Development and external validation of a nomogram for predicting overall survival of patients with non-endometrioid endometrial cancer: A population-based analysis.

Objectives: The main objective of this study was to identify the key predictors and construct a nomogram that can be used to predict the overall survival of individuals with non-endometrioid endometrial cancer. Methods: A total of 2686 non-endometrioid endometrial cancer patients confirmed between 1988 and 2018 were selected from the Surveillance, Epidemiology, and End Results database. They were divided into a training cohort and an internal validation cohort. Independent risk factors were chosen by Cox regression analyses. A predictive nomogram model for overall survival was constructed based on above factors. A Chinese cohort of 41 patients was collected to be an external validation cohort. Results: Eight variables were estimated as independent predictors for overall survival. A nomogram was established using these factors. The C-index for predicting the overall survival of patients with non-endometrioid endometrial cancer from the nomogram was 0.734, 0.700, and 0.767 in training, internal, and external validation cohort, respectively. Calibration plots and decision curve analysis showed that the nomogram was valuable for further clinical application. Conclusion: We constructed a nomogram which can be used as an effective tool to predict the 3- and 5-year overall survival of Non-endometrioid endometrial cancer patients.

A high-throughput differential scanning fluorimetry method for rapid detection of thermal stability and iron saturation in lactoferrin.

Thermal stability and iron saturation of lactoferrin (LF) are of great significance not only for the evaluation of the biological activities of LF but also for the optimization of the isolation and drying process parameters. Differential scanning calorimetry (DSC) is a well-established and efficient method for thermal stability and iron saturation detection in LF. However, multiple DSC measurements are typically performed sequentially, thus time-consuming and low throughput. Herein, we introduced the differential scanning fluorimetry (DSF) approach to overcome such limitations. The DSF can monitor LF thermal unfolding with a commonly available real-time PCR instrument and a fluorescent dye (SYPRO orange or Glomelt), and the measured melting temperature of LF is consistent with that determined by DSC. On the basis of that, a new quantification method was established for determination of iron saturation levels using the linear correlation of the degree of ion saturation of LF with DSF measurements. Such DSF method is simple, inexpensive, rapid (<15 min), and high throughput (>96 samples per experiment), and provides a valuable alternative tool for thermal stability detection of LF and other whey proteins.

Molecular engineering of AIE-active boron clustoluminogens for enhanced boron neutron capture therapy.

The development of boron delivery agents bearing an imaging capability is crucial for boron neutron capture therapy (BNCT), yet it has been rarely explored. Here we present a new type of boron delivery agent that integrates aggregation-induced emission (AIE)-active imaging and a carborane cluster for the first time. In doing so, the new boron delivery agents have been rationally designed by incorporating a high boron content unit of a carborane cluster, an erlotinib targeting unit towards lung cancer cells, and a donor-acceptor type AIE unit bearing naphthalimide. The new boron delivery agents demonstrate both excellent AIE properties for imaging purposes and highly selective accumulation in tumors. For example, at a boron delivery agent dose of 15 mg kg-1, the boron amount reaches over 20 µg g-1, and both tumor/blood (T/B) and tumor/normal cell (T/N) ratios reach 20-30 times higher than those required by BNCT. The neutron irradiation experiments demonstrate highly efficient tumor growth suppression without any observable physical tissue damage and abnormal behavior in vivo. This study not only expands the application scopes of both AIE-active molecules and boron clusters, but also provides a new molecular engineering strategy for a deep-penetrating cancer therapeutic protocol based on BNCT.

High-efficiency upconversion luminescence in UCNPs/CsPbBr3 nanocomposites enhanced by silver nanoparticles.

Upconversion nanocomposites with multiple light-emitting centers have attracted great attention as functional materials, but their low efficiency limits their further applications. Herein, a novel, to the best of our knowledge, system for nanocomposites consisting of upconversion nanoparticles (UCNPs) and perovskite quantum dots (PeQDs) assembled with Ag nanoparticles (NPs) is proposed. Upconversion luminescence (UCL) operation from PeQDs is triggered by near-infrared (NIR) sensitization through Förster resonance energy transfer (FRET) and photon reabsorption (PR). Especially, the photoluminescence (PL) emission efficiency is found to be significantly enhanced due to the increased energy transfer efficiency and radiative decay rate in the UCNPs/CsPbBr3 nanocomposites. The results offer new opportunities to improve the UCL properties of perovskites and open new development in the fields of LED lighting, solar cells, biomedicine, and so on.

