Resonance Energy Transfer Biosensor Research Articles

Using FRET to Define Cdk1-Dependent Ordering of Events During Exit from Second Meiotic M-Phase in Oocytes.

Exit from M-phase requires a precise sequence of molecular events for successful completion, with errors in the process resulting in cell death or aneuploidy, a characteristic feature of cancer and the leading cause of pregnancy failure. Exit from the second meiotic division (MII) in oocytes is a unique event triggered by sperm, involving female anaphase II as well as both male and female pronuclear formation. Very little is known about how these events involving two distinct cell types are coordinated. M-phase exit is driven by inactivation of the master cell-cycle regulator, cyclin-dependent kinase 1 (Cdk1), but details of how Cdk1 orchestrates MII exit has remained sketchy due to technical challenges in studying these events. Here we detail a protocol for undertaking in-depth analysis of Cdk1 activity throughout fertilization in live mouse oocytes using a Cdk1 Fluorescence Resonance Energy Transfer (FRET) biosensor. This protocol illustrates the utility of time-lapse imaging and FRET for interrogating experimentally challenging cell-cycle events.

A Putative Frizzled 7-Targeting Compound Acts as a Firefly Luciferase Inhibitor.

The Frizzled family (FZD1-10) of G protein-coupled receptors regulates WNT signaling mediating proliferative input. Dysregulation of FZD7 and exaggerated WNT/β-catenin signaling is frequently observed in intestinal cancers. Therefore, it is attractive to develop therapeutics targeting FZD7 for cancer treatment. Structure-based virtual screening has identified compound 28, which inhibited WNT/β-catenin signaling based on the luciferase-based reporter gene TOPFlash assay. However, upon pharmacological validation, compound 28 rather acts as a potent Firefly luciferase (Fluc) inhibitor (IC50 = 30 nM), matching the reported IC50 for compound 28-mediated inhibition in the TOPFlash assay. Moreover, we employed Fluc-independent assays, a FZD7-focused bioluminescence resonance energy transfer biosensor and quantitative PCR, to emphasize the inability of compound 28 to inhibit the WNT-3A-induced conformational dynamics in FZD7 and transcription of Axin2, a WNT target gene. Thus, we underline the importance of counter screens to validate compounds that interfere with the detection technology used for compound screening.

Electrochemiluminescence resonance energy transfer biosensor based on BNCs@Zn-PTC for ultrasensitive and low-potential detection of CA125 in pancreatic cancer diagnostics

Electrochemiluminescence resonance energy transfer biosensor based on BNCs@Zn-PTC for ultrasensitive and low-potential detection of CA125 in pancreatic cancer diagnostics

Diminished nuclear-localized β-adrenoceptor signalling activates YAP to promote kidney fibrosis in diabetic nephropathy.

Diabetic nephropathy (DN) is a leading cause of chronic kidney disease (CKD), which is characterized by mesangial matrix expansion that involves dysfunctional mesangial cells (MCs). However, the underlying mechanisms remain unclear. This study aims to delineate the spatiotemporal contribution of adrenergic signalling in diabetic kidney fibrosis to reveal potential therapeutic targets. A model of diabetic nephropathy was induced by in db/db mice. Gene expression in kidneys was profiled by RNA-seq analyses, western blot and immunostaining. Subcellular-localized fluorescence resonance energy transfer (FRET) biosensors determined adrenergic signalling microdomains in MCs. Effects of oral rolipram, a phosphodiesterase 4 (PDE4) inhibitor, on the model were measured. Our model exhibited impaired kidney function with elevated expression of adrenergic and fibrotic genes, including Adrb1, PDEs, Acta2 and Tgfβ. RNA-seq analysis revealed that MCs with dysregulated YAP pathway were crucial to the extracellular matrix secretion in kidneys from diabetic nephropathy patients. In cultured MCs, TGF-β promoted profibrotic gene transcription, which was regulated by nuclear-localized β-adrenoceptor signalling. Mechanistically, TGF-β treatment diminished nuclear-specific cAMP signalling in MCs and reduced PKA-dependent phosphorylation of YAP, leading to its activation. In parallel, db/db mouse kidneys showed increased expressions of PDE4B and PDE4D. Treatment with oral rolipram alleviated kidney fibrosis in db/db mice. Diabetic nephropathy impaired nuclear-localized β1-adrenoceptor-cAMP signalling microdomain through upregulating PDE4 expression, promoting fibrosis in MCs via PKA dephosphorylation-dependent YAP activation. Our results suggest PDE4 inhibition as a promising strategy for alleviating kidney fibrosis in diabetic nephropathy.

