Fluorescence Detection Platform Research Articles

A strategy for the accurate detection of glucose in human serum based on the IFE effect of up-transformed nanoparticles

Here, the inner filter effect (IFE) was applied to construct a “turn off” mode up-conversion fluorescence and colorimetric dual-reading detection platform consisting of the chromogenic substance p-phenylenediamine (PPD) and NaYF4:Yb/Er@NaYF4 core–shell up-conversion nanoparticles (UCNPs) for the detection of glucose and hydrogen peroxide (H2O2) with high sensitivity and selectivity. In this strategy, hydrogen peroxide is produced in the process of glucose oxidation by catalyzing glucose oxidase (GOx). PPD is oxidized by H2O2 under the action of horseradish peroxidase (HRP), resulting in the transformation into its oxidation product PPDox, whose absorption peak coincides with the emission peak of UCNP at 540 nm, which can significantly quench the fluorescence of UCNP. The quenching efficiency is related to PPDox production, so the relationship between the glucose content and the fluorescence changes of UCNP can be established to realize the sensitive detection of glucose. UCNPs modified by –COOH and the oxidation product of PPD were negatively charged, which increased the distance between donor and recipient due to electrostatic repulsion. It was not conducive to the occurrence of distance-dependent fluorescence resonance energy transfer (FRET), which was also verified by fluorescence lifetime test. Under the optimal experimental conditions, the linear ranges of hydrogen peroxide and glucose were 2.5 ∼ 50 and 5 ∼ 80 μmol/L with the detection limit (LOD) of 0.32 and 0.83 μmol/L, respectively, which were lower than of some reported approaches. Furthermore, the system could be applied to the detection of glucose in actual serum sample and the results were consistency of commercial glucose meters which indicated that the protocol was reliable and potential applications for clinical diagnosis.

Fe3O4/MXene Nanosphere-Based Microfluidic Chip for the Accurate Diagnosis of Alzheimer’s Disease

Amyloid β protein oligomer (AβO) has been found to be a crucial biomarker of Alzheimer’s disease (AD). Exploring an accurate detection method of AβO is of great significance for early AD diagnosis and treatment. Herein, a fluorescent biosensor for AβO detection is designed. It integrates fluorescence resonance energy transfer (FRET)-based fluorescence detection with a poly(dimethylsiloxane) (PDMS)-based microfluidic chip. The carboxyl fluorescein-modified AβO aptamers are employed as the fluorophore, and three-dimensional Fe3O4/MXene nanospheres are used as the fluorescence quencher for the first time. Moreover, the PDMS-based multichamber microfluidic chip serves as a fluorescence detection platform. The Fe3O4/MXene-based nanosphere-based biosensor shows an excellent linear relationship between the fluorescence intensity and the logarithm of the AβO concentration in the range of 0.10–200 nM. The detection limit is ∼0.05 nM at the ultralow sample volumes of ∼4.50 μL. These results strongly show that the fabricated high-performance biosensor can be widely used in smart healthcare including but not limited to medical diagnosis, health monitoring, and biological studies.

Construction of a simple, localized and homogeneous fluorescence detection platform for T4 PNK activity based on tetrahedral DNA nanostructure-mediated primer exchange reaction

T4 polynucleotide kinase (T4 PNK), which acts as a bifunctional enzyme, is widely involved in important phosphorylation and dephosphorylation processes. Sensitive and specific detection of T4 PNK activity will help to elucidate its critical importance in disease diagnosis and treatment. The conventional detection method uses hairpin or single-strand primers modified with phosphorylation group as the substrates for T4 PNK hydrolysis. However, considering to the large spatial distances and disordered reaction orientation among the substrates and enzymes, the efficiency and sensitivity of the method are somewhat limited. Here, we propose a simple and sensitive signal amplification strategy, integrating the tetrahedral DNA nanostructure with primer exchange reaction (TDN-PER), which ensures different reaction direction and improves the local reaction concentrations. Compared to dispersed single PER, localized TDN-PER provides more primers for initiating signal amplification. Benefiting from the unique structural transformation from dispersion to localization, this strategy provided ultrahigh sensitivity, achieving a detection limit of 1.8 × 10−5 U/mL, while maintaining excellent accuracy and specificity in complex human serum and cell lysates environments. The method successfully evaluated the inhibitory effects of (NH4)2SO4 and Na2HPO4 on T4 PNK activity. Finally, the practical application capability of this strategy was verified in the detection of cell-free extracts, providing a promising analytical platform for T4 PNK-related biological and medical research.