Label-free and sensitive detection of N6-methyladenosine demethylase activity in crude cell extracts and clinical cancer tissues based on demethylation-triggered exponential signal amplification.

The m6A demethylase catalyzes the removal of m6A modification to establish proper RNA methylation patterns, and it has emerged as a promising disease biomarker and a therapeutic target. The reported m6A demethylase assays often suffer from tedious producers, expensive reagents, radioactive risk, limited sensitivity, and poor specificity. Herein, we develop a simple, selective, label-free, and highly sensitive fluorescent biosensor for m6A demethylase assay based on demethylation-triggered exponential signal amplification. In this biosensor, m6A demethylase-catalyzed demethylation can protect the circular DNA from the digestion by DpnI, subsequently triggering hyperbranched rolling circle amplification to achieve exponential signal amplification for producing abundant ssDNA and dsDNA products. The amplified DNA signal can be sensitively and simply detected by SYBR Gold in a label-free manner. This biosensor avoids any antibodies, washing/separation procedures, and fluorophore-/quencher-labeled probes, great simplifying the assay procedures and reducing the assay cost. Moreover, this biosensor achieves good specificity and excellent sensitivity with a detection limit of 1.2 fg/µL, which is superior to conventional ELISA (36.3 pg/µL). Especially, this biosensor enables direct monitoring of m6A demethylase activity in crude cell extracts with high accuracy, and it can be further applied for the screening of m6A demethylase inhibitor, measurement of m6A demethylase activity in different cell lines, and discrimination of m6A demethylase level in clinical cancer and healthy tissues, providing a facile and robust platform for RNA methylation-related biomedical research, disease diagnosis, and drug discovery.

Early and long-term outcomes of young adult patients ≤30 years old with acute type A aortic dissection.

OBJECTIVES: The aim of this study was to investigate the early and long-term outcomes after total arch replacement (TAR) and frozen elephant trunk (FET) implantation in adult patients ≤30 years with acute type A aortic dissection (ATAAD). METHODS: All young adult patients (≤30 years) with ATAAD who underwent TAR and FET between 2009 and 2017 were enrolled. The end points were major organ morbidity and mortality, aortic-related events and reoperation. RESULTS: The mean age of all 83 patients was 25.9 (standard deviation, 3.3) years. The in-hospital mortality was 9.64% (8/83), and 9 (10.8%) patients required re-exploration for bleeding. The aortic-related events risk was 42.7% (32/75) and the aortic reoperation risk was 17.3% (13/75). Overall survival was 85.5% [95% confidence interval (CI), 75.9-91.5%] at 5 years and 75.9% (95% CI, 63.3-84.7%) at 10 years. The cumulative incidence of aortic-related events was 35% (95% CI, 24-47%) at 5 years and 58% (95% CI, 36-75%) at 10 years; the cumulative reoperation rate was 15% (95% CI, 7.9-24%) at 5 years and 17% (95% CI, 9.2-27%) at 10 years. Marfan syndrome significantly increased the aortic-related events (P = 0.036) and reoperation (P = 0.041) risks. CONCLUSIONS: Despite extensive repair in young ATAAD patients, the late aortic dilatation and reoperation risk remain high. The TAR and FET procedures achieved satisfactory early outcomes and reduced late aortic dilatation and reoperation in young patients compared with other records. Close follow-up and aggressive early reintervention are essential for patients with aortic-related risk factors early in life.

Construction of Multiple DNAzymes Driven by Single Base Elongation and Ligation for Single-Molecule Monitoring of FTO in Cancer Tissues.

Fat mass and obesity-associated proteins (FTO) play an essential role in the reversible regulation of N6-methyladenosine (m6A) epigenetic modification, and the overexpression of FTO is closely associated with the occurrence of diverse human diseases (e.g., obesity and cancers). Herein, we demonstrate the construction of multiple DNAzymes driven by single base elongation and ligation for the single-molecule monitoring of FTO in cancer tissues. When target FTO is present, the m6A-RNA is specifically demethylated and subsequently acts as a primer to combine with the padlock probe, initiating single-base elongation and ligation reaction to generate a closed template probe. Upon the addition of phi29 DNA polymerase, a rolling circle amplification (RCA) reaction is initiated to produce large numbers of Mg2+-dependent DNAzyme repeats. Subsequently, the DNAzymes cyclically digest the signal probes, liberating numerous Cy5 molecules that can be precisely counted by single-molecule imaging. Taking advantage of the sequence specificity of the polymerase/ligase-mediated gap-filling and ligation as well as the high amplification efficiency of RCA, this biosensor shows excellent specificity and high sensitivity with a detection limit of 5.96 × 10-16 M. It can be applied to screen FTO inhibitors and quantify FTO activity at the single-cell level. Moreover, the proposed strategy can accurately distinguish the FTO expression level in tissues of healthy individuals and breast cancer patients, providing a new platform for drug discovery, m6A modification-related research, and clinical diagnostics.

