A curcumin direct protein biosensor for cell-free prototyping.

In synthetic biology, biosensors are routinely coupled with a gene expression system for detecting small molecules and physical signals. We reveal a fluorescent complex, based on the interaction of an Escherichia coli double bond reductase (EcCurA), as a detection unit with its substrate curcumin-we call this a direct protein (DiPro) biosensor. Using a cell-free synthetic biology approach, we use the EcCurA DiPro biosensor to fine tune 10 reaction parameters (cofactor, substrate, and enzyme levels) for cell-free curcumin biosynthesis, assisted through acoustic liquid handling robotics. Overall, we increase EcCurA-curcumin DiPro fluorescence within cell-free reactions by 78-fold. This finding adds to the growing family of protein-ligand complexes that are naturally fluorescent and potentially exploitable for a range of applications, including medical imaging to engineering high-value chemicals.

Intensified biochip system using chemiluminescence for the detection of Bacillus globigii spores.

This paper reports the first intensified biochip system for chemiluminescence detection and the feasibility of using this system for the analysis of biological warfare agents is demonstrated. An enzyme-linked immunosorbent assay targeting Bacillus globigii spores, a surrogate species for Bacillus anthracis, using a chemiluminescent alkaline phosphatase substrate is combined with a compact intensified biochip detection system. The enzymatic amplification was found to be an attractive method for detection of low spore concentrations when combined with the intensified biochip device. This system was capable of detecting approximately 1 x 10(5) Bacillus globigii spores. Moreover, the chemiluminescence method, combined with the self-contained biochip design, allows for a simple, compact system that does not require laser excitation and is readily adaptable to field use.

Soft, stretchable, epidermal sensor with integrated electronics and photochemistry for measuring personal UV exposures.

Excessive ultraviolet (UV) radiation induces acute and chronic effects on the skin, eye and immune system. Personalized monitoring of UV radiation is thus paramount to measure the extent of personal sun exposure, which could vary with environment, lifestyle, and sunscreen use. Here, we demonstrate an ultralow modulus, stretchable, skin-mounted UV patch that measures personal UV doses. The patch contains functional layers of ultrathin stretchable electronics and a photosensitive patterned dye that reacts to UV radiation. Color changes in the photosensitive dyes correspond to UV radiation intensity and are analyzed with a smartphone camera. A software application has feature recognition, lighting condition correction, and quantification algorithms that detect and quantify changes in color. These color changes are then correlated with corresponding shifts in UV dose, and compared to existing UV dose risk levels. The soft mechanics of the UV patch allow for multi-day wear in the presence of sunscreen and water. Two evaluation studies serve to demonstrate the utility of the UV patch during daily activities with and without sunscreen application.

Resident neuroelectrochemical interfacing using carbon nanofiber arrays.

Carbon nanofiber electrode architectures are used to provide for long-term, neuroelectroanalytical measurements of the dynamic processes of intercellular communication between excitable cells. Individually addressed, vertically aligned carbon nanofibers are incorporated into multielement electrode arrays upon which excitable cell matrixes of both neuronal-like derived cell lines (rat pheochromocytoma, PC-12) and primary cells (dissociated cells from embryonic rat hippocampus) are cultured over extended periods (days to weeks). Electrode arrays are characterized with respect to their response to easily oxidized neurotransmitters, including dopamine, norepinephrine, and 5-hydroxytyramide. Electroanalysis at discrete electrodes following long-term cell culture demonstrates that this platform remains responsive for the detection of easily oxidized species generated by the cultured cells. Preliminary data also suggests that quantal release of easily oxidized transmitters can be observed at nanofiber electrodes following direct culture and differentiation on the arrays for periods of at least 16 days.

A compact CMOS biochip immunosensor towards the detection of a single bacteria.

Recent use of biological warfare (BW) agents has led to a growing interest in the rapid and sensitive detection of pathogens. Therefore, the development of field-usable detection devices for sensitive and selective detection of BW agents is an important issue. In this work, we report a portable biochip system based on complementary metal oxide semiconductor (CMOS) technology that has great potential as a device for single-bacteria detection. The possibility of single-bacteria detection is reported using an immunoassay coupled to laser-induced fluorescence (LIF) detection. Bacillus globigii spores, which are a surrogate species for B. anthracis spores, were used as the test sample. Enzymatic amplification following immunocomplex formation allowed remarkably sensitive detection of B. globigii spores, and could preclude a complicated optical and instrumental system usually required for high-sensitive detection. Atomic force microscopy (AFM) was employed to investigate whether B. globigii spores detected in the portable biochip system exist in single-cell or multicellular form. It was found that B. globigii spores mostly exist in multicellular form with a small minority of single-cell form. The results showed that the portable biochip system has great potential as a device for single-particle or possibly even single-organism detection.

