Two-Photon FRET/FLIM Imaging of Cerebral Neurons.

Two-photon FRET (Förster resonance energy transfer) and FLIM (fluorescence lifetime imaging microscopy) enable the detection of FRET changes of fluorescence reporters in deep brain tissues, which provide a valuable approach for monitoring target molecular dynamics and functions. Here, we describe two-photon FRET and FLIM imaging techniques that allow us to visualize endogenous and optogenetically induced cAMP dynamics in living neurons with genetically engineered FRET-based cAMP reporters.

Molecular Weights of Polyethyleneimine-Dependent Physicochemical Tuning of Gold Nanoparticles and FRET-Based Turn-On Sensing of Polymyxin B.

Environmental monitoring and the detection of antibiotic contaminants require expensive and time-consuming techniques. To overcome these challenges, gold nanoparticle-mediated fluorometric "turn-on" detection of Polymyxin B (PMB) in an aqueous medium was undertaken. The molecular weight of polyethyleneimine (PEI)-dependent physicochemical tuning of gold nanoparticles (PEI@AuNPs) was achieved and employed for the same. The three variable molecular weights of branched polyethyleneimine (MW 750, 60, and 1.3 kDa) molecules controlled the nano-geometry of the gold nanoparticles along with enhanced stabilization at room temperature. The synthesized gold nanoparticles were characterized through various advanced techniques. The results revealed that polyethyleneimine-stabilized gold nanoparticles (PEI@AuNP-1-3) were 4.5, 7.0, and 52.5 nm in size with spherical shapes, and the zeta potential values were 29.9, 22.5, and 16.6 mV, respectively. Accordingly, the PEI@AuNPs probes demonstrated high sensitivity and selectivity, with a linear relationship curve over a concentration range of 1-6 µM for polymyxin B. The limit of detection (LOD) was calculated as 8.5 nM. This is the first unique report of gold nanoparticle nano-geometry-dependent FRET-based turn-on detection of PMB in an aqueous medium. We believe that this approach would offer a complementary strategy for the development of a highly sophisticated and advanced sensing system for PMB and act as a template for the development of new nanomaterial-based engineered sensors for rapid antibiotic detection in environmental as well as biological samples.

FLIM-FRET Protein-Protein Interaction Assay.

Fluorescence lifetime imaging performed under FRET conditions between two interacting molecules is a sensitive and robust way to quantify intermolecular interactions in cells. The fluorescence lifetime, an inherent property of the fluorophore, remains unaffected by factors such as concentration, laser intensity, and other photophysical artifacts. In the context of FLIM-FRET, the focus lies on measuring the fluorescence lifetime of the donor molecule, which diminishes upon interaction with a neighboring acceptor molecule. In this study, we present a step-by-step experimental protocol for applying FLIM-FRET to investigate protein-protein interactions involving various RAS isoforms and RAS effectors at the live cell's plasma membrane. By utilizing the FRET pair comprising enhanced green fluorescent protein (eGFP) and fluorescent mCherry, we demonstrate that the proximity and possible nanoclustering of eGFP-tagged KRAS4b G12D and mCherry-tagged KRAS4b WT led to a reduction in the donor eGFP's fluorescence lifetime. The donor lifetime of eGFP-tagged KRAS decreases even further when treated with a dimer-inducing small molecule, or in the presence of RAF proteins, suggesting a greater FRET efficiency, and thus less distance, between donor and acceptor.

KRAS4b:RAF-1 Homogenous Time-Resolved Fluorescence Resonance Energy Transfer Assay for Drug Discovery.

Homogenous time-resolved FRET (HTRF) assays have become one of the most popular tools for pharmaceutical drug screening efforts over the last two decades. Large Stokes shifts and long fluorescent lifetimes of lanthanide chelates lead to robust signal to noise, as well as decreased false positive rates compared to traditional assay techniques. In this chapter, we describe an HTRF protein-protein interaction (PPI) assay for the KRAS4b G-domain in the GppNHp-bound state and the RAF-1-RBD currently used for drug screens. Application of this assay contributes to the identification of lead compounds targeting the GTP-bound active state of K-RAS.

A molecular dynamics simulation study of glycine/serine octapeptides labeled with 2,3-diazabicyclo[2.2.2]oct-2-ene fluorophore.

