Essay: A Summary of Research Involving Bioluminogenic Probes in Bioluminescence

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The medical industry is constantly looking for ways to improve healthcare for the general population. One important facet of the healthcare industry is imaging. Being able to see inside the body and to more fully understand what is happening inside the body is one of the most reliable ways to determine a method of treatment for a patient. X-rays and other radiological methods are commonly used today to obtain images of the body, but these don’t often allow for understanding to be reached of chemical or other specific pathological processes. That is where the idea of bioluminescence comes in. According to Urano et. al.1, bioluminescence is a powerful tool that can be used to study complex chemical and pathological processes in the body, and one of its advantages is that it doesn’t require outside lighting in order to achieve luminescence like fluorescence does. Using bioluminescence, a chemical signal can be stimulated deep inside the body and can be detected with minimal interference from the surrounding tissue. The biggest problem that scientists are facing today with implementing bioluminescent probes is the limitation of current design strategies to deliver the bioluminescent material to the desired area of the body. Yasuteru Urano et. al. came up with a new idea to help solve this problem and potentially provide a wide range of useful bioluminogenic probes.
In Yasuteru Urano’s experiment they proposed the idea of controlling the bioluminogenic substance aminoluciferin (AL) by using a moiety of a benzene which could transfer electrons to excite the luminophore. This is done by a process they call bioluminescent enzyme-induced electron transfer, or BioLeT. Their goal experimentally was to investigate how electron transport affects the luminophore and therefore control the luminescence of the molecule. This concept can be observed in figure 1. As molecules are excited chemically, an electron is transferred from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO). When this happens in a fluorophore or luminophore, an electron from the respective group jumps up to the LUMO and en electron from the benzene moiety drops down to fill its spot, releasing energy in the form of light. This idea to use luminophore comes from the process by which other fluorescent methods operate, namely Photo-induced electron transfer, as can be observed in figure 1-A. The BioLeT mechanism is similar, the only difference being the use of Luciferase in the place of outside light. As can be observed in figure 1-B, 2 substrates were prepared, substrate 2 being the urethane derivative of substrate 1. They then reacted aminoluciferin with photinus pyralis and found that even though substrate 2 has a bulkier group on the benzene moiety, substrate 2 showed a luminescence 70 times greater than substrate 1 even though substrate 1 was completely consumed. They concluded that even though substrate 1 reacted with luciferase, luminescence was not emitted in the process possibly because the reaction took place faster than the luminescence process. Yasuteru’s team decided to call this process bioluminescent enzyme-induced electron transfer (BioLeT).
Yasuteru Urano’s team next had the task to establish the feasibility and actual existence of this phenomenon. This had to be accomplished in order for a reliable approach could be made to develop probes that act by the process of BioLeT instead of traditional fluorophores. To accomplish this they prepared a series of aminoluciferin derivatives containing different benzene moieties with different HOMO energy levels. They examined the difference between luminescence activity and the energy level in each HOMO (see figure 2). They found that the energy level of the HOMO on each benzene moiety played a large role in the luminescent activity of each molecule. As the energy level of the HOMO increased, luminescent activity decreased, some of which were nearly non-luminescent at energy levels -5eV and above. This procedure validated the existence of BioLeT. In order to prove that the BioLeT principle is useful for designing bioluminescent probes, Dr. Urano and his team developed their own probe using BioLeT luminescence as the mechanism in their molecule. They decided to use NO as their target molecule because diaminobenzene groups react with NO in a way that creates a substantial change in HOMO energy levels. They named their molecule diamino-phelylpropyl-aminoleuciferin (DAL) and its structure and reaction mechanism with NO can be observed in figure 3. They measured the HOMO energy levels of both DAL and its reaction product, DAL-T, to be -4.68eV and -6.22eV respectively. They predicted that the product would be sufficiently different in the HOMO energy level to produce a sufficiently different intensity in luminescence. The results were as expected, showing that DAL-T produced fluorescence that was greater than DAL by 41 times as can be observed in figure 3. These results support the idea that the excited state regulated by BioLeT is occurring in DAL and DAL-T and that luminescence by DAL is quenched by the luminophore, again supporting the operation of BioLeT.
