Luminescent Proteins: Applications in Microfluidics and Miniaturized Analytical Systems

Dikici, Emre; Rowe, Laura; Moschou, Elizabeth A.; Rothert, Anna; Deo, Sapna K.; Daunert, Sylvia

doi:10.1002/3527609148.ch10

Cited by 4 publications

(3 citation statements)

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“…These devices allow for rapid responses in the order of minutes, with simple and shortened procedure as compared to conventional benchtop protocol. In particular, the use of fluorescent and bioluminescent photoproteins as reporters is expected to facilitate the integration of whole-cell biosensing systems into such miniaturized analytical devices …”

Section: Discussionmentioning

confidence: 99%

“…In particular, the use of fluorescent and bioluminescent photoproteins as reporters is expected to facilitate the integration of whole-cell biosensing systems into such miniaturized analytical devices. 32 This would further enhance environmental on-field applications, as well as biomedical applications at the patient bedside and the physician's office. The use of miniaturized analytical systems would also allow incorporating multiple biosensing systems in one small device, thus providing a portable solution to environmental and clinical multiplexed, on-site analysis.…”

Section: Discussionmentioning

confidence: 99%

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Construction of Spores for Portable Bacterial Whole-Cell Biosensing Systems

2007

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Whole-cell sensing systems based on living genetically engineered bacteria are known to have high sensitivity, selectivity, and rapid response times. Although these systems have found applications in biomedical and environmental analyses, their limited shelf life and transportability still restrict their use for on-site monitoring of analytes. To that end, we have developed a new method for the long-term preservation, storage, and transport of whole-cell biosensing systems that is based on bacterial spores, a dormant form of life. Specifically, we have employed spore-forming bacteria such as Bacillus subtilis and Bacillus megaterium for development of luminescent sensing systems for two model analytes, namely, arsenic and zinc. These sensing cells were converted to spores, which can then be "revived" (germinated) at a later time to generate viable and metabolically active cells. Herein, we demonstrate that these spore-based sensing systems retained their analytical performance, in terms of detection limit, dynamic range, and reproducibility, after storage at room temperature for at least 6 and 8 months, respectively, as well as after three cycles where the cells alternated between being dormant or active, i.e., sporulation-germination cycles. The ability to cycle the sensing cells between active and dormant states prolongs the cell's lifetimes and increases their robustness and ruggedness, thus making them more amenable for field applications. In addition, the small size of spores allows for their easy transport and incorporation in miniaturized portable devices. Finally, we envision that this novel strategy could expand the use of whole-cell biosensors for on-site sensing not only in mild environments but also in harsh environments and locations where there is no easy access to a laboratory, e.g., in developing countries.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Construction of Spores for Portable Bacterial Whole-Cell Biosensing Systems

2007

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“…Aequorin is a bioluminescent protein native to the jellyfish Aequorea victoria , which is composed of the apoprotein and the chromophore coelenterazine . In the presence of Ca 2+ , aequorin undergoes a conformational change that results in an excited state of coelenteramide followed by the emission of light at 469 nm with a quantum yield of Φ of 0.2. , Given that bioluminescence is a rare phenomenon in nature, aequorin demonstrates a very low, virtually zero, bioluminescence background in many environments, such as that of biological fluids, thus increasing the sensitivity of the method .…”

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confidence: 99%

Bioluminescence DNA Hybridization Assay for Plasmodium falciparum Based on the Photoprotein Aequorin

et al. 2007

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A bioluminescence DNA hybridization assay for the detection of Plasmodium falciparum, the most deadly species of malaria, using the photoprotein aequorin as a bioluminescent label has been developed. The current gold standard for the detection of malaria is light microscopy, which can detect down to ∼50 parasites/µL of blood, but has low-throughput, high costs, and requires high skill, which limit the applicability of the method, especially in the developing regions where malaria detection is mostly needed. The utilization of aequorin as a bioluminescence label offers the advantages of high signalto-noise ratio and reliable detection down to attomole levels, allowing for the development of highly sensitive and miniaturized high-throughput bioluminescence assays. Herein, we developed a DNA hybridization assay for the detection of P. falciparum based on the competition between the target DNA and the signal generating DNA streptavidin-aequorin for hybridization with the probe DNA. This bioluminescence hybridization assay demonstrated a detection limit of 3 pg/µL and was employed for the detection of target DNA in standard and spiked human serum samples. The DNA hybridization assay was developed in a microplate format without the need for sample PCR amplification, showing the potential suitability of this method in the parallel analysis of samples by low-trained personnel, such as that typically encountered in developing regions.

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Enabling Aequorin for Biotechnology Applications Through Genetic Engineering

Grinstead

Joel

Zingg

et al. 2015

Bioluminescence: Fundamentals and Applications in Biotechnology - Volume 3

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In recent years, luminescent proteins have been studied for their potential application in a variety of detection systems. Bioluminescent proteins, which do not require an external excitation source, are especially well-suited as reporters in analytical detection. The photoprotein aequorin is a bioluminescent protein that can be engineered for use as a molecular reporter under a wide range of conditions while maintaining its sensitivity. Herein, the characteristics of aequorin as well as the engineering and production of aequorin variants and their impact on signal detection in biological systems are presented. The structural features and activity of aequorin, its benefits as a label for sensing and applications in highly sensitive detection, as well as in gaining insight into biological processes are discussed. Among those, focus has been placed on the highly sensitive calcium detection in vivo, in vitro DNA and small molecule sensing, and development of in vivo imaging technologies. Graphical Abstract.

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Luminescent Proteins: Applications in Microfluidics and Miniaturized Analytical Systems

Cited by 4 publications

References 0 publications

Construction of Spores for Portable Bacterial Whole-Cell Biosensing Systems

Construction of Spores for Portable Bacterial Whole-Cell Biosensing Systems

Bioluminescence DNA Hybridization Assay for Plasmodium falciparum Based on the Photoprotein Aequorin

Enabling Aequorin for Biotechnology Applications Through Genetic Engineering

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