The compound 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) is a versatile fluorophore widely used in Förster resonance energy transfer (FRET) spectroscopy studies due to its remarkable sensitivity, enabling precise donor-acceptor distance measurements, even for short peptides. Integrating time-resolved and FRET spectroscopies with molecular dynamics simulations provides a robust approach to unravel the structure and dynamics of biopolymers in a solution. This study investigates the structural behavior of three octapeptide variants: Trp-(Gly-Ser)3-Dbo, Trp-(GlyGly)3-Dbo, and Trp-(SerSer)3-Dbo, where Dbo represents the DBO-containing modified aspartic acid, using molecular dynamics simulations. Glycine- and serine-rich amino acid fragments, common in flexible protein regions, play essential roles in functional properties. Results show excellent agreement between end-to-end distances, orientational factors from simulations, and the available experimental and theoretical data, validating the reliability of the GROMOS force field model. The end-to-end distribution, modeled using three Gaussian distributions, reveals a complex shape, confirmed by cluster analysis highlighting a limited number of significant conformations dominating the peptide landscape. All peptides predominantly adopt a disordered state in the solvent, yet exhibit a compact shape, aligning with the model of disordered polypeptide chains in poor solvents. Conformations show marginal dependence on chain composition, with Ser-only chains exhibiting slightly more elongation. This study enhances our understanding of peptide behavior, providing valuable insights into their structural dynamics in solution.

Monitoring Real-Time Temperature Dynamics of a Short RNA Hairpin Using Förster Resonance Energy Transfer and Circular Dichroism.

RNA molecules play crucial roles in gene expression regulation and cellular signaling, and these functions are governed by the formation of RNA secondary and tertiary structures. These structures are highly dynamic and subject to rapid changes in response to environmental cues, temperature in particular. Thermosensitive RNA secondary structures have been harnessed by multiple organisms to survey their temperature environment and to adjust gene expression accordingly. It is thus highly desirable to observe RNA structural changes in real time over a range of temperatures. Multiple approaches have been developed to study structural dynamics, but many of these require extensive processing of the RNA, large amounts of RNA input, and/or cannot be applied under physiological conditions. Here, we describe the use of a dually fluorescently labeled RNA oligonucleotide (containing a predicted hairpin structure) to monitor subtle RNA structural dynamics in vitro by Förster resonance energy transfer (FRET) and circular dichroism (CD) spectroscopy. These approaches can be employed under physiologically relevant conditions over a range of temperatures and with RNA concentrations as low as 200 nM; they enable us to observe RNA structural dynamics in real time and to correlate these dynamics with changes in biological processes such as translation.

Assessment of the FRET-based Teen sensor to monitor ERK activation changes preceding morphological defects in a RASopathy zebrafish model and phenotypic rescue by MEK inhibitor.

BACKGROUND: RASopathies are genetic syndromes affecting development and having variable cancer predisposition. These disorders are clinically related and are caused by germline mutations affecting key players and regulators of the RAS-MAPK signaling pathway generally leading to an upregulated ERK activity. Gain-of-function (GOF) mutations in PTPN11, encoding SHP2, a cytosolic protein tyrosine phosphatase positively controlling RAS function, underlie approximately 50% of Noonan syndromes (NS), the most common RASopathy. A different class of these activating mutations occurs as somatic events in childhood leukemias. METHOD: Here, we evaluated the application of a FRET-based zebrafish ERK reporter, Teen, and used quantitative FRET protocols to monitor non-physiological RASopathy-associated changes in ERK activation. In a multi-level experimental workflow, we tested the suitability of the Teen reporter to detect pan-embryo ERK activity correlates of morphometric alterations driven by the NS-causing Shp2D61G allele. RESULTS: Spectral unmixing- and acceptor photobleaching (AB)-FRET analyses captured pathological ERK activity preceding the manifestation of quantifiable body axes defects, a morphological pillar used to test the strength of SHP2 GoF mutations. Last, the work shows that by multi-modal FRET analysis, we can quantitatively trace back the modulation of ERK phosphorylation obtained by low-dose MEK inhibitor treatment to early development, before the onset of morphological defects. CONCLUSION: This work proves the usefulness of FRET imaging protocols on both live and fixed Teen ERK reporter fish to readily monitor and quantify pharmacologically- and genetically-induced ERK activity modulations in early embryos, representing a useful tool in pre-clinical applications targeting RAS-MAPK signaling.

A fluorescent MOF and its synthesized MOF@cotton composite: Ratiometric sensing of vitamin B2 and antibiotic drug molecule.