Dr. Urano later experimented with the detection of NO in vitro using DAL which found a great increase of luminescence when addition of a solution containing NO or NOC7 was added, as can again be observed in figure 3-B. They found that DAL was converted to DAL-T as the product, as expected. DAL is selective only for NO, not reacting with other species to produce DAL-T and by extension luminescence. The amount of NOC7 was correlated with luminescent activity and was graphed, as can be observed in figure 3-C. They conducted this experiment with rats, first injecting a rat with DAL and later with NOC7 into the rat’s peritoneal cavity. Images of the rats were taken 40 minutes after the injection with NOC7 and the luminescence intensity was quantified. The results of this experiment can be seen in figure 4. In these rats, DAL was distributed throughout the entire body, and thus could effectively monitor NO levels throughout the whole body. This experiment illustrates the application of DAL in vivo, and further illustrates the possibility of creating bioluminescent probes using the BioLeT process. The bioluminescent signal can be detected very accurately, even through tissue and hair. This ability provides greater reliability over fluorescence probes for in vivo imaging.
In this article, Dr. Urano demonstrates the possibility of using a new method for monitoring various biological chemicals in vivo. Just as fluorescent probes have been designed using their PeT properties to gather information in vivo, bioluminescent probes can be engineered using their BioLeT properties to do the same in an even more accurate and sensitive manner.
The Work of Yasuteru Urano
Urano’s area of research centers heavily in biochemical science, especially in pharmacology, biomolecular chemistry and applied chemistry. He has largely spent his academic and professional life at the University of Tokyo where he conducted his undergraduate and graduate studies. He obtained his Ph.D. there in 1995 in the Graduate School of Pharmaceutical Studies. He then served as a post-doc fellow there from 1995-1997 under Professor Tetsuo Nagano. He then served as Assistant Professor from 1997-2005 and then Associate Professor from 2005-2009 in the Graduate School of Pharmaceutical Studies. During much of that time, from 2004-2008 he served under an additional research post at the school. He now serves as a Professor in the Graduate School of Medicine and also in the Graduate School of Pharmaceutical Sciences at the University of Tokyo.
One of Dr. Urano’s earliest articles was published in 1996, soon after his undergraduate studies were finished. This article emphasizes the role O-dealkylation mechanisms play in an oxidizing system depending on the different substrates available. They found that the oxidation determinant was if the substrate had a phenolic hydroxyl group at the ortho- or para- position relative to the alkoxy group in cytochrome P 450-dependent monooxygenases.2 Dr. Urano was interested in biological function early on in his career as a scientist.
Dr. Urano published another article in 1998 along with several other authors from the University of Tokyo that was of significance3. It currently has 140 citations. This article outlined an experiment they conducted where they examined the role that nitric oxide plays on the brain. In this experiment they used a fluorescent indicator to directly detect the presence of nitric oxide in samples of rat brain. The samples were from both acute cases and normal. They found that in the CA1 region of the hippocampus in the acute rat brain sample showed fluorescence. This was later confirmed by cell culture from the hippocampus. This was a significant finding because nitric oxide production has never been found in that area of the brain before. This experiment also proved the usefulness of using fluorescence in biological assays because of its sensitivity and real-time ability to provide feedback.
Another article published in the year 1999 improved upon the significance of the previously mentioned one, with 264 citations4. This article continues on the trend that Dr. Urano has developed in fluorescence over the years. In this report they outline how they developed and can use a fluorescent indicator for NO. This indicator is called diaminofluoresceins (DAF5). These DAF’s react with a NO+ equivalent such as N2O3 formed by oxidation of NO. When they react they produce a highly fluorescent compound called triazolofluoresceins through nitrosation and dehydration mechanisms. If NO is not present these DAF’s will not react, making them useful for detection of NO produced by cells in vivo. One issue they ran into was the problem of pH, fluorescent ability is sensitive to changes in pH and the intensity will drop if the pH fluctuates too much. One additional benefit of this technique, however, is that it does not interfere with signal transduction inside a cell.
In an article published in 2000, Dr. Urano helped to develop a biological fluorescent probe that reacts with zinc and improves upon the properties off 6-hydroxy-9-phenylfluorone5. Zinc plays an important role in the body. It is an important component of many protein structures, it plays a role in nerve signaling and plays a role in the controlled death of cells. Even though it is known that zinc plays an important role in the cell, not much is known about the regulation of zinc in a cell, as opposed to calcium, sodium or potassium where much is known. Because of these findings, Dr. Yasuteru and others sought to develop a way to monitor zinc levels in the body in order to observe and learn more about this important element of cellular function. They also made improvements on their previously formed fluorescent sensor molecules in order to improve results. They found that the previous molecules had a slow complex formation rate as well as a small yield, so they decided to utilize fluorescein’s high-yield and created molecules they called ZnAF-1 and ZnAF-2. The structure of ZnAF-1 and ZnAF-2 can be observed in figure 5. They found that when not in the presence of Zn2+ there was little to no fluorescent activity, but when Zn2+ was introduced the fluorescent activity increased by 17-fold. The increase in fluorescent activity can be observed in figure 6.