Ag NPs/PMMA nanocomposite as an efficient platform for fluorescence regulation of riboflavin.

The fluorescence detection platform has broad application in many fields. In this paper, we report a simple and efficient fluorescence detection platform based on the synergistic effects of Ag nanoparticles (Ag NPs) and polymethylmethacrylate (PMMA). Ag NPs were introduced to realize the plasmon enhancement fluorescence and a thin PMMA layer was used to adjust the distance between Ag NPs and riboflavin. The thin PMMA layer not only enhances the fluorescence by enhancing adhesion of substrate, but also optimizes the plasmon enhancement fluorescence effect by serving as the spacer. The fluorescence enhancement factor based on this platform shows a trend of increasing with the decrease of the concentration of riboflavin, and the detection of riboflavin is realized based on this feature, the lowest detectable concentration is as low as 0.27 µM. In addition to the detection based on plasmon enhancement fluorescence, the detection of riboflavin at low concentrations can also be realized by the shift and broadening of the fluorescence peak due to the Ag NPs. The combination of the two ways of plasmon enhancement fluorescence and shift of the fluorescence spectra is used for the detection of riboflavin. These results show that the platform has great potential applications in the field of detection and sensing.

Rapid and direct detection of hepatitis E virus in raw pork livers by recombinase polymerase amplification assays.

Hepatitis E virus (HEV) is a zoonotic pathogen that causes global hepatitis E. Outbreaks of hepatitis E are directly linked to the consumption of pork liver products. Herein reverse transcription recombinase polymerase amplification assays targeting the ORF2 gene were developed for the rapid detection of HEV by integrating the fluorescence detection platform (qRT-RPA) and the visible lateral flow biosensor by naked eyes (LFB RT-RPA). The qRT-RPA assay effectively detected HEV RNA with a limit of detection (LOD) of 154 copies/μl (95%CI: 126–333 copies/µl) in Genie III at 41°C for 20 min. Besides this, the LFB RT-RPA detected the HEV RNA with a LOD of 24 copies/μl (95%CI: 20–57 copies/µl) in an incubator block at 41°C for 20 min. The developed RT-RPA assays also showed good specificity for HEV, with no cross-reactions with any of the other important swine pathogens examined in this work. The performance of the developed RT-RPA assays was validated on 14 HEV RNA-positive and 66 HEV RNA-negative raw pork liver samples identified by a previously described qRT-PCR. Consequently, 11 and 12 samples were HEV RNA-positive as detected by the qRT-RPA and the LFB RT-RPA, respectively. Compared to qRT-PCR, the qRT-RPA and LFB RT- RPA assays revealed a coincidence rate of 96.3 and 97.5% as well as a Kappa value of 0.858 and 0.908, respectively. These results ascertain that the developed RT-RPA assays are effective diagnostic tools for the point-of-care detection of HEV in resource-limited settings.

Development of Fluorescent Aptasensors Based on G-Quadruplex Quenching Ability for Ochratoxin A and Potassium Ions Detection.

G-quadruplexes have received significant attention in aptasensing due to their structural polymorphisms and unique binding properties. In this work, we exploited the fluorescence-quenching properties of G-quadruplex to develop a simple, fast, and sensitive platform for fluorescence detection of ochratoxin A (OTA) and potassium ions (K+) with a label-free fluorophore and quencher strategy. The quenching ability of G-quadruplex was confirmed during the recognition process after the formation of the G-quadruplex structure and the quenching of the labeled fluorescein fluorophore (FAM). The fluorescence-quenching mechanism was studied by introducing specific ligands of G-quadruplex to enhance the quenching effect, to show that this phenomenon is due to photo-induced electron transfer. The proposed fluorescence sensor based on G-quadruplex quenching showed excellent selectivity with a low detection limit of 0.19 nM and 0.24 µM for OTA and K+, respectively. Moreover, we demonstrated that our detection method enables accurate concentration determination of real samples with the prospect of practical application. Therefore, G-quadruplexes can be excellent candidates as quenchers, and the strategy implemented in the study can be extended to an aptasensor with G-quadruplex.