Construction of a Dendritic Nanoassembly-Based Fluorescent Biosensor for Electrostatic Interaction-Independent and Label-Free Measurement of Human Poly(ADP-ribose) Polymerase 1 in Lung Tissues.

Poly(ADP-ribose) polymerase 1 (PARP-1) is responsible for catalyzing the creation of poly(ADP-ribose) polymer and involved in DNA replication and repair. Sensitive measurement of PARP-1 is critical for clinical diagnosis. However, the conventional electrostatic attraction-based PAPR-1 assays usually involve laborious procedures, poor sensitivity, and false positives. Herein, we demonstrate the construction of a dendritic nanoassembly-based fluorescent biosensor for electrostatic interaction-independent and label-free measurement of human PARP-1 in lung tumor tissues. When PARP-1 is present, the specific double-stranded DNA (dsDNA)-activated PARP-1 transfers the ADP-ribosyl group from nicotinamide adenine dinucleotide (NAD+)/biotinylated NAD+ to the PARP-1 itself, resulting in the formation of biotinylated dsDNA-PARP-1-PAR polymer bioconjugates that can be captured by magnetic beads. Upon the addition of TdT, APE1, and NH2-modified T-rich probe, the captured dsDNAs with dual 3'-OH termini initiate TdT-activated APE1-mediated hyperbranched amplification to produce abundant dendritic DNA nanoassemblies that can be stained by SYBR Green I to generate a high fluorescence signal. This biosensor is characterized by a template-free, electrostatic interaction-independent, high sensitivity, and label-free assay. It enables rapid (less than 3 h) measurement of PARP-1 with a limit of detection of 4.37 × 10-8 U/µL and accurate measurement of cellular PARP-1 activity with single-cell sensitivity. Moreover, it is capable of screening potential inhibitors and discriminating the PARP-1 level in normal person tissues and lung cancer patient tissues, with great potential in PARP-1-related clinical diagnosis and drug discovery.

Synergistic luminescence effect and high-pressure optical properties of CsPbBr2Cl@EuMOFs nanocomposites.

Metal-organic frameworks (MOFs) are a class of highly porous materials that have garnered significant attention in the field of optoelectronics due to their exceptional properties. In this study, CsPbBr2Cl@EuMOFs nanocomposites were synthesized using a two-step method. The fluorescence evolution of the CsPbBr2Cl@EuMOFs was investigated under high pressure, revealing a synergistic luminescence effect between CsPbBr2Cl and Eu3+. The study found that the synergistic luminescence of CsPbBr2Cl@EuMOFs remains stable even under high pressure, and there is no energy transfer among different luminous centers. These findings provide a meaningful case for future research on nanocomposites with multiple luminescent centers. Additionally, CsPbBr2Cl@EuMOFs exhibit a sensitive color-changing mechanism under high pressure, making them a promising candidate for pressure calibration via the color change of the MOF materials.

Far-field mid-infrared microscopy via spatial frequency shifting of evanescent waves in photorefractive nematic liquid crystal.

Mid-infrared wavelength has unique advantages in revealing the nanostructures and molecular vibrational signatures. However, the mid-infrared subwavelength imaging is also limited by diffraction. Here, we propose a scheme for breaking the limitation in mid-infrared imaging. With the assistance of orientational photorefractive grating established in nematic liquid crystal, evanescent waves are efficiently shifted back into the observation window. The visualized propagation of power spectra in k-space also proves this point. The resolution has an improvement about 3.2 times higher than the linear case, showing potentials in various imaging areas, such as biological tissues imaging and label-free chemical sensing.

Instability-driven image recovery of 180-degree backscattered polarized-light in turbid water.