In 2001 Dr. Urano and others conducted an in-depth study on fluorescein and its chemical properties6. They concluded that the fluorescent properties of fluorescein come from the benzoic acid moiety to the xanthene ring and are controlled by a photoinduced electron transfer process. They measured the threshold to be around -8.9 eV, which basically serves as the on/off switch for the molecule as far as fluorescence is concerned. Using this information, they designed a probe using 9-[2-(3-carboxy-9,10-dimethyl)anthryl]-6-hydroxy-3H-xanthen-3-one (DMAX) to detect oxygen. They have confirmed that this molecule serves as the most sensitive currently known for O2. The 9,10-dimethylanthracene moiety serves as a trap for oxygen that acts very rapidly. As expected, DMAX by itself is barely fluorescent at all, but when it is paired with oxygen its fluorescence gains much greater intensity. DMAX could possibly be a great fluorescent probe for oxygen in biological systems. The mechanism through which fluorescein works is shown in figure 7. The chemical structure of fluorescein can be observed in figure 8.
In 2001 Dr. Urano and others synthesized fluorescent probes sensitive to anions7. At the time the only anion probes that were known only functioned in organic solvents. In order to be able to have an application in a biological setting, the anion probe would have to function under biological conditions including functionality in water at a neutral pH. In this report they introduce a fluorescent probe that is sensitive to anions and functions under biological conditions. The sensor molecule they designed is called 1-Cd111 with the fluorescent portion being made up of 7-amino-4-trifluoromethylcoumarin. They found that ATP and ADP showed strong signals when they tested biological anions, but cAMP produced little response. The mechanism for this reaction can be observed in figure 9. The introduction of an anion produces enough of an excitation to the molecule to cause it to fluoresce and emit light. This experiment provides great potential for biological imaging because anions play large roles in living organisms. Having the ability to monitor anion activity in real time in a biological setting can produce some very valuable biological and analytical appllications
In 2004 Dr. Urano and others experimented more with NO8. This article currently has over 360 citing references. NO is an important messenger molecule both inside and outside of cells. Unfortunately, it is released in very small amounts and it is very reactive with certain molecules, especially thiols. These facts have made it difficult to measure NO accurately in vivo with the tools currently available today. Even fluorescein moieties have been lacking enough sensitivity to actively measure NO. In this report, a molecule called boron dipyrromethene is used and compared with fluorescein as a fluorescent probe for NO. The molecule they developed based off of boron dipyrromethene is called DAMBO-PH, short for 8-(3,4-diaminophenyl)-2,6-bis(2-carboxyethyl)-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene, which they found to be a highly sensitive probe for NO. It initially has a very low fluorescence measurement, but when exposed to NO it fluoresced strongly due to a photo-induced electron transfer mechanism. The development of this molecule was significant because they had to do some fine tuning of the molecule in order for it to function well independent of pH and to avoid probe stacking. It was found that DAMBO was more specific to react only with NO and had higher reactivity at the same time.
Dr. Urano conducted even more research regarding NO. One notable article of his from November 2004 was again about improving upon the imaging techniques used to monitor NO activity. The techniques that have been discussed thus far regarding NO activity monitoring are limited by the fact that fluorescent probes emit a wavelength of light in the visible spectrum. This area of wavelengths is absorbed readily by body tissues and prevents observation in deeper tissues because the light is absorbed too quickly by tissue. Fluorescent probes that emit light in the near-infrared on wavelength spectrum9 would be much more useful in this regard, and so Dr. Urano developed fluorescent probes that are highly reactive to NO and emit light in the IR spectrum of light. He created the probes with two parts. The first is tricarbocyanine, an IR emitting fluorophore, and o-phenylenediamine as the NO reactive portion. They called the chemical group diaminocyanines (DAC’s) which are classified into two types: DAC-P’s which are DAC’s with two propyl groups attached to allow the DAC through a cell membrane easily and DAC-S’s with sulfonate groups to make them highly soluble in water. The chemical scheme of these molecules is shown in figure 10. The absorbance spectra and emittance spectra of DAC-S is shown in figure 11 with the presence of varying amounts of NO. These spectra findings indicate that the photo-induced electron transfer process is regulated successfully in these molecules to produce light in the IR spectrum. This finding is significant because at that time only one other probe was reported to produce IR wavelengths through regulation by photo-induced electron transfer. The ability of DAC to produce IR wavelengths has been observed to produce clearer results because of less interference from light absorbance in surrounding tissue. Figure 12 shows the results of administration of NOC13 in the presence of DAC-P in a rat kidney. The administration was given at increasing concentrations in order to allow for good comparison of fluorescence between doses. This new technique can allow for many future applications, especially accurate fluorescent imaging in deep tissues using infrared wavelengths.
