Fluorescence Resonance Energy Transfer Detection Research Articles

Remarkable fluorescence enhancement of acridine orange and rhodamine B through immobilization on zirconia nanotube array film and its application on nitrite sensing

Zirconia nanotube array films (ZNAF) prepared by anodic oxidation method were used as immobilization materials for acridine orange (AO), rhodamine B (RB) and AO-RB systems. A comparative study on their fluorescence emission intensity, fluorescence resonance energy transfer (FRET) and fluorescence detection of nitrite in aqueous solutions and on immobilization films with ZNAF as carriers was carried out. Results demonstrate that the solution pH values and immobilization on ZNAF have a great influence on the performance of these fluorescent molecules. Compared with aqueous solutions, the fluorescence emission intensity of AO and RB is considerably increased by immobilization, which is 8.0 and 4.2 times higher than the original, respectively. The energy transfer efficiency (E) of the AO-RB system increases from 40.9 % to 84.8 % by loading it on ZNAF. Moreover, after immobilization onto ZNAF, the fluorescence detection performance of nitrite is also significantly improved. The limit of detection decreases from 0.95 ng/mL to 0.22 ng/mL and the sensitivity increases from 939.18 to 15,031.68 mL/μg through loading AO onto ZNAF.

A DNA octahedral amplifier for endogenous circRNA detection and bioimaging in living cells and its biomarker study.

The discovery of reliable biomarkers is essential for early diagnosis, treatment, and prognosis assessment of diseases. Many research studies have shown that circRNA is a potential biomarker for diagnosis and prognosis of diseases. However, in situ monitoring circRNA in live cells is still a challenge at present, which brings a major limitation to the development and verification of circRNA as a disease biomarker. In this study, a catalytic hairpin assembly (CHA) reaction-based DNA octahedral amplifier (DOA) was developed for fluorescence resonance energy transfer (FRET) detection and bioimaging of circRNA in living cells. The DOA was first produced by self-assembling a DNA octahedron with six customized single-stranded DNAs, and two hairpins H1 (Cy3) and H2 (Cy5) were then hybridized to four vertices of the DNA octahedron. Idiopathic pulmonary fibrosis (IPF)-related circHIPK3 was used as the target. Once the CHA reaction from H1 and H2 on DOA was activated by a sequence-specific back-splice junction (BSJ) of circHIPK3, a significant FRET signal can be obtained from Cy3 to Cy5. The circHIPK3 was subsequently released to cause the next CHA reaction. Because the DOA has the advantages of the spatial-confinement effect, resistance to nuclease degradation and easy penetration into cells, rapid and excellent signal amplification FRET detection and bioimaging of endogenous circHIPK3 can be achieved in various cells. This study provides a high-precision assay platform to explore the possibility of using circRNA as a biomarker, and it is valuable for circRNA-related early diagnosis and treatment of diseases.

HTS driven by fluorescence lifetime detection of FRET identifies activators and inhibitors of cardiac myosin.

Small molecules that bind to allosteric sites on target proteins to alter protein function are highly sought in drug discovery. High-throughput screening (HTS) assays are needed to facilitate the direct discovery of allosterically active compounds. We have developed technology for high-throughput time-resolved fluorescence lifetime detection of fluorescence resonance energy transfer (FRET), which enables the detection of allosteric modulators by monitoring changes in protein structure. We tested this approach at the industrial scale by adapting an allosteric FRET sensor of cardiac myosin to high-throughput screening (HTS), based on technology provided by Photonic Pharma and the University of Minnesota, and then used the sensor to screen 1.6 million compounds in the HTS facility at Bristol Myers Squibb. The results identified allosteric activators and inhibitors of cardiac myosin that do not compete with ATP binding, demonstrating high potential for FLT-based drug discovery.

Aptamer-based DNA-catalyzed amplification strategy for sensitive fluorescence resonance energy transfer detection of Acinetobacter baumannii