Although the incoherent modulation instability has been proven to be effective for the recovery of forward-scattering images, the similar attempt of backscatter is still non-ideal. In this paper, considering the preservation properties of polarization and coherence in 180° backscatter, we propose an instability-driven nonlinear imaging method based on polarization modulation. A coupling model is established using Mueller calculus and mutual coherence function, in which the instability generation and image reconstruction are both analyzed. Experimental results clearly show the enhancement of imaging quality. This method is general and has potential for echo detection in various scattering environments.

Suppressed Phase Separation of Mixed-Halide Perovskite Quantum Dots Confined in Mesoporous Metal Organic Frameworks.

Mixed-halide perovskite quantum dots (PeQDs) are the most competitive candidates in designing solar cells and light-emitting devices (LEDs) due to their tunable bandgap and high-efficiency quantum yield. However, phase separation in mixed-halide perovskites under illumination can form rich iodine and bromine regions, which change its optical responses. Herein, we synthesize PeQDs combined with mesoporous zinc-based metal organic framework (MOF) crystals, which can greatly improve the stability of anti-anion exchange, including photo-, thermal, and long-term stabilities under illumination. This unique structure provides a solution for improving the performance of perovskite optoelectronic devices and stabilizing mixed-halide perovskite devices.

Construction of a Single-Molecule Biosensor for Antibody-Free Detection of Locus-Specific N6-Methyladenosine in Cancer Cells and Tissues.

N6-Methyladenosine (m6A) has emerged as a key post-transcriptional regulator in mRNA metabolism, and its dysregulation is associated with multiple human diseases. Herein, we construct a single-molecule fluorescent biosensor for antibody-free detection of locus-specific m6A in cancer cells and tissues. A 5'-biotinylated capture probe and a 3'-hydroxylated assistant probe are designed for the recognition of specific m6A-mRNA. The m6A-sensitive endoribonuclease MazF can identify and cleave the unmethylated mRNA, and the retained intact m6A-mRNA can hybridize with assistant probes and capture probes to achieve sandwich hybrids. The sandwich hybrids are immobilized on magnetic beads (MBs) to initiate the terminal deoxynucleotidyl transferase (TdT)-assisted polymerization, facilitating the continuous incorporation of Cy5-dATP to form long Cy5-polyA tails for the production of an on-bead amplified fluorescence signal. After magnetic separation and exonuclease digestion, numerous Cy5 fluorophores are released and subsequently measured by single-molecule detection. Especially, this biosensor is implemented simply and isothermally without the involvement of either radiolabeling or m6A-specific antibody. Moreover, this biosensor shows ultrahigh sensitivity with a detection limit of 2.24 × 10-17 M, and it can discriminate a 0.01% m6A level from a large pool of coexisting counterparts. Furthermore, this biosensor can be used for monitoring cellular m6A-mRNA expression and differentiating the m6A level in the breast cancer patient tissues from that in the healthy person tissues, providing a new avenue for clinical diagnosis and epitranscriptomic research.

Peptidoglycan-Targeting Staphylolytic Enzyme Lysostaphin as a Novel and Efficient Protease toward Glycine-Rich Flexible Peptide Linkers.

Glycine-rich flexible peptide linkers have been widely adopted in fusion protein engineering; however, they can hardly be cleaved for the separation of fusion partners unless specific protease recognition sites are introduced. Herein, we report the use of the peptidoglycan-targeting staphylolytic enzyme lysostaphin to directly digest the glycine-rich flexible linkers of various lengths including oligoglycine linkers and (G4S)x linkers, without the incorporation of extra amino acids. Using His-MBP-linker-LbCpf1 as a model substrate, we show that both types of linkers could be digested by lysostaphin, and the digestion efficiency improved with increasing linker length. The enzyme LbCpf1 retained full activity after tag removal. We further demonstrated that the proteolytic activity of lysostaphin could be well maintained under different environmental conditions and in the presence of a series of chemical reagents at various concentrations that are frequently used in protein purification and stabilization. In addition, such a digestion strategy could also be applied to remove the SUMO domain linked to LwCas13a via an octaglycine linker. This study extends the applications of lysostaphin beyond an antimicrobial reagent and demonstrates its potential as a novel, efficient, and robust protease for protein engineering.

Pressure-induced distinct excitonic properties of 2D perovskites with isomeric organic molecules for spacer cations.