Near-field scanning optical microscopy for bioanalysis at nanometer resolution.

The nondestructive imaging of biomolecules in nanometer domains in their original location and position as adsorbed or deposited on a surface is of garners considerable experimental interest. Near-field scanning optical microscopy (NSOM) is an emerging technique with its astonishing resolving power of <100-nm domains, and nondestructive nature compared with other scanning probe microscopic techniques is an emerging technique to achieve this goal. At the single-molecule level of resolution, it is possible to use the NSOM as a critical tool for visualization of proteins on surfaces to obtain more fundamental information about their orientation and locality without disturbing their original orientation and position, and level of interaction with the surface. Several areas of science and medicine can benefit from this type of study especially for biomedical and biochip applications. To illustrate possible applications, imaging of green fluorescent proteins and biomolecules associated with multidrug resistance proteins in tumor cells will be demonstrated using NSOM.

SERS nanosensors and nanoreporters: golden opportunities in biomedical applications.

This article provides an overview of recent developments and applications of surface-enhanced Raman scattering (SERS) nanosensors and nanoreporters in our laboratory for use in biochemical monitoring, medical diagnostics, and therapy. The design and fabrication of different types of plasmonics-active nanostructures are discussed. The SERS nanosensors can be used in various applications including pH sensing, protein detection, and gene diagnostics. For DNA detection the 'Molecular Sentinel' nanoprobe can be used as a homogenous bioassay in solution or on a chip platform. Gold nanostars provide an excellent multi-modality theranostic platform, combining Raman and SERS with two-photon luminescence (TPL) imaging as well as photodynamic therapy (PDT), and photothermal therapy (PTT). Plasmonics-enhanced and optically modulated delivery of nanostars into brain tumor in live animals was demonstrated; photothermal treatment of tumor vasculature may induce inflammasome activation, thus increasing the permeability of the blood brain-tumor barrier. The imaging method using TPL of gold nanostars provides an unprecedented spatial selectivity for enhanced targeted nanostar delivery to cortical tumor tissue. A quintuple-modality nanoreporter based on gold nanostars for SERS, TPL, magnetic resonance imaging (MRI), computed tomography (CT), and PTT has recently been developed. The possibility of combining spectral selectivity and high sensitivity of the SERS process with the inherent molecular specificity of bioreceptor-based nanoprobes provides a unique multiplex and selective diagnostic modality. Several examples of optical detection using SERS in combination with other detection and treatment modalities are discussed to illustrate the usefulness and potential of SERS nanosensors and nanoreporters for medical applications.

Application of a miniature biochip using the molecular beacon probe in breast cancer gene BRCA1 detection.

We report for the first time the application of a biochip using the molecular beacon (MB) detection scheme. The usability of this biochip novel detection system for the analysis of the breast cancer gene BRCA1 is demonstrated using molecular beacon probes. The MB is designed for the BRCA1 gene and a miniature biochip system is used for detection. The performance of the biochip-MB detection system is evaluated. The optimum conditions for the MB system for highest fluorescence detection sensitivity are investigated for the detection system. The detection of BRCA1 gene is successfully demonstrated in solution and the limit of detection (LOD) is estimated as 70 nM.

Screening for the breast cancer gene (BRCA1) using a biochip system and molecular beacon probes immobilized on solid surfaces.

We describe the use of a biochip based on complementary metal oxide semiconductor (CMOS) technology for detection of specific genetic sequences using molecular beacons (MB) immobilized on solid surfaces as probes. The applicability of this miniature detection system for screening for the BRCA1 gene is evaluated using MB probes, designed especially for the BRCA1 gene. MB probes are immobilized on a zeta-probe membrane by biotin-streptavidin immobilization. Two immobilization strategies are investigated to obtain optimal assay sensitivity. The MB is immobilized by manual spotting on zeta-probe membrane surfaces with the use of a custom-made stamping system. The detection of the BRCA1 gene using an MB probe is successfully demonstrated and expands the use of the CMOS biochip for medical applications.

Plasmonic nanoprobes: from chemical sensing to medical diagnostics and therapy.