Development of mKate3/HaloTag7 (JFX650) and CFP/YFP Dual-Fluorescence (or Förster) Resonance Energy Transfer Pairs for Visualizing Dual-Molecular Activity.

Although several imaging strategies for dual fluorescence (or Förster) resonance energy transfer (FRET) biosensors have been reported, their implementation is challenging because of the limited performance of fluorescent proteins and the spectral overlap of FRET biosensors. These processes often require additional data calibration to eliminate artifacts. Many CFP/YFP FRET biosensors have been developed. In this study, we introduced the mKate3/HT7(JFX650) FRET pair, which effectively formed two pairs of FRET pairs for dual-FRET imaging when combined with the CFP/YFP FRET pair. The FRET donor mKate3 exhibited higher brightness than its predecessor mKate. The FRET acceptor, HT7(JFX650), is a HaloTag7 protein covalently conjugated with a far-red JFX650-THL ligand. The pair comprising mKate3 and HT7(JFX650) represents an excellent FRET dyad, exhibiting a high FRET efficiency ratio. To use the FRET pair for dual FRET biosensor imaging, we constructed PKA and K+ biosensors based on the mKate3/HT7(JFX650) FRET pair. These biosensors can be used along with CFP/YFP biosensors to simultaneously detect the responses of intracellular PKA/Src, PKA/Ca2+, and K+/Ca2+ under different stimuli. The findings revealed that dual FRET biosensors, which are based on the combination of CFP/YFP and mKate3/HT7 (JFX650), exhibit adequate compatibility and can be used to visualize multiple molecular activities in a live cell.

GIBBERELLIN PERCEPTION SENSOR 2 reveals genesis and role of cellular GA dynamics in light-regulated hypocotyl growth.

The phytohormone gibberellic acid (GA) is critical for environmentally sensitive plant development including germination, skotomorphogenesis, and flowering. The Förster resonance energy transfer biosensor GIBBERELLIN PERCEPTION SENSOR1, which permits single-cell GA measurements in vivo, has been used to observe a GA gradient correlated with cell length in dark-grown, but not light-grown, hypocotyls. We sought to understand how light signaling integrates into cellular GA regulation. Here, we show how the E3 ligase CONSTITUTIVE PHOTOMORPHOGENESIS1 (COP1) and transcription factor ELONGATED HYPOCOTYL 5 (HY5) play central roles in directing cellular GA distribution in skoto- and photomorphogenic hypocotyls, respectively. We demonstrate that the expression pattern of the GA biosynthetic enzyme gene GA20ox1 is the key determinant of the GA gradient in dark-grown hypocotyls and is a target of COP1 signaling. We engineered a second generation GPS2 biosensor with improved orthogonality and reversibility. GPS2 revealed a previously undetectable cellular pattern of GA depletion during the transition to growth in the light. This GA depletion partly explains the resetting of hypocotyl growth dynamics during photomorphogenesis. Achieving cell-level resolution has revealed how GA distributions link environmental conditions with morphology and morphological plasticity. The GPS2 biosensor is an ideal tool for GA studies in many conditions, organs, and plant species.

A Reliable System for Quantitative G-Protein Activation Imaging in Cancer Cells.