Acinetobacter baumannii (A. baumannii) is a common pathogen that causes hospital-acquired infections and is resistant to a wide variety of antibiotics. Consequently, the rapid and highly sensitive detection of A. baumannii is required during the early stages of infection. Therefore, we developed a DNA-catalyzed amplification mechanism based on aptamers, combined with a novel fluorescence resonance energy transfer (FRET) method based on graphene oxide (GO), for the detection of A. baumannii. In the presence of A. baumannii, an aptamer bound to A. baumannii, releasing the template strand, which triggered an entropy-driven catalysis (EDC) reaction. One EDC product was then used as the catalyst for catalytic hairpin assembly (CHA) on a GO nanosheet. Finally, the GO released a huge amount of FAM-labeled DNA duplices, which could be detected with FRET. This strategy circumvented the extraction of nucleic acids and was easy to execute, with a detection time of ≤1.5 h. The detection of A. baumannii with this method ranges from 5 cfu/mL to 1 × 105 cfu/mL, with a detection limit of 1.1 cfu/mL. The method was sufficiently sensitive and specific to detect A. baumannii rapidly in cerebrospinal fluid. In summary, our strategy provides a new option for the early detection and point-of-care testing (POCT) of A. baumannii infections, allowing their earlier and more precise treatment.

Exploration of SARS-CoV-2 3CLpro Inhibitors by Virtual Screening Methods, FRET Detection, and CPE Assay.

COVID-19 caused by a novel coronavirus (SARS-CoV-2) has been spreading all over the world since the end of 2019, and no specific drug has been developed yet. 3C-like protease (3CLpro) acts as an important part of the replication of novel coronavirus and is a promising target for the development of anticoronavirus drugs. In this paper, eight machine learning models were constructed using naïve Bayesian (NB) and recursive partitioning (RP) algorithms for 3CLpro on the basis of optimized two-dimensional (2D) molecular descriptors (MDs) combined with ECFP_4, ECFP_6, and MACCS molecular fingerprints. The optimal models were selected according to the results of 5-fold cross verification, test set verification, and external test set verification. A total of 5766 natural compounds from the internal natural product database were predicted, among which 369 chemical components were predicted to be active compounds by the optimal models and the EstPGood values were more than 0.6, as predicted by the NB (MD + ECFP_6) model. Through ADMET analysis, 31 compounds were selected for further biological activity determination by the fluorescence resonance energy transfer (FRET) method and cytopathic effect (CPE) detection. The results indicated that (+)-shikonin, shikonin, scutellarein, and 5,3′,4′-trihydroxyflavone showed certain activity in inhibiting SARS-CoV-2 3CLpro with the half-maximal inhibitory concentration (IC50) values ranging from 4.38 to 87.76 μM. In the CPE assay, 5,3′,4′-trihydroxyflavone showed a certain antiviral effect with an IC50 value of 8.22 μM. The binding mechanism of 5,3′,4′-trihydroxyflavone with SARS-CoV-2 3CLpro was further revealed through CDOCKER analysis. In this study, 3CLpro prediction models were constructed based on machine learning algorithms for the prediction of active compounds, and the activity of potential inhibitors was determined by the FRET method and CPE assay, which provide important information for further discovery and development of antinovel coronavirus drugs.

Synthesis of gold nanoclusters-loaded lysozyme nanoparticles for ratiometric fluorescent detection of cyanide in tap water, cyanogenic glycoside-containing plants, and soils

The modification of protein-stabilized gold nanoclusters with fluorophores has been intensively applied for the ratiometric detection of biomolecules, metal ions, and anions. This study developed a straightforward strategy to prepare lysozyme nanoparticle-encapsulated gold nanoclusters (LysNP-AuNCs) as a dual-emission probe for the ratiometric sensing of cyanide through fluorescence resonance energy transfer (FRET) without the conjugation of additional fluorophores. The reduction of gold ion precursors with lysozyme generated lysozyme-stabilized AuNCs under an alkaline pH, which were demonstrated to self-assemble into nanoaggregates during the formation of AuNCs. The aggregated lysozyme molecules on the AuNCs were treated with glutaraldehyde, triggering the conversion of the aggregated lysozymes into blue-emitting lysozyme nanoparticles. As a result, the AuNCs were well distributed inside a single lysozyme nanoparticle, as demonstrated by transmission electron microscopy. The presence of cyanide triggered the etching of the AuNCs in the LysNP-AuNCs, leading to the suppression of FRET from lysozyme nanoparticle to AuNCs. The LysNP-AuNC probe was implemented for FRET detection of cyanide with a linear range of 3–100 μM. Additionally, the selectivity of the LysNP-AuNC probe for cyanide toward other anions was remarkably high. The practicality of the proposed probe was evaluated by quantifying cyanide in tap water and soils and monitoring the liberation of hydrogen cyanide from cyanogenic glycoside-containing foods.

Position-Dependent Effect of Guanine Base Damage and Mutations on Telomeric G-Quadruplex and Telomerase Extension.

