costs; however, they usually lack reusability, which precludes the continuous use of a single sensor. We have developed a threshold-responsive colorimetric system that enables repeated analyte measurements by a single colorimetric sensor. The threshold responsive algorithm automatically adjusts the sensor exposure time to the analyte and measurement frequency according to the sensor response. The system registers the colorimetric sensor signal change rate, prevents the colorimetric sensor from reaching saturation, and allows the sensor to fully regenerate before the next measurement is started. The system also addresses issues common to colorimetric sensors, including the response time and range of detection. We demonstrate the benefits and feasibility of this novel system, using colorimetric sensors for ammonia and carbon dioxide gases for continuous monitoring of up to (at least) 60 detection cycles without signs of analytical performance degradation of the sensors.

Identification of Antibody Biomarker Using High-Density Nucleic Acid Programmable Protein Array.

A novel protein microarray technology, called high-density nucleic acid programmable protein array (HD-NAPPA), enables the serological screening of thousands of proteins at one time. HD-NAPPA extends the capabilities of NAPPA, which produces protein microarrays on a conventional glass microscope slide. By comparison, HD-NAPPA displays proteins in over 10,000 nanowells etched in a silicon slide. Proteins on HD-NAPPA are expressed in the individual isolated nanowells, via in vitro transcription and translation (IVTT), without any diffusion during incubation. Here we describe the method for antibody biomarker identification using HD-NAPPA, including four main steps: (1) HD-NAPPA array protein expression, (2) primary antibodies (serum/plasma) probing, (3) secondary antibody visualization, and (4) image scanning and data processing.

A Microdroplet-Based Colorimetric Sensing Platform on a CMOS Imager Chip.

Interest in mobile chemical sensors is on the rise, but significant challenges have restricted widespread adoption into commercial devices. To be useful these sensors need to have a predictable response, easy calibration, and be integrable with existing technology, preferably fitting on a single chip. With respect to integration, the CMOS imager makes an attractive template for an optoelectronic sensing platform. Demand for smartphones with cameras has driven down the price and size of CMOS imagers over the past decade. The low cost and accessibility of these powerful tools motivated us to print chemical sensing elements directly on the surface of the photodiode array. These printed colorimetric microdroplets are composed of a nonvolatile solvent so they remain in a uniform and homogeneous solution phase, an ideal medium for chemical interactions and optical measurements. By imaging microdroplets on the CMOS imager surface we eliminated the need for lenses, dramatically scaling down the size of the sensing platform to a single chip. We believe the technique is generalizable to many colorimetric formulations, and as an example we detected gaseous ammonia with Cu(II). Limits of detection as low as 27 ppb and sensor-to-sensor variation of less than 10% across multiple printed arrays demonstrated the high sensitivity and repeatability of this approach. Sensors generated this way could share a single calibration, greatly reducing the complexity of incorporating chemical sensors into mobile devices. Additional testing showed the sensor can be reused and has good selectivity; sensitivity and dynamic range can be tuned by controlling droplet size.

Identification of Antibody Targets for Tuberculosis Serology using High-Density Nucleic Acid Programmable Protein Arrays.

Better and more diverse biomarkers for the development of simple point-of-care tests for active tuberculosis (TB), a clinically heterogeneous disease, are urgently needed. We generated a proteomic Mycobacterium tuberculosis (Mtb) High-Density Nucleic Acid Programmable Protein Array (HD-NAPPA) that used a novel multiplexed strategy for expedited high-throughput screening for antibody responses to the Mtb proteome. We screened sera from HIV uninfected and coinfected TB patients and controls (n = 120) from the US and South Africa (SA) using the multiplex HD-NAPPA for discovery, followed by deconvolution and validation through single protein HD-NAPPA with biologically independent samples (n = 124). We verified the top proteins with enzyme-linked immunosorbent assays (ELISA) using the original screening and validation samples (n = 244) and heretofore untested samples (n = 41). We identified 8 proteins with TB biomarker value; four (Rv0054, Rv0831c, Rv2031c and Rv0222) of these were previously identified in serology studies, and four (Rv0948c, Rv2853, Rv3405c, Rv3544c) were not known to elicit antibody responses. Using ELISA data, we created classifiers that could discriminate patients' TB status according to geography (US or SA) and HIV (HIV- or HIV+) status. With ROC curve analysis under cross validation, the classifiers performed with an AUC for US/HIV- at 0.807; US/HIV+ at 0.782; SA/HIV- at 0.868; and SA/HIV+ at 0.723. With this study we demonstrate a new platform for biomarker/antibody screening and delineate its utility to identify previously unknown immunoreactive proteins.

A Contra Capture Protein Array Platform for Studying Post-translationally Modified (PTM) Auto-antigenomes.

Aberrant modifications of proteins occur during disease development and elicit disease-specific antibody responses. We have developed a protein array platform that enables the modification of many proteins in parallel and assesses their immunogenicity without the need to express, purify, and modify proteins individually. We used anticitrullinated protein antibodies (ACPAs) in rheumatoid arthritis (RA) as a model modification and profiled antibody responses to ∼190 citrullinated proteins in 20 RA patients. We observed unique antibody reactivity patterns in both clinical anticyclic citrullinated peptide assay positive (CCP+) and CCP- RA patients. At individual antigen levels, we detected antibodies against known citrullinated autoantigens and discovered and validated five novel antibodies against specific citrullinated antigens (osteopontin (SPP1), flap endonuclease (FEN1), insulin like growth factor binding protein 6 (IGFBP6), insulin like growth factor I (IGF1) and stanniocalcin-2 (STC2)) in RA patients. We also demonstrated the utility of our innovative array platform in the identification of immune-dominant epitope(s) for citrullinated antigens. We believe our platform will promote the study of post-translationally modified antigens at a breadth that has not been achieved before, by both identifying novel autoantigens and investigating their roles in disease development. The developed platforms can potentially be used to study many autoimmune disease-relevant modifications and their immunogenicity.