Fluorescence resonance energy transfer (FRET) biosensors have proven to be an indispensable tool in cell biology and, more specifically, in the study of G-protein signalling. The best method of measuring the activation status or FRET state of a biosensor is often fluorescence lifetime imaging microscopy (FLIM), as it does away with many disadvantages inherent to fluorescence intensity-based methods and is easily quantitated. Despite the significant potential, there is a lack of reliable FLIM-FRET biosensors, and the data processing and analysis workflows reported previously face reproducibility challenges. Here, we established a system in live primary mouse pancreatic ductal adenocarcinoma cells, where we can detect the activation of an mNeonGreen-Gαi3-mCherry-Gγ2 biosensor through the lysophosphatidic acid receptor (LPAR) with 2-photon time-correlated single-photon counting (TCSPC) FLIM. This combination gave a superior signal to the commonly used mTurquoise2-mVenus G-protein biosensor. This system has potential as a platform for drug screening, or to answer basic cell biology questions in the field of G-protein signalling.

An intra-stimuli resonance energy transfer-based electrochemiluminescence biosensor for DNA methylation detection

Aberrant DNA methylation, as one of the promising biomarkers, is intimately connected to a variety of genetic and tumor diseases. Herein, we constructed an innovative intra-stimuli electrochemiluminescence resonance energy transfer (ECL-RET) biosensor for methylated DNA detection with high sensitivity and accuracy. Glucose oxidase (GOD) was modified at the terminal of assist DNA as functional conjugation (assist DNA-GOD), which then hybridized with methylated target DNA and hairpin DNA to form Y-shaped DNA ternary conjugate. Ru(bpy)32+ encapsulated silica nanospheres (RuSiO2 NPs) were uniformly coated with silver nanoparticles (Ag NPs) and the formed RuSiO2@Ag nanocomposites were introduced to the terminal of hairpin DNA via amide bond. The combination of the ECL donor (RuSiO2 NPs) and acceptor (Ag NPs) on one nanostructure yielded an intra-molecular ECL-RET. Afterward, the presence of H2O2 as the product of GOD catalyzing glucose induced the etching of Ag NPs and subsequent recovery of ECL signal from RuSiO2 NPs. Thus, due to the dependence of the ECL enhancement on the target concentration, this method achieved the quantitative analysis of methylated target DNA with good selectivity, repeatability, and practical utility.

Identification of Riluzole derivatives as novel calmodulin inhibitors with neuroprotective activity by a joint synthesis, biosensor, and computational guided strategy

The development of new molecules for the treatment of calmodulin related cardiovascular or neurodegenerative diseases is an interesting goal. In this work, we introduce a novel strategy with four main steps: (1) chemical synthesis of target molecules, (2) Förster Resonance Energy Transfer (FRET) biosensor development and in vitro biological assay of new derivatives, (3) Cheminformatics models development and in vivo activity prediction, and (4) Docking studies. This strategy is illustrated with a case study. Firstly, a series of 4-substituted Riluzole derivatives 1–3 were synthetized through a strategy that involves the construction of the 4-bromoriluzole framework and its further functionalization via palladium catalysis or organolithium chemistry. Next, a FRET biosensor for monitoring Ca2+-dependent CaM-ligands interactions has been developed and used for the in vitro assay of Riluzole derivatives. In particular, the best inhibition (80%) was observed for 4-methoxyphenylriluzole 2b. Besides, we trained and validated a new Networks Invariant, Information Fusion, Perturbation Theory, and Machine Learning (NIFPTML) model for predicting probability profiles of in vivo biological activity parameters in different regions of the brain. Next, we used this model to predict the in vivo activity of the compounds experimentally studied in vitro. Last, docking study conducted on Riluzole and its derivatives has provided valuable insights into their binding conformations with the target protein, involving calmodulin and the SK4 channel. This new combined strategy may be useful to reduce assay costs (animals, materials, time, and human resources) in the drug discovery process of calmodulin inhibitors.