Nanozyme-based sensitive ratiometric fluorescence detection platform for glucose

Development and application of carbon-based nanozymes are attracting wide interest in recent years. Abnormal glucose level can be threat to human health. Sensitive and accurate sensing methods for glucose are still of great urgency. In this work, we constructed a sensitive nanozyme-based ratiometric fluorescence sensing platform for glucose. The sensing system composed of copper-doped carbon-based nanozyme (CuAA) with superb peroxidase-like activity and Mg/N doped carbon quantum dots (Mg–N-CQDs) with distinguished fluorescence property. Efficient tandem catalysis of glucose oxidase (Glu Ox) and CuAA, and inner-filter effect (IFE) between 2, 3-diaminophenazine (DAP) and Mg–N-CQDs played crucial roles in this sensing system. The oxidization of glucose was catalyzed by Glu Ox firstly to produce H2O2. In the presence of H2O2, fluorescent DAP was formed from non-fluorescent substrate o-phenylenediamine (OPD) with assistance of CuAA, resulting in generation of emission at 558 nm. Meanwhile, the emission at 444 nm from Mg–N-CQDs was quenched efficiently by DAP through IFE. The ratiometric fluorescence signal I558/I444 increased linearly with glucose concentration in the scope of 2–400 μmol L−1, the limit of detection (LOD) was 1.56 μmol L−1. It was also practicable to apply the method to the determination of glucose in human serum, satisfactory recoveries and RSDs were acquired.

Rapid detection of porcine encephalomyocarditis virus (EMCV) by isothermal reverse transcription recombinase polymerase amplification assays

In this study, we combined reverse transcription recombinase polymerase amplification assay with the fluorescence detection platform (qRT-RPA) and lateral flow biosensor (LFB RT-RPA) to allow for rapid detection of porcine encephalomyocarditis virus (EMCV). Primers and probes were designed to target the highly conserved region of 3D gene of porcine EMCV. The optimal reaction condition of qRT-RPA and LFB RT-RPA was set as 42 °C for 20 min. The assays were highly specific to EMCV and no cross-reactions were observed with seven other porcine viruses. With a 10-fold serially diluted EMCV genomic RNA as template, the limit of detection was 1.0 × 102 and 1.0 × 101 copies for qRT-RPA assay and LFB RT-RPA assay, respectively. A total of 92 samples from different sources were examined using qRT-RPA, LFB RT-RPA and qRT-PCR. We found 100% diagnostic agreement between qRT-RPA (23/92) and qRT-PCR (23/92), and 97.83% diagnostic agreement between LFB RT-RPA (25/92) and qRT-PCR (23/92). There was no significant difference in performance between the RT-RPA assays developed in this study and a previously described qRT-PCR. However, RT-RPA assays were rapid and easy to perform while LFB RT-RPA exhibited higher sensitivity for EMCV than qRT-PCR. Therefore, the developed EMCV RT-RPA assays provide an attractive and promising tool for effective detection of EMCV in low-resource settings.

A regenerable and reducing false-positive fluorescent switch for detection of β-amyloid 1−42 oligomers

The conventional fluorescence analysis methods for disease identification are vulnerable to the restriction with false-positive. Here, a fluorescent switch with high efficiency and regeneration by the black phosphorus (BP) nanosheets-regulated was developed to overcome false-positive issue in the assay of β-amyloid1−42 oligomers (Aβ) process. The Aβ was rapidly recognized using the fluorescent emitter‐nitrogen-doped carbon nanodots (N-CDs) under the regulation of BP nanosheets, while the N-CDs alone cannot recognize Aβ without the introduction of BP. The fluorescence analysis methods exhibited a wide sensing range of 0.25–15.0 ng/mL and a low detection limit of 83 pg/mL for Aβ analysis, which was superior to the reported fluorescence analysis method. Further, BP nanosheets were recycled, demonstrating the fluorescent switch with highly efficient, stable, and regenerable. This provides a new idea for developing high-efficiency and high-precision fluorescence detection platform.