More research on fluorescein and developments on creating finely tunable fluorescent probes10 was shown in an article published in October 2004 by Dr. Urano. In this article he shows how he isn’t afraid to think outside the box and challenge other people’s ideas. Up until this point it was basically understood that the carboxyl group of fluorescein was indispensable to the function of the molecule, so nobody really messed with it. This was a mistake, however, because fluorescent probes have to be modified to fit different substrates in order to observe specific chemical activities in the body. Being able to modify the fluorescein molecule to fit your need was essential in order to achieve the desired goal. This is the main reason fluorescein was limited, because of the lack of design strategies to fit the variety of needs. In this article, Dr. Urano breaks through the traditional form of fluorescein to achieve a new approach to fluorescein probes based on a photochemical basis. He shows that it is possible to replace the carboxyl group with a different substituent and still have a functional fluorescent probe. He illustrates the feasibility of this idea by using this method to create a probe that is sensitive to beta-galactosidase which is the most widely used reporter enzyme. As can be observed in figure 13, he first replaced the carboxyl group with a methyl group and fluorescence was still achieved. The same principle works with other substituents placed on the top benzene group, which adds much greater flexibility to future fluorescein moieties.
In an article published in December of 200611 Dr. Urano discusses further the greater options that become available when the carboxyl group is replaced on the benzene moiety of fluorescene. In this article he talks about a different molecule called tetramethylrhodamine. This molecule has good potential for use as a fluorescent probe because of its resistance to pH, its excellent photochemical properties and longer excitation time and wavelengths. Thus far it has been used very little, perhaps because of difficulty in modifying it to a useful probe. Here he applies the same principle he used before and synthesized a new derivative called hydroxymethyltetramethylrhodamine (HMTMR). They found that HMTMR displayed a large amount of absorbance and fluorescence in protic solvents but little in aprotic or basic solvents. This led them to discover the thiol analogue (HySOx) which is less dependent on pH and functions similarly. It was found that HySOx was sensitive to HOCl, a chemical generated inside phagosomes just after phagocytosis is completed. They then applied HySOx to help visualize phagocytosis of pathogens. As can be observed in figure 14, the activation of these phagosomes could be observed visually as the HOCl contained in the phagosomes reacts with HySOx and causes excitation and luminescence. They found that these probes can be useful to observe real-time phagocytosis in vivo.
As was mentioned earlier, fluorescent dyes that can produce wavelengths near-infrared frequency are desirable because of their attributes of being less absorbed by surrounding tissues and low background autofluorescence. These properties make imaging much clearer and more accurate. These dyes have been utilized to diagnose various different diseases, such as arteriosclerosis, rheumatoid arthritis and cancer. The problem that is being faced with these probes is that most of them don’t function well in aqueous media because the molecules stack on each other and prevent efficient emission of luminescence. In 2012 Dr. Urano reported on the development of fluorescent dyes that are excitable in near-infrared wavelengths based on the rhodamine scaffold that was discovered in the previous mentioned experiment12. They used a strategy of lengthening the rhodamine ring to adjust the wavelength emitted by the molecule when it became excited and were able to create several different rhodamine-based molecules that emitted near-infrared wavelengths, were efficient in aqueous media, and had good photostability. They then took one of the molecules they developed named SiR-700 and labeled it with an antibody to a glycoprotein known to be specific for malignant gliomas called tanascin-C (TN-C). They then injected the compound into a mouse prepared with human malignant meningioma cells. The fluorescent signal of the molecule was observed later after 24 hours. The fluorescent imaging can be observed in figure 15. The signal amazingly remained observable for 10 days. This displays the huge developments he has made in creating probes that are resistant to photobleaching and can last for a long time in vivo and still display an accurate image.
Dr. Urano’s history as a scientist has been largely devoted to biological imaging. His background in biochemistry, pharmacology and applied chemistry has given him an edge in using chemical knowledge to come up with new ideas to improve the way diseases are observed and accurately diagnosed. With each passing year he has improved upon his previous studies and experiments to produce greater results that have very useful applications, especially in the field of medicine.