Coreactant-free aggregation-induced electrochemiluminescence system based on the novel zinc-luminol metal-organic gel for ultrasensitive detection of PiRNA-823

Aggregation-induced electrochemiluminescence (AIECL) technology has aroused widespread interest due to the significant improve in ECL response by solving the problems of aggregation-caused quenching and poor water solubility of the luminophore. However, the existing AIECL emitters still suffer from low ECL efficiency, additional coreactants and complex synthesis steps, which greatly limit their applications. Herein, luminol, as a kind of AIE molecule, was assembled with Zn2+ nodes to obtain a novel microflower-like Zinc-luminol metal-organic gel (Zn-MOG) by one-step method. In the light of the strong affinity of N atoms in luminol ligand to Zn2+, Zn-MOG with vigorous viscosity and stability can be formed immediately after vortex oscillation, overcoming the main difficulties of the complicated synthesis steps and poor film-forming performance encountered in current AIECL materials. Impressively, an AIECL resonance energy transfer (RET) biosensor was constructed using Zn-MOG as a donor and Alexa Fluor 430 as an acceptor in combination with DNA-Fuel-driven target recycling amplification for the ultrasensitive detection of PiRNA-823. The fabricated biosensor exhibited a wide linear relationship in the range of 100 aM to 100 pM and a detection limit as low as 60.0 aM. This work is the first to realize the construction of ECL emitters using the AIE effect of luminol, which provides inspiration for the design of AIECL systems without adding coreactants.

Unravelling molecular dynamics in living cells: Fluorescent protein biosensors for cell biology.

Genetically encoded, fluorescent protein (FP)-based Förster resonance energy transfer (FRET) biosensors are microscopy imaging tools tailored for the precise monitoring and detection of molecular dynamics within subcellular microenvironments. They are characterised by their ability to provide an outstanding combination of spatial and temporal resolutions in live-cell microscopy. In this review, we begin by tracing back on the historical development of genetically encoded FP labelling for detection in live cells, which lead us to the development of early biosensors and finally to the engineering of single-chain FRET-based biosensors that have become the state-of-the-art today. Ultimately, this review delves into the fundamental principles of FRET and the design strategies underpinning FRET-based biosensors, discusses their diverse applications and addresses the distinct challenges associated with their implementation. We place particular emphasis on single-chain FRET biosensors for the Rho family of guanosine triphosphate hydrolases (GTPases), pointing to their historical role in driving our understanding of the molecular dynamics of this important class of signalling proteins and revealing the intricate relationships and regulatory mechanisms that comprise Rho GTPase biology in living cells.

Highly sensitive and specific graphene oxide-based FRET aptasensor for quantitative detection of human soluble growth stimulating gene protein 2

Soluble growth stimulation expressed gene 2 (sST2) is a new generation biomarker in the diagnosis and prognosis of heart failure (HF). Here, the sST2-specific aptamers were selected from a random ssDNA library with the full length of 88 nucleotides (nt) via target-immobilized magnetic beads (MB)-based systematic evolution of ligands by exponential enrichment (SELEX) technology. After eight rounds of selection, six aptamers with the most enrichment were selected. Among, the aptamer L1 showed the high-affinity binding to sST2 with the lowest Kd value (77.3 ± 0.05 nM), which was chosen as the optimal aptamer for further molecular docking. Then, the aptamer L1 was used to construct a graphene oxide (GO) - based fluorescence resonance energy transfer (FRET) biosensor for sST2, which exhibits a linear detection range of 0.1–100 μg/ml and a detection limit of 3.7 ng/ml. The aptasensor was applied to detect sST2 in real samples, with a good correlation and agreement with the traditional enzyme-linked immunosorbent assay (ELISA) when quantitative analyzing the sST2 concentration in serum samples from HF patients. The results show that not only an efficient strategy for screening the practicable aptamer, but also a rapid and sensitive detection platform for sST2 were established.

Genetically Encoded Fluorescence Resonance Energy Transfer Biosensor for Live-Cell Visualization of Lamin A Phosphorylation at Serine 22.