Spacer organic cations in two-dimensional (2D) perovskites play vital roles in inducing structural distortion of the inorganic components and dominating unique excitonic properties. However, there is still little understanding of spacer organic cations with identical chemical formulas, and different configurations have an impact on the excitonic dynamics. Herein, we investigate and compare the evolution of the structural and photoluminescence (PL) properties of [CH3(CH2)4NH3]2PbI4 ((PA)2PbI4) and [(CH3)2CH(CH2)2NH3]2PbI4 ((PNA)2PbI4) with isomeric organic molecules for spacer cations by combining steady-state absorption, PL, Raman and time-resolved PL spectra under high pressures. Intriguingly, the band gap is continuously tuned under pressure and decreased to 1.6 eV at 12.5 GPa for (PA)2PbI4 2D perovskites. Simultaneously, multiple phase transitions occur and the carrier lifetimes are prolonged. In contrast, the PL intensity of (PNA)2PbI4 2D perovskites exhibits an almost 15-fold enhancement at 1.3 GPa and an ultrabroad spectral range of up to 300 nm in the visible region at 7.48 GPa. These results indicate that the isomeric organic cations (PA+ and PNA+) with different configurations significantly mediate distinct excitonic behaviors due to different resilience to high pressures and reveal a novel interaction mechanism between organic spacer cations and inorganic layers under compression. Our findings not only shed light on the vital roles of isomeric organic molecules as organic spacer cations in 2D perovskites under pressure, but also open a route to rationally design highly efficient 2D perovskites incorporating such spacer organic molecules in optoelectronic devices.

Synthesis, structure, and luminescence properties of Europium/Cerium/Terbium doped strontium-anorthite-type green phosphor for solid state lighting.

Novel phosphor exploration and luminescence property regulation are two important strategies in pursing high performance phosphors for white light emitting diodes, and have attracted great attention from the researchers. Herein, novel green phosphors Sr2Ga2SiO7:Eu2+ and Sr2Ga2SiO7:Ce3+,Tb3+ had been obtained by high-temperature solid-state reactions and their luminescence properties had been investigated in detail. Powder X-ray diffraction and Rietveld structure refinement results verified the phase purity and gave the crystal structure of the prepared samples. Due to the electric dipole transition between inter configurations of 4fN and 4fN-15d1, Sr2Ga2SiO7:Eu2+ and Sr2Ga2SiO7:Ce3+ exhibited intense broad excitation and emission bands, giving out green and blue emitting light under UV excitation, respectively. By codoping Tb3+ with Ce3+ in the host and utilizing the energy transfer, tunable blue to green emission had been obtained. The energy transfer mechanism had been determined to be electric dipole-quadrupole interaction through dynamic luminescence analysis using I-H model. The prepared phosphors exhibited good thermal stability with integral emission intensity at 150 °C remaining more than 80 % of the emission intensity at 25 °C. Moreover, by coating Sr2Ga2SiO7:Eu2+ and Sr2Ga2SiO7:Ce3+,Tb3+ on UV chips, green LED devices had been obtained. The investigation results indicated that the Eu2+ singly doped and Ce3+-Tb3+ codoped Sr2Ga2SiO7 might be potential UV excited green phosphors for solid state lighting.

Multi-color UCNPs/CsPb(Br1-xIx)3 for upconversion luminescence and dual-modal anticounterfeiting.

Advanced hybrid materials have attracted extensive attention in optoelectronics and photonics application due to their unique and excellent properties. Here, the multicolor upconversion luminescence properties of the hybrid materials composed of CsPbX3(X = Br/I) perovskite quantum dots and upconversion nanoparticles (UCNPs, core-shell NaYF4:25%Yb3+,0.5%Tm3+@NaYF4) is reported, achieving the upconversion luminescence with stable and bright of CsPbX3 perovskite quantum dots under 980 nm excitation. Compared with the nonlinear upconversion of multi-photon absorption in perovskite, UCNPs/CsPbX3 achieves lower power density excitation by using the UCNPs as the physical energy transfer level, meeting the demand for multi-color upconversion luminescence in optical applications. Also, the UCNPs/CsPbX3 combined with ultraviolet curable resin (UVCR) shows excellent water and air stability, which can be employed as multicolor fluorescent ink for screen printing security labels. Through the conversion strategy, the message of the security labels can be encrypted and decrypted by using UV light and a 980 nm continuous wave excitation laser as a switch, which greatly improves the difficulty of forgery. These findings provide a general method to stimulate photon upconversion and improve the stability of perovskite nanocrystals, which will be better applied in the field of anti-counterfeiting.