Here, we demonstrated the synthesis of a zinc based luminescent MOF, 1 (NDC = 2,6- naphthalenedicarboxylate) for the ratiometric detection of biomarker riboflavin (RBF; vitamin B2) in water dispersed medium. Further, this MOF detected two other antibiotic drug molecules, nitrofurantoin (NFT) and nitrofurazone (NZF). The detection of these analytes is very quick (∼seconds), and the limit of detection (LOD) for RBF, NZF and NFT are calculated as 16.58 ppm, 47.63 ppb and 56.96 ppb, respectively. The detection of these analytes was also comprehended by solid, solution, cost-effective paper strip method i.e., triphasic identification capabilities. The sensor is reusable without losing its detection efficacy. The sensor further showed the recognition abilities of these antibiotics in real field samples (river water, urine and tablet) and RBF in vitamin B2 pills and food samples (milk and cold drinks). The sensing merit of 1 urged us to fabricate of 1@cotton fabric composite, which exhibited the colorimetric detection of these analytes. In-depth experimental analysis suggested that the occurrence of photo-induced electron transfer (PET), fluorescence resonance energy transfer (FRET), and the inner filter effect (IFE) are the possible sensing mechanisms for the recognition of the antibiotics drug. The FRET mechanism is responsible for the recognition of RBF. The sensing mechanism is further supported by the theoretical analysis and the excited lifetime measurement.

A combined experimental and theoretical approach for doxycycline sensing using simple fluorescent probe with distinct fluorescence change in wide range of interferences.

Overuse of doxycycline (DOXY) can cause serious problems to human health, environment and food quality. So, it is essential to develop a new sensing methodology that is both sensitive and selective for the quantitative detection of DOXY. In our current research, we synthesized a simple fluorescent probe 4,4'-bis(benzyloxy)-1,1'-biphenyl (BBP) for the highly selective detection of doxycycline by through fluorescence spectroscopy. The probe BBP displayed ultra-sensitivity towards doxycycline due to Forster resonance energy transfer (FRET). Fluorescence spectroscopy, density functional theory (DFT), 1H NMR titration, UV-Vis, and Job's plot were used to confirm the sensing mechanism. The charge transfer between the probe and analyte was further examined qualitatively by electron density differences (EDD) and quantitively by natural bond orbital (NBO) analyses. Whereas the non-covalent nature of probe BBP towards DOXY was verified by theoretical non-covalent interaction (NCI) analysis as along with Bader's quantum theory of atoms in molecules (QTAIM) analysis. Furthermore, probe BBP was also practically employed for the detection of doxycycline in fish samples, pharmaceutical wastewater and blood samples.

Integrating magnetic tweezers and single-molecule FRET: A comprehensive approach to studying local and global conformational changes simultaneously.

This chapter presents the integration of magnetic tweezers with single-molecule FRET technology, a significant advancement in the study of nucleic acids and other biological systems. We detail the technical aspects, challenges, and current status of this hybrid technique, which combines the global manipulation and observation capabilities of magnetic tweezers with the local conformational detection of smFRET. This innovative approach enhances our ability to analyze and understand the molecular mechanics of biological systems. The chapter serves as our first formal documentation of this method, offering insights and methodologies developed in our laboratory over the past decade.

Design and Synthesis of Novel Squaraine-Based Fluorescent Probe for Far-Red Detection of Chymotrypsin Enzyme.

Chymotrypsin, a crucial enzyme in human digestion, catalyzes the breakdown of milk proteins, underscoring its significance in both health diagnostics and dairy quality assurance. Addressing the critical need for rapid, cost-effective detection methods, we introduce a groundbreaking approach utilizing far-red technology and HOMO-Förster resonance energy transfer (FRET). Our novel probe, SQ-122 PC, features a unique molecular design that includes a squaraine dye (SQ), a peptide linker, and SQ moieties synthesized through solid-phase peptide synthesis. Demonstrating a remarkable quenching efficiency of 93.75% in a tailored H2O:DMSO (7:3) solvent system, our probe exhibits absorption and emission properties within the far-red spectrum, with an unprecedented detection limit of 0.130 nM. Importantly, our method offers unparalleled selectivity towards chymotrypsin, ensuring robust and accurate enzyme detection. This pioneering work underscores the immense potential of far-red-based homo-FRET systems in enabling the sensitive and specific detection of chymotrypsin enzyme activity. By bridging the gap between cutting-edge technology and biomedical diagnostics, our findings herald a new era of enzyme sensing, promising transformative advancements in disease diagnosis and dairy quality control.