A CRISPR-Cas12a-Assisted Fluorescence Platform for Rapid and Accurate Detection of Nocardia cyriacigeorgica.

Nocardia cyriacigeorgica has gradually become a common pathogen in clinical microbial infections. Identification of Nocardia at the species level is essential to assess the susceptibility and pathogenicity of antimicrobials. However, there is no suitable method for rapid and accurate laboratory detection of N. cyriacigeorgica. In this study, we combined PCR amplification with the CRISPR-Cas12a system to establish a novel detection platform, named CRISPR-PCR, and applied it to the detection of N. cyriacigeorgica in clinical samples. The Cas12a protein exhibited collateral cleavage activity following CRISPR RNA binding to specific targets, then indiscriminately cleaved nearby single-stranded DNA, and this was evaluated for diagnostic nucleic acid detection by measuring the fluorescence signal using a fluorescence reader. The assay takes only 2 h, including DNA extraction for 20 min, nucleic acid pre-amplification for 70 min, and fluorescence detection for 20 min. The limit of detection for N. cyriacigeorgica was 10-3 ng and the specificity was 100%. Thus, the N. cyriacigeorgica CRISPR-PCR assay is a rapid and specific method for detecting N. cyriacigeorgica, and the CRISPR-PCR fluorescence detection platform has great potential for detection of other pathogens.

Study on visual multicolor intelligent detection of tetracycline antibiotics in various environmental samples by palygorskite-based fluorescent nano-probe

Tetracycline is abused in animal husbandry due to the role of animal growth hormone, which causes serious pollution to the natural environment and is more likely to enter the human body through the food chain, thus causing harm to human health. Therefore, the detection of tetracycline is particularly important. At present, there are many problems in the detection of tetracycline, such as high cost, low sensitivity and narrow detection range. This research mainly introduces a new fluorescent nano-probe Atta-Eu-FITC based on palygorskite to realize the detection of tetracycline in environmental samples. The nano-probe has the advantages of high sensitivity (detection limit 19 nM), wide detection range (detection linear range 0–25 μM), high visualization and strong anti-interference ability. To improve the portability and practicability of the detection, a portable fluorescent test paper and a low background fluorescent agarose gel detection platform were prepared to realize the fast sensing of tetracycline. Combined with the smartphone chroma analysis application (App), the real-time and on-site detection of tetracycline can be realized. Furthermore, the fluorescent nano-probe can also be applied for TC sensing in practical complex samples with good anti-interference ability and accuracy.

Highly selective fluorescent detection platform based on isoquinoline Schiff base ligand monitors glutathione in biological systems

Aberrant levels of intracellular glutathione (GSH) have been held responsible for various disease and pathological processes, and existing evidence underscores the significance of GSH detection in biological systems. The recognition, discrimination, and precise detection of GSH in the cellular matrix and living organisms can revolutionize our understanding of the physiological and pathological processes of GSH. Herein, we reported a new isoquinoline Schiff base fluorescence probe, N'-((7-(diethylamino)-2-oxo-2H-chromene)methylene) isoquinoline-3-carbohydrazide (NCMC), which can selectively recognize GSH by its ensemble complex NCMC-Cu2+ with a low nanomolar level detection limit (4.16 × 10−8 M) in the aqueous environment. Moreover, the binding behavior and “ON-OFF-ON” fluorescence probing pattern were illustrated in detail by UV–vis titration, ESI-MS spectroscopy, and density functional theory calculations. Notably, the nontoxic NCMC-Cu2+ ensemble platform showed great potential for detecting GSH distribution in cells and zebrafish larvae, and can also be applied in differentiating endogenous GSH in both cancer cells and normal cells, which may represent a promising GSH fluorescence probe in bioimaging and disease diagnosis.

