A clinical role for Förster resonance energy transfer in molecular diagnostics of disease

KEYWORDS:

• Clinical diagnostics
• in-vitro diagnostics
• immunoassays
• FRET
• imaging
• biosensors

1. Why choosing FRET for molecular diagnostics?

The large popularity of FRET-based applications started in the early 90s, driven by significant advances in new fluorophores, detection methods, and instrumentation [1]. Implementation of FRET techniques into molecular diagnostics has started synchronously, with a continuous increase over the last 30 years. Owing to its strong distance dependence in the biological interaction range (circa 1 to 20 nm), FRET can quantify almost any target of interest (proteins, nucleic acids, metabolites, drugs, toxins, human cells, microbes, and other pathogens) from various types of clinical specimen (body fluids, cells, tissues) [2,3]. FRET can also monitor molecular dynamics in biophysics and molecular biology, such as DNA-DNA, protein–protein, or protein–DNA interactions, and protein conformational changes [4]. FRET-based biosensors have been utilized to monitor cellular dynamics not only in heterogeneous cellular populations, but also at the single-cell level in real time.

Examples of FRET application in clinical diagnostics include:

1. Simple and rapid molecular diagnostics via homogeneous immunoassays;

2. High throughput molecular diagnostics via microarrays;

3. Highly sensitive molecular diagnostics via real-time PCR;

4. Molecular dynamic diagnostics via live cell imaging.

2. Methodologies

FRET has been widely integrated in various methodologies of in-vitro diagnostics (IVD) research and clinical applications. Different commercially available real-time PCR platforms and fluorescence plate readers use FRET-based kits for molecular diagnostics and a myriad of advanced FRET techniques have been developed for further improving clinical diagnostics regarding simplicity, sensitivity, specificity, and multiplexing.

2.1. Immunoassays

Immunoassays are based on antibody–antigen interactions that allow for highly specific analyte detection. Heterogeneous immunoassays, such as ELISA, are most widely used in bioanalysis and diagnostics. However, they require multiple incubation and washing steps. In contrast, FRET can perform homogeneous assays, which enable single-step, rapid, and direct detection of the assembly of antibody−antigen complexes in solution and require only minimal expertise of the end-user. In particular, time-resolved (TR) or time-gated (TG) FRET based on lanthanide-to-dye pairs provides autofluorecence-free detection [5]. Such assays have become standard techniques for diagnostics of various biomarkers for cancer, respiratory, pulmonary, cardiovascular, and infectious diseases, endocrinology and metabolic disorders, and prenatal screening and are commercially available under brand names such as KRYPTOR/TRACE (BRAHMS/ThermoFisher), HTRF (Cisbio), or LANCE (PerkinElmer). Recent trends of FRET-based immunoassay development include multiplexing, the use of nanoparticles (NPs) such as quantum dots (QDs), or the implementation of nanobodies or small artificial binding proteins [6,7].

2.2. Real-time PCR

There is an established IVD market using real-time PCR technologies. FRET probes have been the most popular choices in real-time PCR, which include hydrolysis probes (e.g., Taqman probes), hybridization probes, molecular beacons, and scorpion primers [8]. The first two platforms developed for real-time PCR were the LightCycler (Roche) and the ABI 7700 (Applied Biosystems) [9]. New and improved models have superseded these two instruments and several other manufacturers (e.g., Stratagene, Cepheid, Corbett, Eppendorf and BioRad) have introduced their own platforms. Technical improvements include multiplexing and increased throughput, and also faster thermocycling times, improved flexibility, expanded optical systems, and more user-friendly software [10]. Today, there are multiple choices of instruments available for dedicated diagnostic purposes. Real-time PCR-based analyses can identify single-nucleotide polymorphism (SNP) alleles and provide quantitative detection, such as gene copy number variation based on quantitation cycle values. Owing to technical limitations of PCR techniques, such as relatively low specificity, rather extensive guidelines to obtain reliable results, and proprietary primer sequences for commercial kits, FRET has also been applied to many other DNA amplification technologies. Isothermal amplification methods, such as rolling circle amplification (RCA) [11] and hybridization chain reaction (HCR) [12], and hairpin-mediated quadratic isothermal amplification [13], are an ideal match for FRET because they also simplify the assay procedure and can be applied for cellular imaging. SNiPer genotyping technology (Amersham Pharmacia Biotech) is a FRET-based RCA detection method. The advantage of this approach is the avoiding of errors during PCR amplification of genomic DNA [14]. These isothermal amplification systems potentially have a high capacity in SNP detection or can provide sensitive quantification of extremely similar microRNAs (miRNAs).