Other studies done in this field
Intra-cellular processes are very important to be able to understand, especially when it comes to genes that get turned on that result in disease, such as cancer, autoimmunity and chronic inflammatory diseases. Dr.’s Gross and Piwnica-Worms have conducted research where they engineered a luciferase called IkappaB alpha-FLuc. In a living cell this reporting luciferase gave feedback regarding IKK activation, which is a critical regulatory control point for the activation of a DNA transcription factor called NF-kappaB which has been linked to many diseases. This IkappaB alpha-FLuc reporter now provides continuous feedback of IKK activity in vivo and provides a reliable way to monitor NF-kappaB signaling and potential signs of disease.
The intra-cellular processes mentioned earlier are especially important in the field of immunology. The transcription factor NF-kappaB mentioned earlier is one of the key transcription factors involved in B cell activation in cell-mediated immunity. Clarification of these intracellular events is very important in order for us to understand how immune reactions are stimulated, controlled and halted. As Negrin and Contag mention in their article, the pathophysiology regarding grafts is a subject that is currently being researched. Bioluminescence imaging has great potential to be a reliable modality for observing cell trafficking and gaining greater understanding of biological systems relating to immunity and grafts.
Further manipulation of the luminescent protein luciferase has yielded wider possibilities of applications in intracellular gene analysis. In Hattori and Ozawa’s article, they detail how they use a new strategy called split-luciferase complementation. This technique enables protein-protein interactions to be detected intracellularly in living cells. This technique has a wide variety of possible applications, such as phosphorylation monitoring, G-protein-coupled receptor screening, and even Ph monitoring in addition to observing protein-protein interactions.
Dragulescu-Andrasi et. al. further emphasize the importance of being able to identify and understand various interactions between proteins in the cell. They describe a system using bioluminescence resonance energy transfer that makes it possible to identify protein-protein interactions not only in culture, but in deep tissue in a living organism. They outline the potential usages of this system to investigate protein-protein interactions in the context of target validation and drug screening in a patient. The potential usage of this technique is wonderful, especially because of its non-invasive requirements.
Wehrman et. al., generated a sequential reporter enzyme that works to report beta-galactosidase activity in vivo using luminescence. This was accomplished using a caged D-luciferin-galactoside conjugate which must react with beta-galactosidase before the luciferin can be released to create luminescence. They state that one benefit of this method, besides the non-invasiveness that comes with using bioluminescence, is the ability for the probe to remain fully active even when it is outside of the cell since beta-galactosidase is able function without the aid of intracellular enzymes. Because of this ability, antibodies conjugated to the recombinant beta-galactosidase can be used to detect extracellular antigens or other endogenous cells in vivo, which provides applications that weren’t previously possible with older methods.
In an experiment conducted by Hickson et. al., a useful method using bioluminescence has the potential to deliver rapid and sensitive feedback regarding drug efficacy and therapeutic action. Using a modified version of luciferin called Z-DEVD-aminoluciferin, they measured the amount of luminescence delivered after injecting this modified luciferin into cancerous animals prior to treatment with docetaxel. They also had a control group who were not injected with docetaxel. Z-DEVD-aminoluciferin is cleaved by caspase-3, which is released in apoptotic cells, which in turn releases the luciferin and results in luminescence. Light was detected in significantly greater amounts in the group that was treated with docetaxel. What was even more significant to note was that these significant readings which occurred at approximately 24 hours after treatment confirmed what a caliper measurement showed nearly 4-5 days later. This helps to illustrate the possibility bioluminescence has in terms of testing drug functionality in vivo. Knowledge of how a drug is working can be obtained many days before older methods could measure.
Van de Bittner et. al. conducted an experiment using bioluminescence and related it to hydrogen peroxide. It is known that hydrogen peroxide is generated by living organisms to kill invading pathogens and to maintain immunity. The specifics on hydrogen peroxide’s general effects on aging and health are largely unknown largely attributed to the difficulties of being able to study it in vivo. In their experiment they report on their use Peroxy Caged Luciferin-1 which serves as a bioluminescent probe that selectively reacts with hydrogen peroxide, causes the release of luciferin and consequently creates luminescence. Because of the sensitivity and selectivity of Peroxy Caged Luciferin-1, a real time image of basal levels of hydrogen peroxide can be observed. The effects of hydrogen peroxide, in addition to all kinds of other chemicals, on aging, disease and health can now be observed where previously it was impossible.

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