Biosensors

SenseOmics Inc. has designed and produced proteins for the highly sensitive and selective detection of target analytes.  Based on the structural changes that certain proteins undergo upon binding with their corresponding ligand, highly stable “tailor made” proteins are designed to detect these ligands in a highly specific manner. Our systems take advantage of naturally occurring biological molecules, which produce a structural change in response to binding various analytes, and incorporate them into a rational sensing architecture.  Through the use of either wild type or genetically engineered mutant proteins as biorecognition elements, sulfate binding protein (SBP), glucose binding protein (GBP), calmodulin (CaM), and phosphate binding protein (PBP) can be used in sensing platforms for sulfate, glucose, chlorpromazine (a member of the phenothiazine class of drugs), and phosphate, respectively.   By employing genetic engineering tools, a fluorescent reporter is site-selectively incorporated into the protein. In the presence of the target ligand analyte, the protein undergoes a structural change in conformation that results in a change in the fluorescence signal emitted by the reporter.  The concentration of target analyte present in a sample can be directly related to the change in fluorescence signal. Analogously, if electrochemical detection is desired, an electroactive reporter can be employed instead of a fluorescent one.  In this case, the concentration of target analyte can be determined by registering the variation in the electrochemical signal of the reporter in the absence and presence of the analyte.

This technology can also be adapted to the design of YES/NO answer assays.  By creating a change of color in the presence of the target analyte, detection can be accomplished. Depending upon platform used, sample volumes from the microliter to picoliter range can be detected. With new advances in instrumentation, biological technology, and protein immobilization strategies, our sensing schemes should find applications in high-throughput screening assays, clinical diagnostic monitoring, and also environmental analysis.

The following table shows some of SenseOmics’ biosensors. As shown in the table, the SenseOmics biosensors detect low analyte concentrations.

Photoproteins

Certain marine organisms produce calcium-activated photoproteins, such as aequorin, that allow them to emit light, usually in the blue region, for purposes such as defense, feeding, and breeding.  The mechanism of emission of light in the blue region is the result of an internal chemical reaction.  Since there is no need for excitation from external irradiation for the emission of bioluminescence, the signal produced has virtually no background.  This allows for detection limits at extremely low levels, making these photoproteins attractive labels for analytical applications. 

SenseOmics Inc. has developed variants of the photoprotein aequorin, which are more active and more stable than currently available.

The jellyfish Aequorea victoria

The structure of aequorin consists of four helix-loop-helix domains, three of which can bind calcium. These domains create a central hydrophobic cavity, in which aequorin's chromophore, coelenterazine, resides. In the presence of molecular oxygen aequorin undergoes a conformational change following the binding of calcium. This conformational change allows for the rearrangement of the coelenterazine into an excited electronic state. This excited molecule, coelenteramide, subsequently relaxes to its ground state, and emits a photon of light at 469 nm, giving aequorin its bioluminescence. In a similar manner, obelin also emits luminescent light from its chromophore, coelenterazine, following incubation with oxygen and addition of calcium.  These photoproteins can be employed in a variety of bioanalytical applications.  They can be used in calcium sensing, as a label in immunoassays, nucleic acid hybridization assays, and in biosensor applications.

Structure of Aequorin

IMMUNOASSAYS

To develop the assay, the analyte of interest is either chemically or genetically conjugated to aequorin, depending upon the characteristics of the analyte. The aequorin-analyte will bind to analyte-specific antibody with a similar affinity as the natural analyte. Addition of calcium will cause the bound aequorin-analytes to emit a flash of light. The bioluminescence light emitted will, therefore, correspond to, in a dose-dependent manner, amounts of native analyte in the sample. The constitutes the basis for the development of highly sensitive homogeneous and heterogeneous binding assays.

An example of this is our aequorin immunoassay for prostacyclin in plasma. Prostacyclin, a potent vasodilator with antiplatelet and antiproliferative properties, is an effective treatment for primary pulmonary hypertension and pulmonary arterial hypertension associated with scleroderma and scleroderma-like symptoms. Levels of 6-keto-prostaglandin F_1α (6-keto-PGF_1α) can be directly correlated with levels of Prostacyclin. Therefore, 6-keto-PGF_1α has become the indicator of choice to measure Prostacyclin levels. The Aquamax-6-keto-prostaglandin F_1α Conjugate can be used in immunoassays for the measurement of the 6-keto-PGF_1α directly in plasma without any pretreatment of the sample resulting in a much simpler method with faster assay time over standard assays
 

Dose-response curve for prostacyclin

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Last modified: 09/19/2007