2.3. Amplification-free genetic analysis

The rapid development of analytical techniques with extremely low limits of detection and emerging materials with superior photophysical properties (high brightness, photostability, large Stokes shift, narrow emission peak) have resulted in technologies that do not require amplification of nucleic acids [15]. Single-pair (sp)FRET techniques (analysis of single donor-acceptor pairs instead of many donor-acceptor ensembles) have been one major approach for accomplishing higher sensitivities [16,17]. Wabuyele et al. utilized spFRET in combination with a reverse molecular beacon (Cy5-to-Cy5.5 FRET pair) to detect a point mutation in the KRAS oncogene with 600 copies (50 aM) of human genomic DNA [18]. Nanomaterials such as QDs, metal NPs, graphene oxides, and upconversion NPs (UCNPs) have also been widely explored as FRET donors or acceptors for DNA or RNA based diagnostics. Zhang et al. showed that the QD-to-AF647 spFRET strategy with a 5 fM detection limit of target DNA was capable of detecting KRAS point mutations in clinical samples from ovarian cancer patients [19]. This method was later extended to a multiplexed format to detect the gap gene of HIV-1 and env gene of HIV-2 [20]. Such technologies are continuing to be improved for better sensitivity, specificity, and higher order multiplexing.

2.4. Microarrays

Microarrays have enabled a variety of important high throughput applications and are widely adopted in gene expression profiling and SNP detection [21]. FRET-based microarrays have also been used in protease detection [22]. Lei et al. demonstrated a FRET-peptide assay to profile multiplexed matrix metalloproteinase (MMP) activities in various cell lines and clinical thyroid tissue samples of papillary thyroid carcinoma (PTC) and thyroid nodules (TN) patients [23]. Another promising application of FRET in microarrays is to create different barcodes for probing distinct binding events. Due to the innumerable available luminophores (e.g., organic dyes, NPs, lanthanide complexes) and the different properties of luminescence, namely color, lifetime, intensity, and polarization, FRET between luminophores with different emission spectra, lifetimes, and brightness placed at defined distances to each other has the potential to provide extremely high numbers of barcodes for higher-order multiplexed microarray application in diagnostics [24,25]. Theoretically, the combination of only five colors, five lifetimes, and five intensities can result in 5^5 × 5 = 5²⁵ = 298 × 10¹⁵ (~300 quadrillion) distinguishable codes, if these codes can be spatially separated. For a multiplexed assay in solution, a 125-fold (5 × 5 × 5) coding would be experimentally more realistic but already very valuable for most real world applications.