Telomeres are hot spots for mutagenic oxidative and methylation base damage due to their high guanine content. We used single-molecule fluorescence resonance energy transfer detection and biochemical assays to determine how different positions and types of guanine damage and mutations alter telomeric G-quadruplex structure and telomerase activity. We compared 15 modifications, including 8-oxoguanine (8oxoG), O-6-methylguanine (O6mG), and all three possible point mutations (G to A, T, and C) at the 3' three terminal guanine positions of a telomeric G-quadruplex, which is the critical access point for telomerase. We found that G-quadruplex structural instability was induced in the order C < T < A ≤ 8oxoG < O6mG, with the perturbation caused by O6mG far exceeding the perturbation caused by other base alterations. For all base modifications, the central G position was the most destabilizing among the three terminal guanines. While the structural disruption by 8oxoG and O6mG led to concomitant increases in telomerase binding and extension activity, the structural perturbation by point mutations (A, T, and C) did not, due to disrupted annealing between the telomeric overhang and the telomerase RNA template. Repositioning the same mutations away from the terminal guanines caused both G-quadruplex structural instability and elevated telomerase activity. Our findings demonstrate how a single-base modification drives structural alterations and telomere lengthening in a position-dependent manner. Furthermore, our results suggest a long-term and inheritable effect of telomeric DNA damage that can lead to telomere lengthening, which potentially contributes to oncogenesis.

Concurrent Exposure of Neutralizing and Non-neutralizing Epitopes on a Single HIV-1 Envelope Structure.

The trimeric envelope spikes on the HIV-1 virus surface initiate infection and comprise key targets for antiviral humoral responses. Circulating virions variably present intact envelope spikes, which react with neutralizing antibodies; and altered envelope structures, which bind non-neutralizing antibodies. Once bound, either type of antibody can enable humoral effector mechanisms with the potential to control HIV-1 infection in vivo. However, it is not clear how the presentation of neutralizing vs. non-neutralizing epitopes defines distinct virus populations and/or envelope structures on single particles. Here we used single-virion fluorescence correlation spectroscopy (FCS), fluorescence resonance energy transfer (FRET), and two-color coincidence FCS approaches to examine whether neutralizing and non-neutralizing antibodies are presented by the same envelope structure. Given the spatial requirements for donor-acceptor energy transfer (≤10 nm), FRET signals generated by paired neutralizing and non-neutralizing fluorescent Fabs should occur via proximal binding to the same target antigen. Fluorescent-labeled Fabs of the neutralizing anti-gp120 antibodies 2G12 and b12 were combined with Fabs of the non-neutralizing anti-gp41 antibody F240, previously thought to mainly bind gp41 “stumps.” We find that both 2G12-F240 and/or b12-F240 Fab combinations generate FRET signals on multiple types of virions in solution. FRET efficiencies position the neutralizing and non-neutralizing epitopes between 7.1 and 7.8 nm apart; potentially fitting within the spatial dimensions of a single trimer-derived structure. Further, the frequency of FRET detection suggests that at least one of such structures occurs on the majority of particles in a virus population. Thus, there is frequent, overlapping presentation of non-neutralizing and neutralizing epitope on freely circulating HIV-1 surfaces. Such information provides a broader perspective of how anti-HIV humoral immunity interfaces with circulating virions.

Time-Resolved FRET Detection of the Myosin Super-Relaxed off State in Cardiac Thick Filament

We developed time-resolved fluorescence resonance energy transfer (FRET) sensors to detect the structural state of myosin in cardiac thick filaments. Cardiac myosin thick filaments are hypothesized to isomerize between an active state, termed RX (relaxed) that interacts with actin and generates force in the sarcomere, and an inactive state, termed SRX (super-relaxed) that does not bind actin and exhibits inhibited ATP hydrolysis compared to soluble myosin without actin. Regulation of the transition between these states is hypothesized to underlie the Frank-Starling law of the heart, and dysregulation of the transition to lead to genetically-driven hypertrophic cardiomyopathy; however, exactly how remains unclear, because the structural basis of the SRX is poorly understood. To investigate the structural transition between RX and SRX, we developed a time-resolved FRET assay that measures the intermolecular distance between two adjacent myosin regulatory light chains. Based on proposed structural models, formation of the SRX increases FRET while disruption decreases FRET. We tested this model with known modulators of the SRX biochemical kinetics, including temperature and ionic strength, and with two physiologically relevant SRX modulators, RLC phosphorylation and myosin-binding protein-C. This work was supported by National Institute of Health grants AR032961 and AR057220 (DDT), and American Heart Association Scientist Development Grant (JMM).

