FRET-Based Biosensors in Diagnosing Biomedical Pathologies
Fluorescence resonance energy transfer (FRET), also referred to as Förster resonance energy transfer, is a physical phenomenon whereby energy from a donor fluorophore is transferred to an acceptor fluorophore. This form of resonance energy transfer is strongly dependent on close localisation of the donor and acceptor fluorophore, wherein a typical distance range for FRET to occur efficiently is 1-10nm. Given this unique distance feature, FRET is often labelled as a ‘molecular ruler’.
In a biological setting, conjugation of a FRET donor and FRET acceptor to distinct domains of a protein or to two interacting partners allows for both conformational changes and protein-protein interaction dynamics to be accurately assessed. FRET has the added feature that it can be used at the single molecule level, providing unique insight into protein function compared to conventional assays that rely on analysing a protein ensemble.
Beyond this more structural/molecular approach, however, FRET has shown significant promise in biosensor applications, where its high sensitivity, specificity and rapid nature of response have rendered it a novel strategy in point-of-care and therapeutic testing. In many cases, this involves the inclusion of FRET pairs in widely employed techniques such as enzyme-linked immunosorbent assays (ELISAs) as well as in biomarker technologies in early recognition of cancer.
FRET and Cancer Biomarkers
A biomarker commonly refers to any naturally occurring biological molecule or gene that is associated with a particular disease pathology and can be readily detected. Techniques that are both highly sensitive and rapid are critical in forming an early diagnosis in an oncology background.
MicroRNAs (miRNAs) are one particular biomarker often associated with cancer diagnosis. miRNAs are small, and critically, non-coding RNA molecules that are typically involved in the regulation of gene expression. Fluorescently labelling miRNAs with the relevant probes has enabled detection of miRNAs in screening for hepatocellular carcinoma. This relies on implementing FRET technology in a current diagnostic tool referred to as liquid biopsy using circulating microvesicles and exosomes, wherein the miRNAs reside within the microvesicles collected. The ease of liquid biopsy collection makes this a formidable technique in screening procedures.
Implementation of FRET technology within biomarker sensors has also been successfully used to detect overexpression of a specific protein kinase in epithelial cancer. Protein kinases are capable of regulating the activity of their target proteins by reversibly modifying them with a specific type of molecular ‘tag’. Many of these kinases function in the regulatory control of proteins involved in the life cycle of the cell, often referred to as the cell cycle. Dysregulation of the cell cycle is one of the hallmarks of cancer, meaning early detection of oncoproteins, that is proteins contributing to tumorigenic growth, provides the best possible chance of a positive prognosis.
FRET and Immunoassays
Put simply, an immunoassay is a type of test capable of detecting a specific biological molecule in a sample using an antibody-antigen interaction. Typically, ELISAs form the current gold standard for immunoassay-based detection. FRET-based antibody applications offer great potential given the ease of conjugation of functionalised groups, such as the probes required for an efficient FRET response, to the antibody scaffold.
Heart muscle cells, known as cardiac myocytes, consist of a bundled network of myofibrils that themselves are made up of multiple myofilaments. Within these myofilaments is a repeating unit called a sarcomere comprised of thick myosin filaments and thin actin filaments. Interactions between actin and myosin allow the sarcomere length to shorten and, ultimately therefore, allow the cardiac myocyte to contract and co-ordinate the pumping action of the heart.
Cardiac troponin I is a protein responsible for mediating the interaction between calcium and the actin and myosin in cardiac myocytes. Elevated cardiac troponin I levels are known to be indicative of cardiac injury and potential acute myocardial infarction. Antibodies against cardiac troponin I have been used in modified FRET-based ELISA set ups to detect raised levels of the protein, paving the way toward early diagnosis of at-risk individuals.
The examples detailed provide only a glimpse of the detection capabilities of this exciting technology. With the highly sensitive, specific and rapid nature of FRET-based biosensors, it seems highly likely we will see frequent diagnostic tests and new patent filings emerging from this exciting field in the future.
J A Kemp has extensive expertise in protecting innovations at the forefront of biomedical testing, including in Antibodies and Biologics, Biotechnology and Life Sciences, Diagnostics and Personalised Medicine and Medical Devices Technology.