Construction of a Simple, One-Pot and Homogeneous Fluorescence Detection Platform for T4 Pnk Activity Based on Tetrahedral DNA Nanostructure-Mediated Primer Exchange Reaction

Construction of a Simple, One-Pot and Homogeneous Fluorescence Detection Platform for T4 Pnk Activity Based on Tetrahedral DNA Nanostructure-Mediated Primer Exchange Reaction

An efficient dual functional Raman and Fluorescence detection platform achieved by controlling the electromagnetic enhanced field in three-dimensional Ag/ZnO composited arrays

Integrating SERS and MEF into a single platform is a challenge. Here, we design a three-dimensional electromagnetic field via decorating Ag nanoparticles onto ZnO nanorod arrays to realize dual-function detection by SERS and MEF simultaneously.

Optimization of Fluorescence Labeling of Trace Analytes: Application to Amino Acid Biosignature Detection with Pacific Blue.

Fluorescence labeling of biomolecules and fluorescence detection platforms provide a powerful approach to high-sensitivity bioanalysis. Reactive probes can be chosen to target specific functional groups to enable selective analysis of a chosen class of analytes. Particularly, when targeting trace levels of analytes, it is important to optimize the reaction chemistry to maximize the labeling efficiency and minimize the background. Here, we develop methods to optimize the labeling and detection of Pacific Blue (PB)-tagged amino acids. A model is developed to quantitate labeling kinetics and completeness in the circumstance where analyte labeling and reactive probe hydrolysis are in competition. The rates of PB hydrolysis and amino acid labeling are determined as a function of pH. Labeling kinetics and completeness as a function of PB concentration are found to depend on the ratio of the hydrolysis time to the initial labeling time, which depends on the initial PB concentration. Finally, the optimized labeling and detection conditions are used to perform capillary electrophoresis analysis demonstrating 100 pM sensitivities and high-efficiency separations of an 11 amino acid test set. This work provides a quantitative optimization model that is applicable to a variety of reactive probes and targets. Our approach is particularly useful for the analysis of trace amine and amino acid biosignatures in extraterrestrial samples. For illustration, our optimized conditions (reaction at 4 °C in a pH 8.5 buffer) are used to detect trace amino acid analytes at the 100 pM level in an Antarctic ice core sample.

Aptamer Conjugated Indium Phosphide Quantum Dots with a Zinc Sulphide Shell as Photoluminescent Labels for Acinetobacter baumannii.

Acinetobacter baumannii is a remarkable microorganism known for its diversity of habitat and its multi-drug resistance, resulting in hard-to-treat infections. Thus, a sensitive method for the identification and detection of Acinetobacter baumannii is vital. However, current methods used for the detection of pathogens have not improved in the past decades and suffer from long process times and low detection limits. A cheap, quick, and easy detection mechanism is needed. In this work, we successfully prepared indium phosphide quantum dots with a zinc sulphide shell, conjugated to a targeting aptamer ligand, to specifically label Acinetobacter baumannii. The system retained both the photophysical properties of the quantum dots and the folded structure and molecular recognition function of the aptamer, therefore successfully targeting Acinetobacter baumannii. Confocal microscopy and transmission electron microscopy showed the fluorescent quantum dots surrounding the Acinetobacter baumannii cells confirming the specificity of the aptamer conjugated to indium phosphide quantum dots with a zinc sulphide shell. Controls were undertaken with a different bacteria species, showing no binding of the aptamer conjugated quantum dots. Our strategy offers a novel method to detect bacteria and engineer a scalable platform for fluorescence detection, therefore improving current methods and allowing for better treatment.

One facile fluorescence strategy for sensitive determination of baicalein using trypsin-templated copper nanoclusters

Herein, we established a fluorescent detection platform for baicalein (Bai) based on copper nanoclusters, which were prepared by using copper sulfate as the precursor, trypsin (Tryp) as the template and hydrazine hydrate as the reducing agent. The entire preparation and testing process were rapid, facile and green. Many characterization methods, such as UV–vis absorption spectroscopy, fluorescence spectroscopy, fourier transform infrared spectroscopy (FT-IR), fluorescence lifetime, transmission electron microscope (TEM) and X-ray photoelectron spectroscopy (XPS), were applied for the analysis of trypsin-templated copper nanoclusters (Cu NCs@Tryp). The Cu NCs@Tryp released green fluorescence at maximum emission wavelength of 457 nm under maximum excitation wavelength of 377 nm. More importantly, the fluorescence of Cu NCs@Tryp was efficiently quenched by Bai. According to this phenomenon, a facile, rapid and selective turn-off fluorescence probe for Bai sensing was developed. Under the optimized testing conditions, the ln(F0/F) value and concentration of Bai displayed excellent linear relationship changing from 0.5 to 60 μM (R2 = 0.9969), and the detection limit was 0.078 μM. Furthermore, the Cu NCs@Tryp has been successfully employed to measure the amount of Bai in bovine serum samples with satisfactory recoveries.