Extensive phosphorylation at serine 22 (pSer22) on lamin A is the hallmark of cell mitosis, which contributes to the breakdown of nuclear envelope. In the interphase, pSer22 lamin A exists in low abundance and is involved in mechanotransduction, virus infection, and gene expression. Numerous evidences emerge to support lamin A regulation on cell function and fate by phosphorylation. However, live-cell imaging tools for visualizing the dynamics of pSer22 lamin A are yet to be established. Herein, we developed a novel lamin A phosphorylation sensor (LAPS) based on fluorescence resonance energy transfer (FRET) with high sensitivity and specificity. We observed the dynamic lamin A phosphorylation during the cell cycle progression in single living cells: the increase of pSer22 modification when cells entered the mitosis and recovered upon the mitosis exit. Our biosensor also showed the gradual reduction of pSer22 modification during cell adhesion and in response to hypotonic environment. By applying LAPS, we captured the propagation of pSer22 modification from inside to outside of the inner nuclear membrane, which further led to the breakdown of nuclear envelope. Meanwhile, we found the synchronous phosphorylation of pSer22 lamin A and H3S10ph at mitosis entry. Inhibition of Aurora B, the responsible kinase for H3S10ph, could shorten the mitotic period without obvious effect on the pSer22 modification level of lamin A. Thus, LAPS allows the spatiotemporal visualization of the lamin A pSer22, which will be useful for elucidating the molecular mechanisms underlying cell mitosis and mechanoresponsive processes.

Magnetic-responsive upconversion luminescence resonance energy transfer (LRET) biosensor for ultrasensitive detection of SARS-CoV-2 spike protein

Upconversion nanoparticles (UCNPs) are ideal donors for luminescence resonance energy transfer (LRET)-based biosensors due to their excellent upconversion luminescence properties. However, the relatively large size of antibodies and proteins limits the application of UCNPs-based LRET biosensors in protein detection because the large steric hindrance of proteins leads to low energy transfer efficiency between UCNPs and receptors. Herein, we developed a magnetic responsive UCNPs-based LRET biosensor to control the coupling distance between antibody-functionalized UCNPs (Ab-UCNPs) as donors and antibody-PEG linker-magnetic gold nanoparticles (Ab-PEG-MGNs) as acceptors for ultrasensitive and highly selective detection of SARS-CoV-2 spike proteins. Our results showed that this platform reversibly shortened the coupling distance between UCNPs and MGNs and enhanced the LRET signal with a 10-fold increase in the limit of detection (LOD) from 20.6 pg/mL without magnetic modulation to 2.1 pg/mL with magnetic modulation within 1 h. The finite-difference time-domain (FDTD) simulation with cyclic distance change confirmed the distance-dependent LRET efficiency under magnetic modulation, which supported the experimental results. Moreover, the applications of this magnetic-responsive UCNP-based LRET biosensor could be extended to other large-size biomolecule detection.

Remodelling of cAMP dynamics within the SERCA2a microdomain in heart failure with preserved ejection fraction caused by obesity and type 2 diabetes.

Despite massive efforts, we remain far behind in our attempts to identify effective therapies to treat heart failure with preserved ejection fraction (HFpEF). Diastolic function is critically regulated by sarcoplasmic/endoplasmic reticulum (SR) calcium ATPase 2a (SERCA2a), which forms a functional cardiomyocyte (CM) microdomain where 3',5'-cyclic adenosine monophosphate (cAMP) produced upon β-adrenergic receptor (β-AR) stimulation leads to phospholamban (PLN) phosphorylation and facilitated Ca2+ re-uptake. To visualize real-time cAMP dynamics in the direct vicinity of SERCA2a in healthy and diseased myocytes, we generated a novel mouse model on the leprdb background that stably expresses the Epac1-PLN Förster resonance energy transfer biosensor. Mice homozygous for the leprdb mutation (db/db) developed obesity and type 2 diabetes and presented with a HFpEF phenotype, evident by mild left ventricular hypertrophy and elevated left atria filling pressures. Live cell imaging uncovered a substantial β2-AR subtype stimulated cAMP response within the PLN/SERCA2a microdomain of db/db but not healthy control (db/+) CMs, which was accompanied by increased PLN phosphorylation and accelerated calcium re-uptake. Importantly, db/db CMs also exhibited a desensitization of β1-AR stimulated cAMP pools within the PLN/SERCA2a microdomain, which was accompanied by a blunted lusitropic effect, suggesting that the increased β2-AR control is an intrinsic compensatory mechanism to maintain PLN/SERCA2a-mediated calcium dynamics and cardiac relaxation. Mechanistically, this was due to a local loss of cAMP-degrading phosphodiesterase 4 associated specifically with the PLN/SERCA2a complex. These newly identified alterations of cAMP dynamics at the subcellular level in HFpEF should provide mechanistic understanding of microdomain remodelling and pave the way towards new therapies.

