Several biomedical research applications make the most of the natural fluorescence response of amino acids – the essential building blocks of all proteins. These protein fluorescence responses to light have been employed for everything from pharmaceutical manufacturing and cancer treatments through to biowarfare defense. Investigating this biomedical spectroscopy niche is a jump into the deep end of cutting-edge science.
Spectroscopy is an indispensable technology that makes these biomedical applications, and so many others, possible. Avantes specializes in the development of high-resolution, high-sensitivity spectrometers and is a reliable option for hundreds of researchers and original equipment manufacturers in biomedical applications.
Proteins and Amino Acids
Proteins are complex organic compounds made of chains of amino acids. They are the most abundant organic molecule in all living things on Earth. These molecules have many roles to play within cells and organisms. For instance, they act as catalysts in biochemical reactions, as hormones to regulate the physiological processes, and they also defend the body against disease. Most of these proteins have a weak intrinsic fluorescent response to UV excitation, and depending on the species, different proteins react to excitation at specific wavelengths and fluoresce at different wavelengths.
Tyrosine, tryptophan, and albumin are the three common amino acids which are responsible for the majority of inherent fluorescence. They have a common structure that contains hydrogen-based ring structures. These ring structures, known as aromatic hydroxyl groups, absorb UV radiation and emit a weak fluorescent signal at differing wavelengths.
The amino acid compounds have different quantum yields, and distinct absorption and emission profiles. They can also change the spectral profile by reacting with each other; for example, the presence of tryptophan will quench the fluorescent signal of tyrosine because of resonant energy transfer of a similar excitation wavelength. Tryptophan and tyrosine can be excited at 280 nm and 274 nm, respectively. However, the fluorescence response is considerably different; tyrosine’s response can be detected at 303 nm while tryptophan fluoresces at 348 nm. Alternatively, when albumin is excited at 257 nm, it will fluoresce at 282 nm.
Since amino acids are the common building blocks of many more complicated proteins and are prevalent in all life on Earth, their reactions are often used as a representative sample for these complex proteins in research applications. It is also possible to monitor the changes in the reaction as a process control function.
Fluorescent Response in Action
Fluorescence finds an extensive range of applications in life science and biomedical fields, and Avantes equipment can be found at the forefront of new scientific frontiers.
Weapons to Fight Cancer
Spectroscopy has been shown to be a useful tool in the fight against cancers. The use of autofluorescence bronchoscopy is the standard for lung cancer detection in which a narrow probe is inserted through the patient’s mouth into the upper bronchial tree. When compared to white light bronchoscopy, autofluorescence has been shown to be far more sensitive in the detection of dysplastic lesions or carcinomas. However, it also has a high rate of returning false positives. At the Department of Respiratory Diseases in Rotterdam, the Netherlands, researchers have investigated the use of absorbance/reflection optical spectroscopy in order to enhance specificity without losing sensitivity.
The researchers used a specially designed probe to feed fiber light sources through the 2.8 mm bronchoscope channel. The mucosa tissue was irradiated with a tungsten/halogen broadband white light (predecessor to the AvaLight-HAL-S-Mini) and a blue laser calibrated to 407 nm was introduced through fiber optics. The autofluorescence of healthy tissue had an emission peak wavelength of 500 nm. The fluorescence and reflectance emissions obtained were collected through a multichannel Avantes spectrometer installation equivalent to two AvaSpec-ULS2048CL-EVOs in parallel.
An area exhibiting an abnormal fluorescence profile can be quickly targeted for additional spectral measurements which can be obtained in less than one second. When compared to healthy tissues, diseased tissue showed drastically lower emission intensity at shorter wavelengths. The combination of autofluorescence imagine and optical reflectance spectroscopy considerably enhanced the positive predictive value than autofluorescence alone without sacrificing sensitivity.
Both the excitation and fluorescence wavelengths at 407 and 500 nm respectively are within the range of 360-600 nm, wherein blood is a primary absorber. This indeed creates a challenge for this application but not an insurmountable one. Researchers were sure that the modified bronchoscope with additional fibers was viable to deploy during a standard bronchoscopy procedure.
Protein Contamination Detection on Surfaces
Research over the past 10 years has proven that traditional methods of sterilization, including chemical and thermal processes, are not adequate to ensure complete inactivation of all pathogenic biomolecules, particularly proteins. This is a serious concern in a surgical setting where the decontamination of typically reusable medical instruments, like scalpels, can directly affect patient care.
Prions are a class of proteins which is of particular concern. These proteins are folded “wrong”, manifesting in lethal neurological degenerative diseases like the human variant of mad cow disease known as Creutzfeldt-Jakob disease, Fatal Familial Insomnia, and Kuru. These diseases though rare are caused by infectious proteins that spread their wrong folding patterns to new proteins in a way that is epidemiologically similar to a viral infection. Proteins are the compound most resistant to present methods of decontamination and as a result, transmission of these “contagious” neurological diseases presents a serious danger.
The latest technique of decontamination being investigated needs a low pressure inductively coupled plasma discharge. Spectroscopic instruments are employed for process control and monitoring test results.
Detection of Dangerous Biological Compounds
At the Swedish Defense Research Agency (FOI), researchers are studying the use of fluorescence spectroscopy as the first line of detection in bioaerosol detection systems. Due to the dangerous nature of biological warfare agents (BWAs) themselves, both tryptophan and tyrosine – amino acids likely to be a part of any biological agent – are used in testing instead of these BWAs. An ideal system would be able to monitor tiny particle concentrations in real time and detect compounds present with a high degree of specificity.
The system being tested by the Swedish research team forces ambient air through a nozzle so that it is confined to a single particle beam and passed through the optical chamber. The use of a continuous wave blue laser at 404 nm received by an Avantes spectrometer, acts as a trigger. At this stage, fluorescence and scattering are analyzed. A pulsed UV laser is triggered at 263 nm, when a compound is present at pre-determined detection levels. The resultant laser-induced fluorescence can be further investigated to categorize individual aerosol particles.
Recently, Avantes US Engineers reproduced a series of experiments conducted with samples of bovine serum albumin, tyrosine, and tryptophan to demonstrate the protein detection capabilities. The team used traditional absorbance methods with an AvaSpec-ULS3648-USB2 spectrometer configured for UV (190-400 nm) and a continuous wave (CW) illumination source (AvaLight-DHc). Absorption measurements recorded between 190 nm and 320 nm correlated the changes in concentration to changes in absorption. During this experiment, fluorescence was not measured.
For each of the three sample analytes, the team running this experiment prepared three different concentrations: 0.25 mg/ml, 0.5 mg/ml, and 1 mg/ml, and took broadband absorption spectra profiles for each.
The absorption profiles of the amino acids correlated directly with the concentration of amino acids in solution. Since fluorescence is a function of absorbance and quantum yield, it can also be estimated and would be expected to correlate with concentration
Trust Avantes for UV Absorbance and Fluorescence Applications
A key enabling technology for many advances in biomedical technologies and dozens of other industries and applications are absorbance and fluorescence measurements. Avantes has been the trusted source for users’ high-quality spectroscopy systems and instrumentation for 20 years and will remain their spectroscopy partner for decades to come.
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This information has been sourced, reviewed and adapted from materials provided by Avantes BV.
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