Biochemical processes within the body generate specific biomolecular products that can indicate the activity of said process, and various disease states can induce or inhibit the generation of particular biomolecules. As the systemic or local concentration of these molecules can be used to infer information relating to a patient’s health or disease state, they are termed biomarkers, i.e., biological markers.
Colorimetric sensors undergo a color change, or more specifically, a change in peak emission and/or absorption wavelength, only in the presence of the biomarker of interest, confirming their presence quantitatively based on the intensity of color change.
The application of several types of colorimetric sensors to biomarker evaluation and their mechanism of action is discussed in this article.
The Science Behind Colorimetric Sensing
A wide range of substances can change their optical properties upon bonding with biomarker molecules, including changes in absorbance, reflectance, luminescence, fluorescence, refractive index, light scattering, and optothermal measurements. Typically, colorimetric sensors undergo a color change within the visible spectra (400 – 800 nm wavelength) and can thus be appreciated conveniently using the naked eye, though also include many only detectable using additional optical apparatus.
A number of specific chemical mechanisms may be taken when a colorimetric sensor encounters the target biomarker, frequently involving altered electron arrangement within the newly formed complex and, thus, differing optical properties. Colorimetric sensors are widely exploited in the clinic and in lifescience research, regularly utilized in the diagnosis of various diseases where altered biomarker presentation is expected, and also exploited in discovering new biomarkers.
Biomarkers: Unveiling Health Secrets
Biomarkers are utilized extensively in disease detection, prognosis, and the evaluation of ongoing treatment in a wide variety of cases, often sub-categorized as diagnostic, prognostic, and therapeutic biomarkers, respectively. Similarly, genetic biomarkers may also be utilized in indicating the risk and occurrence of genetic diseases or other issues.
Numerous biological molecules, such as proteins, polysaccharides, and nucleotides, may be biomarkers and can be collected from the sera or other biological compartments for evaluation by colorimetric assay. These assays typically consist of an array of colorimetric sensors each sensitive to specific biomarkers, to which collected samples can be exposed.
Therefore, color changes in particular regions of the assay array indicate the presence of the biomarker. Colorimetric sensors may also be utilized in vivo in combination with diagnostic techniques such as computed tomography or positron emission tomography to infer the presence of biomarkers and their spatial and, in some cases temporal positioning.
Colorimetric Sensing Techniques in Action
Enzymatic methods of colorimetric sensing are widely employed in medical laboratories, as many of the biomolecular biomarkers of interest innately engage with readily available enzymes. Enzyme-linked colorimetric assays have, therefore been adapted to detect a range of biomarkers.
For example, glucose can be detected colorimetrically using glucose oxidase, which produces gluconate and hydrogen peroxide. Hydrogen peroxide then reacts with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) to produce a colored compound, facilitated by peroxidase enzyme. ABTS is used in several enzyme-linked assays where hydrogen peroxide is produced, and its color change from mild yellow (342 nm) to intense turquoise (419 nm) is triggered upon oxidation.
In another example, urea can be colorimetrically sensed using the urease enzyme to produce ammonium carbonate, which can then be further processed without the aid of additional enzymes using phenol and hypochlorite to produce a colored compound.
Some types of colorimetric sensors do not require sequential processing to produce the indicative color, namely those incorporating fluorescent components. Fluorescence can be engineered to begin or cease upon bonding with a biomarker, typically by breaking or forming bonds with an adjacent quenching molecule.
Nanoparticles capable of surface plasmon resonance, primarily those constructed from gold or silver, are also useful as colorimetric sensors. Surface plasmon resonance is the collective oscillation of conduction band electrons belonging to the nanoparticle in phase with incident light, significantly enhancing absorption at the resonance wavelength. When a collection of nanoparticles of the same material, size, and shape are gathered together, this absorption is sufficient to lend color to the medium in which they are suspended.
For example, gold nanoparticles 10 nm in diameter typically engage in surface plasmon resonance with light 520 nm wavelength and thus absorb strongly in the blue region. The subtraction of blue light from white light gives the medium an overall red hue. The specific wavelength of light with which plasmonic materials are in resonance depends on the material, the size, and the shape of the nanoparticle. It’s immediate dielectric surroundings and thus is highly sensitive to changes in the surrounding molecular composition and nanoparticle aggregation.
Gold nanoparticles are already used as colorimetric sensors in simple strip-flow assays, such as pregnancy tests or the widely disseminated COVID-19 testing kits. These devices contain nanoparticles bound with enzymes or other molecules that form bonds only with the biomarker of interest, such as pregnancy-related hormones or the SARS-CoV-2 spike protein, and thereby form a colored region where nanoparticles were successfully bound. Alternatively, successful biomarker interaction may cause nanoparticles to aggregate, changing the shape and size of the particle and inducing red shift of the absorption spectra, causing a gold nanoparticle colloid to appear more blue.
Nanoparticles are particularly useful in vivo when combined with other diagnostic apparatus, as discussed. Materials such as gold possess a high z number and thus act as excellent contrast agents within these techniques. Additionally, light within the near-infra red region is optimally penetrating through biological tissue, known as the “tissue transparency window”, and is capable of being detected several centimeters into the body by the proper light detectors. When excited by lasers, nanoparticles of the appropriate material and morphology can emit light of this wavelength, thus acting as precise in vivo colorimetric indicators.
The Future of Colorimetric Biomarker Sensing
In the future, the range of diseases and the specificity with which their progress and treatment effectiveness can be assessed will continue to broaden as the significance of additional biomarkers is realized and suitable biomarker probes are identified. While colorimetric sensors incorporating color shifts noticeable to the human eye are convenient, many small shifts in optical properties require equipment to appreciate.
Hand held spectrometers are increasingly becoming available to the clinical market and will allow a much broader range of colorimetric sensors to be utilized at the bedside. Additionally, miniaturization of assays using microfluidic devices will allow multiplexed detection of a wider range of biomarkers in single assays, possibly in out-of-lab settings using samples collected in a minimally invasive manner.
Ultimately, colorimetric sensors could be utilized in point-of-care diagnostics or even by patients themselves, possibly with results interpreted with the aid of smartphone apps via the Internet of Things.
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