CRISPR/Cas12a controlled chemiluminescence resonance energy transfer sensor for detection of carcinoembryonic antigen

A CRISPR-Cas12a controlled chemiluminescence resonance energy transfer (CRET) biosensor was constructed for the detection of cancer biomarker carcinoembryonic antigen (CEA). The CRET probe, a single-strand DNA (ssDNA) with hemin/G-quadruplex on one end and CdSe/ZnS quantum dots (QDs) nanosphere on the other end, was used as the substrate for the trans-cleavage of CRISPR/Cas12a system. The hemin/G-quadruplexes catalyzed the oxidation of luminol in the presence of H2O2. The generating chemiluminescence could excite the CdSe/ZnS QDs encapsulated in the nanosphere, producing CRET. When target CEA specifically identified dual aptamers on the surface of magnetic beads (MBs), the terminal sequence of Aptamer 2 triggered the hybridization chain reaction (HCR), whose product initiated the trans-cleavage activity of CRISPR/Cas12a and destroyed the CRET system. As a result, the luminescence of QDs nanosphere was declined. With HCR, CRISPR-Cas12a trans-cleavage, and mimic DNAzyme catalytic amplification, the biosensor gave a detection limit down to 5.6 pg/mL. Due to the application of CRISPR/Cas12a, the CRET sensor with the characteristics of easy operation and no external light source required was more suitable for point-of-care testing (POCT) detection of various targets.

Development of a high-throughput fluorescence resonance energy transfer assay for biotin in food

A new fluorescence resonance energy transfer (FRET) biosensor to evaluate the presence of biotin has been developed and optimized. The biosensor is based on the direct evaluation of BirA enzyme from Escherichia coli homodimerization as a result of biotin binding. BirA monomer was genetically fused either to fluorescent protein mTurquoise2 (mTur) or to cp173Venus (Venus). In the presence of biotin, BirA dimerization brings mTur and Venus molecules close enough for an energy transfer. The analytical performances of the biosensor in the presence biotin using the purified fusion proteins which expressed in Escherichia coli have been evaluated and optimized. The biotin assay can be performed in 96-well microplate format with a 10 min incubation. The FRET signal increased with the biotin concentration in the range of 0.4 nmol/L - 4 nmol/L, and the linear equation was Y = 0.235 + 0.333X (R2 = 0.991), which was slightly affected by other water-soluble vitamins and trace metal elements. The FRET biosensor was used to detect biotin in infant formula samples, and the results were in good agreement with those obtained from the microbial method, indicating that the developed FRET biosensor was applicable to the biotin detection in food.

Plasmon‐enhanced FRET biosensor based on Tm3+/Er3+ co‐doped core‐shell upconversion nanoparticles for ultrasensitive virus detection

AbstractOutbreaks of infectious viruses offer a formidable challenge to public healthcare systems and early detection of viruses is essential for preventing virus propagation. In this work, an ultrasensitive plasmon‐enhanced fluorescence resonance energy transfer (FRET) biosensor based on core‐shell upconversion nanoparticle (csUCNP) and gold nanoparticle (AuNP) for accurate detection of SARS‐CoV‐2 viral RNA is presented. In this biodetection assay, the Tm3+/Er3+ co‐doped csUCNP NaGdF4:Yb/Tm@NaYF4:Yb/Er acts as an energy donor and AuNP serves as an energy acceptor. The upconversion emission of Tm3+ and the design of the core‐shell structure led to a simultaneous surface plasmon effect of AuNP. The localized surface plasmon resonance (LSPR) arising from collective oscillations of free electrons significantly enhanced FRET efficiency between Er3+ and AuNP. The as‐prepared biosensor obtained a limit of detection (LOD) as low as 750 am, indicating that the integration of FRET and surface plasmon into one biodetection assay significantly boosted the sensitivity of the biosensor. In addition, samples extracted from clinical samples are also utilized to validate the effectiveness of the biosensor. Therefore, this innovative plasmon‐enhanced FRET biosensor based on Tm3+/Er3+ co‐doped csUCNP may pave the way for rapid and accurate biodetection applications.