Mix-and-Detection Assay with Multiple Cyclic Enzymatic Repairing Amplification for Rapid and Ultrasensitive Detection of Long Noncoding RNAs in Breast Tissues.

Long noncoding RNAs (lncRNAs) are valuable biomarkers and therapeutic targets, and they play essential roles in various pathological and biological processes. So far, the reported lncRNA assays usually suffer from unsatisfactory sensitivity and time-consuming procedures. Herein, we develop a mix-and-read assay based on multiple cyclic enzymatic repairing amplification (ERA) for sensitive and rapid detection of mammalian metastasis-associated lung adenocarcinoma transcript 1 (lncRNA MALAT1). In this assay, we design two three-way junction (3WJ) probes including a 3WJ template and a 3WJ primer to specifically recognize lncRNA MALAT1, and the formation of a stable 3WJ structure induces cyclic ERA to generate triggers. The resulting triggers subsequently hybridize with a free 3WJ template and act as primers to initiate new rounds of cyclic ERA, generating abundant triggers. The hybridization of triggers with signal probes forms stable double-stranded DNA duplexes that can be specifically cleaved by apurinic/apyrimidinic endonuclease 1 to produce a high fluorescence signal. This assay can be carried out in a mix-and-read manner within 10 min under an isothermal condition (50 °C), which is the rapidest and simplest method reported so far for the lncRNA MALAT1 assay. This method can sensitively detect lncRNA MALAT1 with a limit of detection of 0.87 aM, and it can accurately measure endogenous lncRNA MALAT1 at the single-cell level. Moreover, this method can distinguish lncRNA MALAT1 expression in breast cancer patient tissues and their corresponding healthy adjacent tissues. Importantly, the extension of this assay to different RNAs detection can be achieved by simply replacing the corresponding target recognition sequences.

Facile synthesis of boric acid-functionalized magnetic covalent organic frameworks and application to magnetic solid-phase extraction of trace endocrine disrupting compounds from meat samples.

A facile approach was proposed for the preparation of boric acid-functionalized core-shell structured magnetic covalent organic framework (COF) nanocomposites through employing the Fe3O4 nanoparticles as magnetic core, boric acid-functionalized COFs as the shell via sequential post-synthetic modification (denoted as Fe3O4@COF@BA). The synthesized nanocomposites showed large specific surface area, high magnetic responsiveness, and desirable chemical and thermal stability. Combined with HPLC-MS/MS, the as-prepared Fe3O4@COF@BA composite was applied as a sorbent for magnetic solid-phase extraction (MSPE) of endocrine disrupting compounds (EDCs) from meat samples. Under optimal conditions, the method displays low limits of detection (LODs, 0.08-0.72 µg kg-1) and good precision with relative standard deviations (RSD) lower than 5.4 %. The approach was successfully employed for the extraction and detection of EDCs in blank and spiked beef, chicken and pork samples with recovery ranging from 88.8 to 104.2 %.

Artificial clickase-triggered fluorescence "turn on" based on a click bio-conjugation strategy for the immunoassay of food allergenic protein.

Herein, based on an artificial clickase-catalyzed bio-conjugation strategy, we established a sensitive fluorescent clickase-linked immunosorbent assay (FCLISA) platform using an oligonucleotide-molecular beacon (Oligo-MB) hairpin structure as a fluorescence switch for detection of food allergenic protein. Firstly, a highly stable Cu(I)-containing nanocube was prepared for usage as an artificial clickase, which could catalyze the bio-conjugation of two short oligonucleotides (i.e., Oligo-A and Oligo-B labeled by a 5'-alkyne and a 3'-azide group, respectively) through clickase-catalyzed azide/alkyne cycloaddition reaction. Subsequently, the formed long-chain oligonucleotide (Oligo-A-B) could hybridize with Oligo-MB hairpin to open hairpin structure, leading to its fluorescence turn on. By using clickase as an alternative enzymatic label in conventional ELISAs, the established FCLISA showed high sensitivity and accuracy in detection of casein, achieving a limit of detection as low as 1.5 × 10-8 g/mL. Additionally, FCLISA has been challenged by detecting the casein in real samples, indicating a great potential in food safety assay.