Confocal microscopic dual-laser dual-polarization FRET (2polFRET) at the acceptor side for correlating rotations at different distances on the cell surface

Relationship of donor and acceptor fluorescence anisotropies as well as efficiency of fluorescence resonance energy transfer (FRET) has been investigated in a confocal microscope in the context of FRET systems comprised of donor and acceptor-labeled MHCI and MHCII receptors on the surface of Kit-225 K6 human T-cells. The measurements have been carried out in a 2-laser, 5-signal platform where the total donor fluorescence intensity and 2 acceptor fluorescence intensities with their anisotropies – one at the donor's excitation wavelength, the other at the acceptor's excitation wavelength – have been detected. This configuration enabled the determination of FRET efficiency and correlating it with the two acceptor fluorescence anisotropies as a kind of calibration. Estimations for the FRET-enhanced donor fluorescence anisotropy, the directly excited acceptor fluorescence anisotropy, and the fluorescence anisotropy of sensitized emission have been obtained. Procedures for determining FRET by measuring only the total donor intensity and the acceptor intensity and its anisotropy, or two acceptor intensities and their anisotropies have been elaborated, the errors of which have been estimated based on the fluorescence anisotropy values obtained in the calibration with the method of flow cytometric energy transfer (FCET).The combined detection of the donor and acceptor fluorescence anisotropies enabled also the determination of the lower and upper limits of the orientation factor for FRET (κ2). An increase in range for κ2 with increasing FRET efficiency has been observed, with average κ2 values different from the dynamic random average of 2/3. These observations call for the need of κ2 determination in proximity measurements, where the donor and acceptor orientations are not predictable.An increasing range of κ2 with increasing intermolecular proximity of the MHCI and MHCII receptors has been observed. This indicates that molecular flexibility in the clusters of the MHCI and MHCII receptors reduces with increasing cluster density, i.e. a “fluidity gradient” exists in the clusters. More specifically, the local density dependent flexibility can also be taken as a direct proof for that the association of these receptors is non-random, but mediated by some type of physical interaction, a finding as a benefit of FRET detection by polarization spectroscopy.Two new quantities – the quenched donor fluorescence anisotropy and a fluorescence anisotropy analogue, the “dissymmetry index” of the polarized FRET efficiency components – have also been introduced for the characterization of the orientational dynamics of the excited state during FRET.

High-Throughput Spectral and Lifetime-Based FRET Screening in Living Cells to Identify Small-Molecule Effectors of SERCA

A robust high-throughput screening (HTS) strategy has been developed to discover small-molecule effectors targeting the sarco/endoplasmic reticulum calcium ATPase (SERCA), based on a fluorescence microplate reader that records both the nanosecond decay waveform (lifetime mode) and the complete emission spectrum (spectral mode), with high precision and speed. This spectral unmixing plate reader (SUPR) was used to screen libraries of small molecules with a fluorescence resonance energy transfer (FRET) biosensor expressed in living cells. Ligand binding was detected by FRET associated with structural rearrangements of green fluorescent protein (GFP, donor) and red fluorescent protein (RFP, acceptor) fused to the cardiac-specific SERCA2a isoform. The results demonstrate accurate quantitation of FRET along with high precision of hit identification. Fluorescence lifetime analysis resolved SERCA’s distinct structural states, providing a method to classify small-molecule chemotypes on the basis of their structural effect on the target. The spectral analysis was also applied to flag interference by fluorescent compounds. FRET hits were further evaluated for functional effects on SERCA’s ATPase activity via both a coupled-enzyme assay and a FRET-based calcium sensor. Concentration-response curves indicated excellent correlation between FRET and function. These complementary spectral and lifetime FRET detection methods offer an attractive combination of precision, speed, and resolution for HTS.

Regulation of plasma factor XIII levels in healthy individuals; a major impact by subunit B intron K c.1952 + 144 C > G polymorphism