Mercury (II) sensing using a simple turn-on fluorescent graphene oxide based aptasensor in serum and water samples.

A simple, highly sensitive, and selective fluorometric aptasensing platform based on aptamer and graphene oxide (GO) is proposed for the determination of mercury (II) ion (Hg2+). In the designed assay, two aptamer probes, a carboxy-fluorescein (FAM) labeled aptamer (aptamer A) and its complementary (aptamer B) with partial complement containing several mismatches and GO as the quencher were used. In the absence of Hg2+, both A and B aptamers were adsorbed on the surface of GO by π-π-stacking, leading to fluorescence quenching of FAM due to fluorescence resonance energy transfer (FRET). Upon exposure to Hg2+, the A and B aptamer strands bind Hg2+ and form T-Hg2+-T complexes, leading to the formation of a stable double-stranded aptamer. The double-stranded aptamer is detached from the GO surface, resulting in the recovery of FAM fluorescence. The fluorescence intensity (FI) of the developed sensor was correlated with the Hg2+ concentration under optimized experimental conditions in two wide linear ranges, even in the presence of 10 divalent cations as interferences. The linear ranges were obtained from 200.0 to 900.0 fM and 5.0 to 33.0 pM, a limit of detection (LOD) of 106.0 fM, and a limit of quantification (LOQ) of 321.3 fM. The concentration of Hg2+ was determined in five real samples containing three water and two serum samples, using spiking and standard addition methods and the results were compared with the spiked amounts and atomic absorption (AAS) as standard method respectively, with acceptable recoveries. Furthermore, in the standard addition method, to overcome the effects of matrix influence of real samples in quantitative predictions, the excitation-emission matrix (EEM) data for samples was simultaneously analyzed by multivariate curve resolution with alternating least squares (MCR-ALS) as a second-order standard addition method (SOSAM).

Development of highly sensitive dual-enhanced fluorescence quenching immunochromatographic test strips based on Pt nanoprobes.

The fluorescence-quenching method is crucial in vitro analysis, particularly for immunochromatographic test strips (ICTs) using noble metal nanoparticles as probes. However, ICTs still fall short in meeting the requirements for the detection of traces biomarkers due to the noble metal nanoparticles can only quench fluorescence of the dyes within a confined distance. Interestingly, noble metal nanoparticles, such as Pt NPs cannot only perform fluorescence-quenching ability based on the Förster resonance energy transfer (FRET), but also show perfect oxidase-like catalytic performance on many kinds of substrates, such as 3,3',5,5' -tetramethylbenzidine (TMB). We observed that the oxTMB (the oxidation products of TMB) exhibited notable effectiveness in quenching Cy5 fluorescence by the strong inner filter effect (IFE), which obviously improved the fluorescence-quenching efficiency with extremely low background signal. Through the dual-enhanced fluorescence quenching mechanism, the fluorescence quenching constant (Kn) was 661.24-fold that of only Pt NPs on the NC membrane. To validate the feasibility of this technique, we employed two types of biomarkers, namely microRNA (miR-15a-5p) and the signature protein (PSA). The sensitivity of miR-15a-5p was 9.286 × 10-18 mol/L and 17.5-fold more than that based on Pt NPs. As for the PSA, the LOD (0.6265 pg/mL) was 15.5-fold enhancement more sensitive after catalysis. Overall, the dual-enhanced fluorescence quenching rFICTs could act as a practical detection for biomarker in real samples.

Rapid FRET Assay for the Early Detection of Alpha-Synuclein Aggregation in Parkinson's Disease.

Alpha-synuclein (α-Syn) is a key protein of Parkinson's disease (PD). Oligomers formed by misfolding and aggregation of α-Syn can cause many pathological phenomena and aggravate the development of PD. Therefore, sensitive and accurate detection of oligomers is essential to understanding the pathology of PD and beneficial to screening and developing new drugs against PD. Here, we demonstrated a simple and sensitive method to detect the early aggregation of α-Syn via Förster resonance energy transfer (FRET) technology. We performed systematic investigations of the FRET sensitizations, efficiencies, and donor-to-acceptor distances during α-Syn aggregation, which was proved to be more sensitive to reflect small distance changes in the early stage of α-Syn aggregation, especially for α-Syn oligomers. The FRET assays were also applied to study the influence of Ser129 phosphorylation (pS129) on the aggregation rate of α-Syn. Our results showed that pS129 modification promotes α-Syn aggregation and enhances the ability of preformed fibrils to induce monomer aggregation. pS129 also increased the cytotoxicity of α-Syn. These results are of great significance for a better understanding of the pathological mechanisms of PD and future PD drug development.