2.5. Cell imaging

Owing to its unique functional distance range, FRET can visualize diagnostically relevant molecular events in living or fixed cells that are invisible for other molecular techniques. FRET has morphologic applications in anatomic pathology, and has been a key technique to study enzyme activity using live-cell imaging [26,27]. The continuous detection of enzyme activities (proteinases, kinases, phosphatases, nucleases, telomerase, polymerase, etc.) is particularly important in medical diagnostics. Standard approaches to look at enzymes are analysis of transcript levels (i.e. mRNAs) or protein levels, however, neither of them can reflect the specific activity of the enzyme under a given condition. Activity-based probes have been designed to detect enzyme activity, but as the probe destroys the enzyme activity upon covalent attachment, time resolution of dynamic processes is limited [28]. FRET can provide dynamic and continuous detection of enzyme activity in live-cells. Fluorescent proteins (FP) and dyes remain the most powerful and popular choices for FRET pairs because of targeted expression (for FPs) and the relatively small sizes, ease of bioconjugation, and availability (for dyes). New nanomaterials with superior photophysical properties have great potential to advance FRET imaging but are still limited to research labs because further improvements (e.g., in cell delivery and localization) and validations (e.g., concerning cytotoxicity and cellular interactions) are necessary before clinical implementation [26].

3. Clinical relevance of FRET-based molecular diagnostic

FRET-based molecular diagnostic assays/platforms are key techniques in genetic diagnostics of diseases and infectious pathogens, prediction of cancer risk, disease progression and early diagnostics, and prenatal screening, to name only a few. The following section provides representative examples of FRET applications in the various fields.

3.1. Oncology

FRET can be used in all types of cancer biomarkers such as proteins, SNPs, DNAs, miRNAs, tumor cells, and protein-protein interactions. Most single biomarkers have not reached the level of cancer specificity and sensitivity required for routine clinical use, and thus, biomarker discovery is moving to panels of multiple biomarkers [6,29].

3.1.1. Oncogenes

The therascreen EGFR RGQ PCR Kit is a molecular diagnostic kit for non-small cell lung cancer by detecting exon 19 deletions, L858R, L861Q, G719X, S768I, exon 20 insertions, and the resistance mutation T790M in the EGFR gene using real-time PCR with Scorpions detection technology [30]. Another approach, based on QD-to-dye FRET, enables multiplexed detection of methylation at PYCARD, CDKN2B, and CDKN2A genes in patient sputum samples that contain low concentrations of methylated DNA [31]. Besides oncogenes, FRET has been used for diagnostics of other genetic diseases such as cystic fibrosis, thrombosis, Alzheimer’s disease, McCune-Albright syndrome, schizophrenia, rheumatoid arthritis, and Atherosclerosis.

3.1.2. MiRNAs

The discovery of many different miRNAs specific for breast, colorectal, esophageal, gastric, liver, lung, ovarian, and pancreatic cancers, have strongly driven the implementation of miRNAs as potential cancer biomarkers [32]. FRET probes have been largely explored in reverse transcription quantitative PCR (RT-qPCR) and new isothermal amplification strategies, such as RCA and HCR to perform single or multiplexed miRNA detections [11,12].

3.1.3. Protein cancer biomarkers

FRET based immunoassays can be found for various FDA-approved protein cancer biomarkers [33] such as alpha-fetoprotein (AFP, for management of testicular cancer) [34], prostate specific antigen (PSA, for prostate cancer diagnosis and monitoring) [35], estrogen receptor (ER, prognosis, response to therapy in breast cancer) [36], CA15-3 (monitoring disease response to therapy in breast cancer) and CA125 (monitoring disease progression, response to therapy in ovarian). An important focus of assay development is multiplexing. Our group demonstrated a multiplexed lung cancer immunoassay using five tumor markers, which could allow a distinction of small cell (SCLC) from non-small cell (NSCLC) lung carcinoma by FRET from Tb to multiple dyes [29]. Using FRET from Tb to different QDs, we also developed a duplexed EGFR/HER2 immunoassays, which may aid to more specific lung and breast cancer diagnostics [6].

3.1.4. Protein-protein interactions

The power of FRET is its ability to combine cellular-level localization with dynamic information about molecular interactions [37]. Weitsman et al. revealed the HER receptor dimerization patterns on breast cancer tissue using fluorescence lifetime imaging microscopy (FLIM)-FRET, and showed that these patterns may be more important than the expression levels of single receptors in cancer prognosis and prediction of response to targeted therapies [38].