Rapid and sensitive detection of Salmonella in milk based on hybridization chain reaction and graphene oxide fluorescence platform

Salmonella is a foodborne pathogen that has contributed to numerous food safety accidents worldwide, making it necessary to detect contamination at an early stage. A pair of specific primers based on the invA gene of Salmonella was designed for PCR. Target double-stranded DNA (dsDNA) from PCR was purified and denatured at high temperature to obtain target single-stranded DNA (ssDNA). Two carboxyfluorescein-labeled hairpin probes (H1-FAM and H2-FAM) were designed with complementary portions to the ssDNA sequence so that binding could trigger H1-FAM and H2-FAM hybridization chain reaction (HCR) to produce a long dsDNA complex. In this study, graphene oxide (GO) was used in the development of a homogeneous fluorescence detection platform for Salmonella. Using this HCR-GO assay platform, Salmonella detection was completed in 3.5 h. Salmonella was reliably and specifically detected with a limit of detection (LOD) of 4.2 × 101 cfu/mL in pure culture. Moreover, this new HCR-GO assay platform was successfully applied to the detection of Salmonella in artificially contaminated milk with a LOD of 4.2 × 102 cfu/mL.

MnO2 Nanospheres Assisted by Cysteine Combined with MnO2 Nanosheets as a Fluorescence Resonance Energy Transfer System for "Switch-on" Detection of Glutathione.

Manganese dioxide nanosheets combined with cysteine-assisted emitting manganese dioxide nanospheres (Cys-MnO2 nanospheres) is fabricated for the first time as an "off-on" fluorescence detection platform for glutathione (GSH). In this sensing system, Cys-MnO2 nanospheres served as energy donors, while MnO2 nanosheets were used as both energy acceptors and recognition units. MnO2 nanosheets can effectively quench the fluorescence of Cys-MnO2 nanospheres through the fluorescence resonance energy transfer (FRET). The addition of GSH could reduce MnO2 nanosheets into Mn2+, disrupting the FRET process and restoring the fluorescence of Cys-MnO2 nanospheres. Under the optimum conditions, the "switch-on" platform we established has a wide response to GSH with a range of 5-50 μM and 150-800 μM, as well as a superior specificity. Importantly, all components of the sensor are nontoxic, biocompatible, easily prepared, and have a high utilization of raw materials. Moreover, the sensing system achieved satisfactory results in human serum, showing a tremendous potential in the field of biomedicine.

An integrated platform for label-free fluorescence detection and inactivation of bacteria based on boric acid functionalized Zr-MOF

Bacterial infections are a major threat to human health. The construction of a multifunctional platform that meets both the demands of sensitive detection and effective bacterial elimination is highly desirable, but still a formidable challenge. Herein, an integrated platform based on Zr-UiO-66-B(OH)2 MOF that can achieve highly effective label-free fluorescence bacterial detection and inactivation is reported. In this design, Zr-UiO-66-B(OH)2 is tightly linked to the surface of the bacteria attributed to the interactions between the boric acid groups of Zr-UiO-66-B(OH)2 and the glycolipids outside the bacteria cells. Taking advantage of the fluorescence “turn-on” property of Zr-UiO-66-B(OH)2 by H2O2, the bacteria could be detected with high sensitivity, with LOD of 1.0 CFU/mL. In addition, the as-obtained Zr-UiO-66-B(OH)2 demonstrated the potential photocatalytic inactivation of bacteria under light irradiation. The photo-induced reactive oxygen species played a dominant role in the antibacterial process. This work could provide a reference for the further development of novel integrated platforms for bacterial discrimination and antibacterial applications.