Bacterial detection based on Förster resonance energy transfer.

The huge economic loss and threat to human health caused by bacterial infection have attracted the public's concern, and there is an urgent need to relieve and improve the tough problem. Therefore, it is significant to establish a facile, rapid, and sensitive method for bacterial detection considering the shortcomings of existing methods. Förster resonance energy transfer (FRET)-based sensors have exhibited immense potential and applicability for bacterial detection given their high signal-to-noise ratio and high sensitivity. This review focuses on the development of FRET-based fluorescence assays for bacterial detection. We summarize the principle of FRET-based assays, discuss the commonly used recognition molecules and further introduce three frequent construction strategies. Based on the strategies and materials, relevant applications are presented. Moreover, some restrictions of FRET fluorescence sensors and development prospects are discussed. Suitable donor-acceptor pairs and stable recognition molecules are the essential conditions for sensors to play their roles, and there is still some room for development. Besides, applying FRET fluorescence sensors to point-of-care detection is still difficult. Future developments could focus on near-infrared fluorescent dyes and simultaneous detection of multiple analytes.

Immunoassay System Based on the Technology of Time-Resolved Fluorescence Resonance Energy Transfer.

To enhance the specificity and sensitivity, cut the cost, and realize joint detection of multiple indicators, an immunoassay system based on the technology of time-resolved fluorescence resonance energy transfer (TR-FRET) was studied. Due to the FRET of the reagent, the donor probe and acceptor probe emitted specific fluorescence to enhance specificity. Long-lifetime specific fluorescence from the acceptor probe was combined with time-resolved technology to enhance sensitivity. A xenon flash lamp and a photomultiplier tube (PMT) were selected as the light source and detector, respectively. A filter-switching mechanism was placed in the light path, so the fluorescence signal from the donor and acceptor was measured alternately. The instrument's design is given, and some specificI parts are described in detail. Key technical specifications of the instrument and procalcitonin (PCT), C-reactive protein (CRP), and interleukin-6(IL-6) were tested, and the test results were presented subsequently. The CV value of the self-designed counting module is better than 0.01%, and the instrument noises for 620 nm and 665 nm are 41.44 and 10.59, respectively. When set at 37 °C, the temperature bias (B) is 0.06 °C, and the temperature fluctuation is 0.10 °C. The CV and bias are between ±3% and 5%, respectively, when pipetting volumes are between 10 µL and 100 µL. Within the concentration range of 0.01 nM to 10 nM, the luminescence values exhibit linear regression correlation coefficients greater than 0.999. For PCT detection, when the concentration ranges from 0.02 ng/mL to 50 ng/mL, the correlation coefficient of linear fitting exceeds 0.999, and the limit of quantification is 0.096 ng/mL. For CRP and IL-6, the detection concentration ranges from 0 ng/mL to 500 ng/mL and 0 ng/mL to 20 ng/mL, respectively, with limits of quantification of 2.70 ng/mL and 2.82 ng/mL, respectively. The experimental results confirm the feasibility of the technical and instrumental solutions.

Highly selective detection of deoxyribonucleic acid in living cells using RecA-green fluorescent protein-single-stranded deoxyribonucleic acid filament fluorescence resonance energy transfer probe.

A fluorescence resonance energy transfer (FRET) method was developed for double-stranded deoxyribonucleic acid (dsDNA) detection in living cells using the RecA-GFP (green fluorescent protein) fusion protein filament. In brief, the thiol-modified single-stranded DNA (ssDNA) was attached to gold nanoparticles (AuNPs); on the contrary, the prepared RecA-GFP fusion protein interacted with ssDNA. Due to the FRET between AuNPs and RecA-GFP, fluorescence of RecA-GFP fusion protein was quenched. In the presence of homologous dsDNA, homologous recombination occurred to release RecA-GFP fusion protein. Thus, the fluorescence of RecA-GFP was recovered. The dsDNA concentration was detected using fluorescence intensity of RecA-GFP. Under optimal conditions, this method could detect dsDNA activity as low as 0.015 optical density (OD) Escherichia coli cells, with a wide linear range from 0.05 to 0.9 OD cells, and the regression equation was ΔF = 342.7c + 78.9, with a linear relationship coefficient of 0.9920. Therefore, it provided a promising approach for the selective detection of dsDNA in living cells for early clinical diagnosis of genetic diseases.