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.

Antiviral antibody profiling by high-density protein arrays.

Viral infections elicit antiviral antibodies and have been associated with various chronic diseases. Detection of these antibodies can facilitate diagnosis, treatment of infection, and understanding of the mechanisms of virus-associated diseases. In this work, we assayed antiviral antibodies using a novel high-density nucleic acid programmable protein array (HD-NAPPA) platform. Individual viral proteins were expressed in situ directly from plasmids encoding proteins in an array of microscopic reaction chambers. Quality of protein display and serum response was assured by comparing intra- and inter-array correlation within or between printing batches with average correlation coefficients of 0.91 and 0.96, respectively. HD-NAPPA showed higher signal-to-background ratio compared with standard NAPPA on planar glass slides and ELISA. Antibody responses to 761 antigens from 25 different viruses were profiled among patients with juvenile idiopathic arthritis and type 1 diabetes. Common and unique antibody reactivity patterns were detected between patients and healthy controls. We believe HD-viral-NAPPA will enable the study of host-pathogen interactions at unprecedented dimensions and elucidate the role of pathogen infections in disease development.

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.

Charge-based detection of small molecules by plasmonic-based electrochemical impedance microscopy.

Charge-based detection of small molecules is demonstrated by plasmonic-based electrochemical impedance microscopy (P-EIM). The dependence of surface plasmon resonance (SPR) on surface charge density is used to detect small molecules (60-120 Da) printed on a dextran-modified sensor surface. Local variations in charge density on an electrode surface are manifest in an optical SPR signal. The SPR response to an applied ac potential measures the sensor surface impedance which is a function of the surface charge density. This optical signal is comprised of a dc and an ac component, and is measured with high spatial resolution. The dc element of the SPR signal represents conventional SPR imaging information. The amplitude and phase of local surface impedance is provided by the ac component. The phase signal of the small molecules is a function of their charge status, which is manipulated by the pH of a solution. Small molecules with positive, neutral, and negative charge are detected by P-EIM. This technique is used to detect and distinguish small molecules based on their charge status, thereby circumventing the mass limitation (~100 Da) of conventional SPR measurement.

Imaging the electrocatalytic activity of single nanoparticles.

The electrocatalytic properties of nanoparticles depend on their size, shape and composition. These properties are typically probed by measuring the total electrocatalytic reaction current of a large number of nanoparticles, but this approach is time-consuming and can only measure the average catalytic activity of the nanoparticles under study. However, the identification of new catalysts requires the ability to rapidly measure the properties of nanoparticles synthesized under various conditions and, ideally, to measure the electrocatalytic activity of individual nanoparticles. Here, we show that a plasmonic-based electrochemical current-imaging technique can simultaneously image and quantify the electrocatalytic reactions of an array of 1.6 × 10(5) platinum nanoparticles printed on an electrode surface, which could facilitate high-throughput screening of the catalytic activities of nanoparticles. We also show that the approach can be used to image the electrocatalytic reaction current and measure the cyclic voltammograms of single nanoparticles.

High density diffusion-free nanowell arrays.

Proteomics aspires to elucidate the functions of all proteins. Protein microarrays provide an important step by enabling high-throughput studies of displayed proteins. However, many functional assays of proteins include untethered intermediates or products, which could frustrate the use of planar arrays at very high densities because of diffusion to neighboring features. The nucleic acid programmable protein array (NAPPA) is a robust in situ synthesis method for producing functional proteins just-in-time, which includes steps with diffusible intermediates. We determined that diffusion of expressed proteins led to cross-binding at neighboring spots at very high densities with reduced interspot spacing. To address this limitation, we have developed an innovative platform using photolithographically etched discrete silicon nanowells and used NAPPA as a test case. This arrested protein diffusion and cross-binding. We present confined high density protein expression and display, as well as functional protein-protein interactions, in 8000 nanowell arrays. This is the highest density of individual proteins in nanovessels demonstrated on a single slide. We further present proof of principle results on ultrahigh density protein arrays capable of up to 24000 nanowells on a single slide.

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.

Piezoelectric printing and probing of Lectin NanoProbeArrays for glycosylation analysis.

Glycans have great potential as disease biomarkers and therapeutic targets. However, the major challenge for glycan biomarker identification from clinical samples is the low abundance of key glycosylated proteins. To demonstrate the potential for glycan analysis with nanoliter amounts of glycoprotein, we have developed a new technology (Lectin NanoProbeArray) based on piezoelectric liquid dispensing for non-contact printing and probing of a lectin array. Instead of flooding the glycoprotein probe on the lectin array surface, as in conventional microarray screening, a piezoelectric printer is used to dispense nanoliters of fluorescently labeled glycoprotein probe over the lectin spots on the array. As a proof-of-concept, the ability of Lectin NanoProbeArrays to precisely identify and reliably distinguish between the closely related glycoforms of fetuin is illustrated here. Sensitivity levels comparable to lectin arrays that use evanescent-field scanners was achieved along with several orders of magnitude reduction in the amount of probe required for glycosylation analysis.