In our group we are interested in using recombinant DNA technology to develop new bioanalytical techniques. A few examples of research projectsthat are currently active within our group are outlined below.

Chemiluminescence assays are ideal for the detection of low levels of biomolecules. In contrast to other sensitive techniques like fluorescence, chemiluminescence and bioluminescence are unaffected by the background fluorescence signal of biological samples. However, the presently used chemiluminescent labels are not ideally suited for use at physiological pH. The use of bioluminescent photoproteins, such as aequorin, as labels can circumvent this limitation of conventional chemiluminescence assays. Aequorin is a calcium-activated photoprotein found in Aequorea victoria (jellyfish), and it can be used as a biological Ca²⁺ indicator. In our laboratory we use aequorin as a bioluminescent label in the developmentof novel competitive binding assays for biomolecules by taking advantage of the selective interaction between biological binders (antibodies, bindingproteins, receptors, etc.) and ligands. Aequorin-ligand conjugates are prepared by genetic engineering of aequorin. Recently, we have demonstrated that aequorin can be detected at attomole levels. Moreover, we were able to detect 4 attomol of biotin (vitamin H) by using aequorin as a label in a homogeneous competitive binding assay. It is anticipated that extremely sensitive assays for other biologically important molecules (such as neuropeptides, hormones, drugs, etc.) will result from these investigations.

We are also interested in employing aequorin to develop assays for biologically important molecules in single cells. It is well established that understanding the effect of chemical and biochemical compounds on different cells is important in a variety of fields (pharmacology, drug design, toxicology, etc.) In addition, the discovery and study of inherent differences in the composition of morphologically identical cells could help elucidate the correlation between a certain cell behavior observed in diseases and the presence of certain biomolecular species within a cellular subclass. Because the composition of cells varies within a given population, conventional biochemical analyses that measure the average of thousands, if not millions, of cells cannot provide this information. Therefore, there is a need for bioanalytical procedures capable of monitoring the biochemical composition of single cells. The levels of most biomolecules in a given single cell are in the femtomole to attomole range. Consequently, the challenge now lies in determining ultra-low levels of important biomolecules in the small volume of a single cell.

Another area of research in our group focuses in the development of fiber optic sensors for the determination of anions and heavy metal pollutants. Along those lines, bacterial-based fiber optic biosensors are developed that can be used in direct and in in-situ environmental monitoring. The sensors incorporate genetically designed bacterial systems that are engineered to bioluminesce only when a targeted heavy metal pollutant is present. These systems are based on the induction of luciferase-based luminescence. Finally, fiber optic biosensors for anions are being designed by using periplasmic binding proteins as the sensing element. It is well established that periplasmic binding proteins undergo a conformational change upon binding, closing around their corresponding ligand anion. This happens by bending around a hinge between the two globular domains of the protein by a mechanism that resembles that of the venus fly-trap. We take advantage of the conformational change to develop fiber optic sensors for anions of interest.

• S. K. Deo, K. Gregory, V. Schauer-Vukasinovic, G. Schrift, L. G. Bachas, and S. Daunert, "Calmodulin-Mediated Reversible Immobilization of Enzymes", Colloid. Surf., B: Biointerfaces, 58, 20-7, 2007.
• X. Qu. S. K. Deo, E. Dikici, C. M. Ensor, M. D. Poon and S. Daunert, "Bioluminescence Immunoassays for Angiotensin II Using Aequorin as a Label", Anal. Biochem. 371, 154-161, 2007.
• J. D. Ehrick, S. M. Stokes, S. Bachas-Daunert, E. A. Moschou, S. K. Deo, L. G. Bachas, and S. Daunert, "Chemically Tunable Lensing of Stimuli-Responsive Hydrogel Microdomes", Adv. Mat. 19, 4024-4027, 2007.
• K. Turner, S. Xu, P. Pasini, S.K. Deo, and S. Daunert, "Hydroxylated Polychlorinated Biphenyls Detection Based on a Genetically Engineered Bioluminescent Whole-Cell Sensing System", Anal. Chem. 79, 5740-5745, 2007.
• L. A Rowe, S. K. Deo, J. Shofner, M. Mirasoli, and S. Daunert, "Aequorin-Based Homogeneous Cortisol Immunoassay for Analysis of Saliva Samples", Bioconjugate Chem. 18, 1772-1777, 2007.
• A. Date, P. Pasini, S. K. Deo, and S. Daunert, "Construction of Spores for Portable Bacterial Whole-Cell Biosensing Systems", Anal. Chem. 79, 9391-9397, 2007.
• L. A. Rowe. A. L. Rothert, C. Logue, C. M. Ensor, S. K. Deo, and S. Daunert, "Spectral Tuning of Ca²⁺-Regulated Photoproteins Using Site-Directed Mutagenesis and Chromophore Analogues", Protein Eng. Des. Sel. 21, 1-9, 2008.
• C. Wang, M. Micic, C. M. Ensor, S. Daunert, and R. M. Leblanc, "Infrared Reflection-Absorption Spectroscopy (IRRAS) and Polarization Modulated-IRRAS (PM-IRRAS) Study of the Aequorin Langmuir Monolayer", J. Phys. Chem. B., 112, 4146-4151. 2008.
• K. M. Teasley Hamorsky, C. M. Ensor, and S. Daunert, "A Bioluminescent Molecular Switch for Glucose", Angew. Chem. Int. Ed., 47, 3718-3721, 2008.
• A. Kumari, P. Pasini, and S. Daunert, "Detection of Bacterial Quorum Sensing N-Acyl Homoserine Lactones in Clinical Samples.", Anal. Bioanal. Chem., 391, 1619-1627, 2008.
• E. Dikici, S. K. Deo, and S. Daunert, "A Whole-Cell Assay for the High Throughput Screening of Calmodulin Antagonists", Anal. Bioanal. Chem., 390, 2073-2079, 2008.
• R. D. Johnson, V. Gavalas, S. Daunert and L. G. Bachas, “Microfluidic Ion-Sensing Devices”, Anal. Chim. Acta., 613, 20-30, 2008.
• L. A. Rowe, K. Combs, S. K. Deo, C. M. Ensor, X. Qu, and S. Daunert "Genetically Modified Semisynthetic Bioluminescent Photoprotein Variants: Simultaneous Dual-Analyte Assay in a Single Well Employing Time Resolution of Decay Kinetics", Anal. Chem. 80, 8424–8430, 2008.
• H.-K. Tsai, E. A. Moschou, L. Kulinsky, S. Daunert and M. J. Madou, "Integrating Biosensors and Drug Delivery: A Step Closer Toward Scalable Responsive Drug Delivery Systems ", Adv. Mater., 21, 656-660, 2009.
• E. Dikici and S. Daunert, “Fluorescent Timers Shine a Light on Protein Trafficking”, Nat. Chem. Biol. 5, 70-71, 2009.
• E. Dikici, X. Qu, L. A. Rowe, L. Millner, C. Logue, S. K. Deo, C. M. Ensor and S. Daunert, "Aequorin Variants with Improved Bioluminescence Properties", PEDS, in press, 2009.  PEDS Advance Access published on January 23, 2009.
• J. D. Ehrick, M. Luckett, S. L. Khatwani, Y. Wei, S. K. Deo, L.G. Bachas, and S. Daunert, "Accordion”-like Response Mechanism in Glucose-Responsive Hydrogels", Macro. Biosci. 9, 2009.