FRETpredict: a Python package for FRET efficiency predictions using rotamer libraries.

Förster resonance energy transfer (FRET) is a widely-used and versatile technique for the structural characterization of biomolecules. Here, we introduce FRETpredict, an easy-to-use Python software to predict FRET efficiencies from ensembles of protein conformations. FRETpredict uses a rotamer library approach to describe the FRET probes covalently bound to the protein. The software efficiently and flexibly operates on large conformational ensembles such as those generated by molecular dynamics simulations to facilitate the validation or refinement of molecular models and the interpretation of experimental data. We provide access to rotamer libraries for many commonly used dyes and linkers and describe a general methodology to generate new rotamer libraries for FRET probes. We demonstrate the performance and accuracy of the software for different types of systems: a rigid peptide (polyproline 11), an intrinsically disordered protein (ACTR), and three folded proteins (HiSiaP, SBD2, and MalE). FRETpredict is open source (GPLv3) and is available at github.com/KULL-Centre/FRETpredict and as a Python PyPI package at pypi.org/project/FRETpredict .

Synergistic synthesis of gold nanoflowers as upconversion near-infrared nanoprobe energy acceptor and recognition unit for improved hydrogen sulfide sensing.

A highly sensitive and selective upconversion near-infrared (NIR) fluorescence and colorimetric dual readout hydrogen sulfide (H2S) nanoprobe was constructed based on the excellent NIR fluorescence emission performance of upconversion nanomaterials (UCNPs), the specific recognition effect of synergistically synthesized gold nanoflowers (trypsin-stabled AuNFs (Try-AuNFs)) and the effective NIR fluorescence quenching capability. In this assay, the sensing strategy included three processes. First of all, the synthesized UCNPs can emit 803 nm NIR fluorescence when they were excited by 980 nm excitation light. Secondly, as a result of the principle of fluorescence resonance energy transfer (FRET), Try-AuNFs can effectively quench the NIR fluorescence of UCNPs at 803 nm, which can effectively improve the signal-to-background ratio of nanoprobes, thereby improving the sensitivity of the probes. Thirdly, in the presence of H2S, the Try protective layer on the surface of Try-AuNFs was specifically penetrated, which will subsequently cleave Try-AuNFs via the strong S-Au bond. As such, the NIR fluorescence of UCNPs will be restored, achieving high selectivity and sensitivity detection of H2S. Under optimized conditions, the linear response range of H2S was 0.1-300 µM, and the detection limit was 53 nM. It is worth noting that the Try on the surface of Try-AuNFs via the synergistic effect can increase the steric hindrance of the probe, and this can effectively prevent the interaction between the probe with biothiols (cysteine (Cys), homocysteine (Hcy)) and other natural amino acids (non-thiol-containing) with resultant in the high selectivity regarding the detection of H2S in human serum, which is unlikely to be achieved by AuNFs synthesized by the gold seed method (Se-AuNFs). This work not only provided a new type of UCNPs fluorescence quencher and recognition unit, but also exemplified that the use of the physical properties (steric hindrance) of protein ligands on the surface of nanoflowers can improve the specificity of the probe. This will provide new ideas for the design of other nanoprobes.

Highly sensitive miRNA-21 detection with enzyme-free cascade amplification biosensor.

In this study, we present an enzyme-free fluorescence biosensor for the highly sensitive detection of miRNA-21, a crucial biomarker in clinical diagnosis. Our innovative approach combines catalytic hairpin assembly (CHA) and entropy-driven amplification into a cascade amplification strategy. MicroRNA initiates the catalytic hairpin assembly reaction, liberating the trigger region needed for the entropy-driven amplification reaction. This triggers a series of strand displacement reactions, resulting in the separation of the fluorescence resonance energy transfer pair and an amplified fluorescence signal from FAM. Our cascade amplification strategy achieves ultra-sensitive microRNA detection, with an impressive limit of detection (LOD) of 1.3 fM, approximately 100-fold lower than CHA alone. Additionally, we successfully applied this biosensor for microRNA quantification in human serum and cell lysates, demonstrating its practicality and potential for early diagnosis.