3.2. Prenatal screening

Real-time PCR (mainly based on FRET chemistries) is the most popular method to perform genetic prenatal screening (analysis of fetal cells from chorionic villus sampling at 10 to 12 weeks’ gestation or amniocentesis at 15 to 18 weeks’ gestation) or for the more recent preimplantation genetic diagnosis for high-risk pregnancies. Su et al. used real-time PCR with FRET hybridization probes to analyze mutations on the HBB gene from a single blastomere by melting curve analysis and they chose the embryos without HBB mutations to transfer, which resulted in the delivery of a normal infant boy [39]. Cell-free DNA testing in prenatal diagnostics has steadily moved beyond aneuploidy, and is proving to be a pioneering technology that meets the clinical need of non-invasiveness and ease of use. Current challenges are test validation and clinical implementation. FRET immunoassays of different serum biomarkers in combination with biophysical markers and maternal history are also used in clinical practice on the KRYPTOR immunoanalyzer system [40].

3.3. Infectious disease diagnostics

The LightCycler SeptiFast test (CE-IVD marked) has multiple sets of dual FRET probes by a combination of both color and melting temperature to identify the 25 most common pathogens known to cause sepsis [9]. Rates of infections caused by methicillin resistant S. aureus (MRSA) and vancomycin resistant Enterococci (VRE) are increasing in hospitals all over the world [41]. Warren et al. described a sensitive and specific test for rapid detection (1.5 h) of MRSA directly from nasal swab specimens, which is based upon real-time PCR using a molecular beacon probe [42]. A multiplex real-time PCR assay for the simultaneous detection of vanA and vanB was performed on a Lightcycler system using FRET hybridization probes [41]. TG-FRET Procalcitonin (PCT) immunoassays are available on the KRYPTOR platform for improved accuracy of diagnosis and risk assessment in bacterial infection and sepsis and to guide antibiotic therapy-related decisions [43].

3.4. Point-of-care (POC) diagnostics

FRET approaches provide ‘mix and measure’ format (binding events and detection are done simply and directly in solution) and simple application for the end user, which are paramount characteristics for POC diagnostics. Handheld fluorometers such as Quantus™ (Promega) and Qubit® (Thermo Fisher Scientific) can be used to perform FRET measurements. Another choice of easily accessible signal detectors can be smartphones, which are ubiquitous throughout much of the world. For example, Petryayeva et al. demonstrated a homogeneous multiplexed enzyme activity assay with RGB imaging from a smartphone. A hand-held UV lamp was used as the light source and a standard smartphone camera enabled multiplexed fluorimetric assays within a single sample volume and across multiple samples in parallel [44].

4. Perspectives

FRET has already found its place in daily clinical practice and will continue to provide diagnostic benefits to medical research and applications. Owing to the versatility of FRET, this technique will continue to advance clinical diagnostics in terms of simplicity, sensitivity, specificity, multiplexing, throughput, miniaturization, and new diagnostic tests and applications. Such advances will not be limited to the methods mentioned in this editorial but will also find implementation into novel technologies, such as next-generation sequencing (NGS) or biologically or technologically amplified detection approaches. Although FRET is only one small tool in the broad technology park of diagnostic methods, it plays an important, specific, and complementary role with still many challenges to face and advances to make for improving medical healthcare.

Declaration of interest

The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Reviewers Disclosure

Peer reviewers on this manuscript have no relevant financial relationships or otherwise to disclose.

Additional information

Funding

We thank the French Agence Nationale de la Recherche (Investissements d’Avenir project ‘Labex NanoSaclay: ANR-10-LABX-0035’ and ANR projects ‘PhenX’ and ‘Neutrinos’), the Institut Universitaire de France (IUF), and the French ‘Institut National Du Cancer’ and ‘Direction générale de l’offre de soins’ (INCa and DGOS; project PRTk 16158 – Gynomir) for financial support.