This article provides an overview of the development and applications of plasmonics-active nanoprobes in our laboratory for chemical sensing, medical diagnostics and therapy. Molecular Sentinel nanoprobes provide a unique tool for DNA/RNA biomarker detection both in a homogeneous solution or on a chip platform for medical diagnostics. The possibility of combining spectral selectivity and high sensitivity of the surface-enhanced Raman scattering (SERS) process with the inherent molecular specificity of nanoprobes provides an important multiplex diagnostic modality. Gold nanostars can provide an excellent multi-modality platform, combining two-photon luminescence with photothermal therapy as well as Raman imaging with photodynamic therapy. Several examples of optical detection using SERS and photonics-based treatments are presented to illustrate the usefulness and potential of the plasmonic nanoprobes for theranostics, which seamlessly combines diagnostics and therapy.

Near-real-time determination of hydrogen peroxide generated from cigarette smoke.

The ability to monitor hydrogen peroxide (H2O2) in aqueous smoke extracts will advance our understanding of the relationship between cigarette smoke-induced oxidative stress, inflammation, and disease and help elucidate the pathways by which the various smoke constituents exert their pathogenic effects. We have demonstrated, for the first time, the measurement of H2O2 production from cigarette smoke without prior separation of the sample. Cigarettes were tested on a commercial smoking machine, such that the whole smoke or gas vapor phase was bubbled through phosphate buffered saline solution at pH 7.4. Aliquots of these solutions were analyzed using an Amplex Red/horseradish peroxidase fluorimetric assay that required only a 2 minute incubation time, facilitating the rapid, facile collection of data. Catalase was used to demonstrate the selectivity and specificity of the assay for H2O2 in the complex smoke matrix. We measured approximately 7-8 microM H2O2 from two reference cigarettes (i.e., 1R4F and 2R4F). We also observed 9x more H2O2 from whole smoke bubbled samples compared to the gas vapor phase, indicating that the major constituent(s) responsible for H2O2 formation reside in the particulate phase of cigarette smoke. Aqueous solutions of hydroquinone and catechol, both of which are particulate phase constituents of cigarette smoke, generated no H2O2 even though they are free radical precursors involved in the production of reactive oxygen species in the smoke matrix.

Detection of cytochrome C in a single cell using an optical nanobiosensor.

In this work, the intracellular measurement of cytochrome c using an optical nanobiosensor is demonstrated. The nanobiosensor is a unique fiberoptics-based tool which allows the minimally invasive analysis of intracellular components. Cytochrome c is a very important protein to the process which produces cellular energy. In addition, cytochrome c is well-known as the protein involved in apoptosis, or programmed cell death. delta-Aminolevulinic acid (5-ALA) was used to induce apoptosis in MCF-7 human breast carcinoma cells. 5-ALA, a photodynamic therapy (PDT) drug in cells was activated by a HeNe laser beam. After the PDT photoactivation, the release of cytochrome c from the mitochondria to the cytoplasm in a MCF-7 cell was monitored by the optical nanobiosensor inserted inside the single cell and followed by an enzyme-linked immunosorbent assay (ELISA) outside the cell. The combination of the nanobiosensor with the ELISA immunoassay improved the detection sensitivity of the nanobiosensor due to enzymatic amplification. Our results lead to the investigation of an apoptotic pathway at the single cell level.

A miniature biochip system for detection of aerosolized Bacillus globigii spores.

The feasibility of using a novel detection scheme for the analysis of biological warfare agents is demonstrated using Bacillus globigii spores, a surrogate species for Bacillus anthracis. In this paper, a sensitive and selective enzyme-linked immunosorbent assay using a novel fluorogenic alkaline phosphatase substrate (dimethylacridinone phosphate) is combined with a compact biochip detection system, which includes a miniature diode laser for excitation. Detection of aerosolized spores was achieved by coupling the miniature system to a portable bioaerosol sampler, and the performance of the antibody-based recognition and enzyme amplification method was evaluated. The bioassay performance was found to be compatible with the air sampling device, and the enzymatic amplification was found to be an attractive amplification method for detection of low spore concentrations. The combined portable bioaerosol sampler and miniature biochip system detected 100 B. globigii spores, corresponding to 17 aerosolized spores/L of air. Moreover, the incorporation of the miniature diode laser with the self-contained biochip design allows for a compact system that is readily adaptable to field use. In addition, these studies have included investigations into the tradeoff between assay time and sensitivity.