BackgroundThe regulation of plasma factor XIII (FXIII) levels in healthy individuals has been only partially explored. The identification of major non-genetic and genetic regulatory factors might provide important information on the contribution of FXIII to the risk of cardio/cerebrovascular diseases. ObjectivesTo determine the effect of age, smoking, BMI, fibrinogen concentration on plasma FXIII activity, complex FXIII antigen (FXIII-A2B2) and total FXIII-B subunit (tFXIII-B) level, to correlate FXIII-B level with the other two FXIII parameters and to assess the variation of FXIII levels in carriers of major FXIII subunit polymorphisms. Methods268 healthy individuals were enrolled in the study. FXIII activity was measured by the ammonia release assay; FXIII-A2B2 and tFXIII-B were determined by ELISAs. FXIII-A p.Val34Leu, FXIII-B p.His95Arg and FXIII-B intron K c.1952+144 C>G polymorphisms were identified by RT-PCR using melting point analysis with fluorescence resonance energy transfer detection. ResultsAll investigated FXIII parameters showed significant positive correlation with age and fibrinogen level; gender and BMI influenced only tFXIII-B. A highly significant positive correlation was demonstrated between tFXIII-B and the other FXIII parameters. FXIII-A p.Val34Leu polymorphism had only slight, if any effect on FXIII levels. The FXIII-B Arg95 allele moderately increased all three FXIII parameters, but the effect became statistically significant only after adjustment. The FXIII-B intron K G allele drastically decreased FXIII levels, and it seemed to be in synergism with the FXIII-A Leu34 allele. ConclusionsPlasma FXIII levels are subjected to multifactorial regulation, in which age, fibrinogen level and FXIII-B intron K polymorphism are major determinants.

VAL34LEU POLYMORPHISM DETECTION BY REAL TIME PCR ASSAY USING

Factor XIII (FXIII) Val34Leu is the most important polymorphism of the A subunit in factor XIII gene. The aim of the present study was to develop an efficient method based on real time PCR with Fluorescence resonance energy transfer (FRET) detection and melting curve analysis for the detection of Val34Leu polymorphism. Specific primers were used to amplify the relevant fragment of the factor XIII-A gene and fluorescence resonance energy transfer hybridization probes were used for detection in a Rotor-Gene Q 5Plex HRM platform. Melting temperature (Tm) for the wild type was at 68.8, while Tm for homozygote mutant was at 63.8; heterozygote demonstrated both peaks. Our results showed that primers and probes propoesd in this study demonstrated a high specificity to identify wild type , heterozygous and homozygous mutantgenotypes. Due to the increasing molecular diagnosis in developing countries and the importance of identifying polymorphisms, this real time PCR assay is of great importance. An important advantage of this approach is the high sensitivity and specificity.

A graphitic carbon nitride based fluorescence resonance energy transfer detection of riboflavin

Fluorescence resonance energy transfer (FRET), which occurs between two luminescent chromophores, can greatly improve the selectivity and sensitivity of a fluorescent assay when a ratiometric signaling with the fluorescence enhancement of the acceptor at the expense of the donor is adopted. In this study, a fluorescence ratiometric detection (FRD) of riboflavin (RF) has been made based on FRET, as the strong overlap occurred between the emission spectrum of graphitic carbon nitride (g-C3N4) and absorption spectrum of RF, in which g-C3N4 acts as the energy donor and RF as the energy acceptor. With increasing concentration of RF, the fluorescence intensity of g-C3N4 emission at 444nm decreased and the fluorescence peak at 523nm for RF increased regularly, making the fluorescence intensity ratio of 523nm to 444nm linearly dependent on the concentration of RF in the range from 0.4μM to 10μM, giving a limit of the detection of 170nM. This method can be used to quantify RF in complex systems such as milk and drink, showing that the novel FRET-based fluorescence ratiometric detection can enable an attractive assay platform for analytes of interest.

DNA Strand-Displacement Digital Logic Circuit with Fluorescence Resonance Energy Transfer Detection

DNA Strand-Displacement Digital Logic Circuit with Fluorescence Resonance Energy Transfer Detection

Fluorescent Protein Based FRET Pairs with Improved Dynamic Range for Fluorescence Lifetime Measurements

Fluorescence Resonance Energy Transfer (FRET) using fluorescent protein variants is widely used to study biochemical processes in living cells. FRET detection by fluorescence lifetime measurements is the most direct and robust method to measure FRET. The traditional cyan-yellow fluorescent protein based FRET pairs are getting replaced by green-red fluorescent protein variants. The green-red pair enables excitation at a longer wavelength which reduces cellular autofluorescence and phototoxicity while monitoring FRET. Despite the advances in FRET based sensors, the low FRET efficiency and dynamic range still complicates their use in cell biology and high throughput screening. In this paper, we utilized the higher lifetime of NowGFP and screened red fluorescent protein variants to develop FRET pairs with high dynamic range and FRET efficiency. The FRET variations were analyzed by proteolytic activity and detected by steady-state and time-resolved measurements. Based on the results, NowGFP-tdTomato and NowGFP-mRuby2 have shown high potentials as FRET pairs with large fluorescence lifetime dynamic range. The in vitro measurements revealed that the NowGFP-tdTomato has the highest Förster radius for any fluorescent protein based FRET pairs yet used in biological studies. The developed FRET pairs will be useful for designing FRET based sensors and studies employing Fluorescence Lifetime Imaging Microscopy (FLIM).