An electrochemiluminescence resonance energy transfer biosensor based on CDs/PAMAM/rGO nanocomposites and Au@Ag2S nanoparticles for PML/RARα fusion gene detection.

In recent years, electrochemiluminescence resonance energy transfer (ECL-RET) with low background signal and high specificity has attracted much attention among researchers. Herein, we established a novel ECL-RET biosensor for PML/RARα fusion gene detection. In this ECL-RET system, carbon dots (CDs) with low toxicity and prominent electrochemical activity were used as donor and Au@Ag2S core-shell nanoparticles (Au@Ag2S NPs) were employed as ECL acceptor. The Au@Ag2S NPs possessed a wide ultraviolet-visible (UV-vis) absorption spectrum between 500 nm and 700 nm, which completely overlapped with the ECL spectrum of CDs. Furthermore, the CDs-decorated poly-amidoamine/reduced graphene oxide (CDs/PAMAM/rGO) nanocomposites were prepared to improve the ECL signals and served as a substrate to stably load capture probe deoxyribonucleic acid (DNA). Based on the ECL-RET biosensing strategy, the Au@Ag2S NPs-labeled assistant probes and target DNA could pair with capture probes to form the sandwich-type DNA structure and the distance between donor and accepter was closed, leading to quenching of theECL signal of CDs. The ECL-RET biosensor represented eminent analytical performance for PML/RARα fusion gene detection with a wide linear relationship from 5 fM to 500 pM and a low detection limit of 0.72 fM, which provided a novel technical means and theoretical basis for detection and diagnosis of acute promyelocytic leukemia.

Development of a N6-methyladenosine-directed single quantum dot-based biosensor for sensitive detection of METTL3/14 complex activity in breast cancer tissues

The METTL3/14 complex is an important RNA N6-Methyladenosine (m6A) methyltransferase in organisms, and the abnormal METTL3/14 complex activity is associated with the pathogenesis and various cancers. Sensitive detection of METTL3/14 complex is essential to tumor pathogenesis study, cancer diagnosis, and anti-cancer drug discovery. However, traditional methods for METTL3/14 complex assay suffer from poor specificity, costly antibodies, unstable RNA substrates, and low sensitivity. Herein, we construct a single quantum dot (QD)-based förster resonance energy transfer (FRET) biosensor for sensitive detection of METTL3/14 complex activity. In the presence of METTL3/14 complex, it catalyzes the methylation of adenine in the substrate probe, leading to the formation of m6A that protects the substrate probes from MazF-mediated cleavage. The hybridization of methylated DNA substrate with biotinylated capture probe initiates polymerization reaction to obtain a biotinylated double-stranded DNA (dsDNA) with the incorporation of numerous Cy5 fluorophores. Subsequently, the Cy5-incorporated dsDNA can self-assembly onto the 605QD surface to form the 605QD-dsDNA-Cy5 nanostructure, causing FRET between 605QD donor and Cy5 acceptor. This biosensor has excellent sensitivity with a limit of detection (LOD) of 3.11 × 10−17 M, and it can measure the METTL3/14 complex activity in a single cell. Moreover, this biosensor can be used to evaluate the METTL3/14 complex kinetic parameters and screen potential inhibitors. Furthermore, it can differentiate the METTL3/14 complex expression in healthy human tissues and breast cancer patient tissues, providing a powerful tool for cancer pathogenesis study, clinical diagnosis, prognosis monitoring, and drug discovery.