Fluorescence Resonance Energy Transfer Detection of Cobalt Ions by Silver Triangular Nanoplates and Rhodamine 6G

In the present paper, the authors studied fluorescence resonance energy transfer (FRET) phenomenon between silver triangular nanoplates and bovine serum albumin (BSA)/Rhodamine 6G fluorescence complex, and established a fluorescence method for the detection of cobalt ions. We found that when increasing the silver triangular nanoplates added to certain concentrations of fluorescent bovine serum albumin (BSA)/Rhodamine 6G complex, the fluorescence of Rhodamine 6G would be quenched up to 80% due to the FRET between the quencher and donor. However, in the presence of cobalt ions, the disassociation of the fluorescent complex from silver triangular nanoplates occurred and the fluorescence of the Rhodamine 6G recovered. The recovery of fluorescence intensity rate (I/I0) has a good relationship with the cobalt ion concentration (cCO2+) added. Thus, the authors developed a fluorescence method for the detection of cobalt ions based on the FRET of silver triangular nanoplates and Rhodamine 6G.

FRET detection of lymphocyte function-associated antigen-1 conformational extension.

Lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18, αLβ2-integrin) and its ligands are essential for adhesion between T-cells and antigen-presenting cells, formation of the immunological synapse, and other immune cell interactions. LFA-1 function is regulated through conformational changes that include the modulation of ligand binding affinity and molecular extension. However, the relationship between molecular conformation and function is unclear. Here fluorescence resonance energy transfer (FRET) with new LFA-1-specific fluorescent probes showed that triggering of the pathway used for T-cell activation induced rapid unquenching of the FRET signal consistent with extension of the molecule. Analysis of the FRET quenching at rest revealed an unexpected result that can be interpreted as a previously unknown LFA-1 conformation.

Fluorescence resonance energy transfer detection methods: Sensitized emission and acceptor bleaching.

The present study compared the advantages and disadvantages of fluorescence resonance energy transfer (FRET) determination technologies, namely, sensitized emission (SE) and acceptor bleaching (AB), in order to analyze the applicability of SE and AB for studies investigating particularly interesting new cysteine histidine-rich protein 1 (PINCH1)/integrin-linked kinase (ILK) interaction. HeLa cells were transfected with cyan fluorescent protein (CFP)-PINCH1 and yellow fluorescent protein (YFP)-ILK to establish a PINCH1/ILK interaction examination model. PINCH1/ILK interactions in different parts of the cells were also examined by SE and AB. The FRET determination technologies SE and AB were able to examine PINCH1/ILK interaction. SE was more sensitive for FRET determination and thus had greater reliability. Therefore, SE is highly commended for membrane protein-protein interaction studies.

Sensitive Assay of Protease Activity on a Micro/Nanofluidics Preconcentrator Fused with the Fluorescence Resonance Energy Transfer Detection Technique

A fast and sensitive assay of protease activity on a micro/nanofluidics preconcentrator combining with fluorescence resonance energy transfer (FRET) detection technique has been developed in a homogeneous real-time format. First, the functionalized nanoprobes are formed by loading dye labeled protein onto gold nanoparticles (AuNPs), in which, the photoluminescence of donor dye was strongly quenched by AuNPs due to FRET mechanisms. For protease activity assay, the nanoprobes are enriched by a micro/nanofluidics preconcentrator. When the target protease is transported to the enriched nanoprobes, cleavage of protein occurs as a consequence of molecular recognition of enzyme to substrate. The release of cleavage fragments from AuNPs nanoprobes leads to the enhancement of fluorescence and enables the protease activity assay on the micro/nanofluidics chip. As a demonstration, digestion of fluorescein isothiocyanate labeled dog serum albumin (FITC-DSA) by trypsin was used as a model reaction. Because of the highly efficient preconcentration and space confinement effect, significantly increased protein cleavage rate and protease assay sensitivity can be achieved with enhanced enzyme activity. The present micro/nanofluidics platform fused with the FRET detection technique is promising for fast and sensitive bioanalysis such as immunoassay, DNA hybridization, drug discovery, and clinical diagnosis.