Fluorescence Assay Strategy Research Articles

A portable smartphone-assisted highly emissive magnetic covalent organic framework-based fluorescence sensor for the detection of Salmonella typhimurium

The core/shell magnetic covalent organic framework doped with gold nanoparticles (MCOF-AuNPs) was prepared by a facile two-step in situ synthesis strategy to achieve high emission. The fluorescence emission was enhanced and overcame the aggregation-induced quenching of COF after the introduction of Fe3O4 and AuNPs. Interestingly, the emission can be quenched by Fe3+ ions. MCOF-AuNPs combined with a specific aptamer (apt-MCOF-AuNPs) can capture Salmonella typhimurium selectively, and Fe3+-induced fluorescence quenching was inhibited based on the competitive binding of Fe3+ ions. A fluorescent assay strategy was constructed with excellent specificity and sensitivity. The detection limit is as low as 4 CFU/mL within 33 min. Furthermore, we developed smartphone-based software as an image reader and analyzer to determine S. typhimurium by capturing the change in fluorescence lightness, which can be assisted for point-of-care testing. Collectively, this innovative design showed a promising on-site detection approach for S. typhimurium.

Ultrasensitive fluorescence detection of microRNA through DNA-induced assembly of carbon dots on gold nanoparticles with no signal amplification strategy.

An ultrasensitive fluorescence assay strategy on the basis of carbon dots (CDs) and cDNA-modified gold nanoparticles (AuNP-cDNA) was developed for the determination of microRNA-21 (miRNA-21) via internal filtering effect (IFE). Positively charged CDs (PEI-CDs), the fluorophores in IFE, were synthesized via a hydrothermal method using polyethyleneimine (PEI) as surface ligand. The maximum emission wavelength is located at 500 nm under the excitation of 410 nm. AuNPs, the absorbers, were modified with single-stranded DNA (cDNA), which is completely complementary to miRNA-21. The fluorescence of PEI-CDs is quenched due to the assembly of PEI-CDs and AuNPs-cDNA. In the presence of miRNA-21, the hybridization between miRNA-21 and cDNA causes the release of PEI-CDs and the recovery of fluorescence intensity.The fluorescence recovery degree is linearly correlated with the logarithm of miRNA-21 concentration in the range of 1-1000 fM. This method can be applied to determine miRNA-21 in real serum samples, and the detection results are in well agreement with those of qRT-PCR. The determination of miRNA-21spiked into diluted human serum samples displays satisfactory recovery within the range88.44-112.7%, which confirmed the reliability for miRNAs detection in real samples.

Aptamer Conformation Switching-Induced Two-Stage Amplification for Fluorescent Detection of Proteins.

Basing on the conformation change of aptamer caused by proteins, a simple and sensitive protein fluorescent assay strategy is proposed, which is assisted by the isothermal amplification reaction of polymerase and nicking endonuclease. In the presence of platelet-derived growth factor (PDGF-BB), the natural conformation of a DNA aptamer would change into a Y-shaped complex, which could hybridize with a molecular beacon (MB) and form a DNA duplex, leading to the open state of the MB and generating a fluorescence signal. Subsequently, with further assistance of isothermal recycling amplification strategies, the designed aptamer sensing platform showed an increment of fluorescence. As a benefit of this amplified strategy, the limit of detection (LOD) was lowered to 0.74 ng/mL, which is much lower than previous reports. This strategy not only offers a new simple, specific, and efficient platform to quantify the target protein in low concentrations, but also shows a powerful approach without multiple washing steps, as well as a precious implementation that has the potential to be integrated into portable, low-cost, and simplified devices for diagnostic applications.

A novel fluorescence “turn off−on” nanosensor for sensitivity detection acid phosphatase and inhibitor based on glutathione-functionalized graphene quantum dots

In this paper, we developed a label-free and sensitive fluorescence sensor for acid phosphatase (ACP) and its inhibitor parathion-methyl (PM) detection based on glutathione-functionalized graphene quantum dots (GQDs@GSH). Upon addition of MnO2 nanosheets, the fluorescence of GQDs@GSH could be efficiently quenched via a fluorescence resonance energy transfer. ACP could easily catalyze the hydrolysis of L-Ascorbic acid-2-phosphate (AAP) to ascorbic acid (AA), which could reduce MnO2 nanosheets to Mn2+ in acidic environment, leading to dramatically increase of the fluorescence intensity of GQDs@GSH. Quantitative detection of ACP in a broad range from 0.1 to 9 mU mL−1 with a detection limit of 0.027 mU mL−1 could be achieved. The feasibility of the proposed sensor in real samples analysis was also studied and satisfactory results were obtained. Furthermore, the fluorescence assay strategy could also be used for the detection of parathion-methyl (PM) as ACP inhibitor.

Label-Free Fluorescent Determination of Sunset Yellow in Soft Drinks Based on an Indicator-Displacement Assay

This work reported a fluorescence sensing platform for Sunset Yellow (SY) determination based on competitive host-guest interaction between cucurbit[7]uril (CB7) and signal probe/target molecules. Luteolin/epigallocatechin gallate (EGCG) and SY were selected as the probe and target molecules, respectively. When luteolin or EGCG entered the CB7 host, its fluorescence significantly improved. However, upon the presence of SY in the performed luteolin·CB7 or EGCG·CB7 complex, this led to a remarkable decrease in fluorescence. This result was due to the fact that the binding constant of CB7/SY (4.9×104 M−1) was greater than that of CB7/luteolin (3.2×103 M−1) or CB7/EGCG (4.8×103 M−1). The fluorescence intensities of CB7/luteolin and CB7/EGCG complexes decreased linearly with increased SY concentration ranges of 0.5–50.0 and 2.0–50.0 μM. The proposed method had detection limits of 0.12 and 0.45 μM and was successfully used to determine SY samples with good recoveries ranging from 96.3% to 103.8%. This competitive mode provided a promising fluorescence assay strategy for potential applications in food safety.

Carbon dots for fluorescent detection of α-glucosidase activity using enzyme activated inner filter effect and its application to anti-diabetic drug discovery

Recently, α-glucosidase inhibitor has been widely used in clinic for diabetic therapy. In the present study, a facile and sensitive fluorescent assay based on enzyme activated inner filter effect (IFE) on nitrogen-doped carbon dots (CDs) was first developed for the detection of α-glucosidase. The N-doped CDs with green emission were prepared by a one-step hydrothermal synthesis and gave the fluorescence quantum yield of 30%, which were used as the signal output. Through α-glucosidase catalysis, 4-nitrophenol was released from 4-nitrophenyl-α-d-glucopyranoside (NGP). Interestingly, the absorption of 4-nitrophenol and the excitation of CDs were completely overlapping. Due to its great molar absorptivity, 4-nitrophenol was capable of acting as a powerful absorber to affect the fluorescent signal of CDs (i.e. IFE). By converting the absorption signals into fluorescence signals, the facile fluorescence assay strategy could be realized for α-glucosidase activity sensing, which effectively avoided the complex modification of the surface of CDs or construction of the nanoprobes. The established IFE-based sensing platform offered a low detection limit of 0.01 U/mL (S/N = 3). This proposed sensing approach has also been expanded to the inhibitor screening and showed excellent applicability. As a typical α-glucosidase inhibitor, acarbose was investigated with a low detection limit of 10−8 M. This developed method enjoyed many merits including simplicity, lost cost, high sensitivity, good reproducibility and excellent selectivity, which also provided a new insight on the application of CDs to develop the facile and sensitive biosensor.

Spectrum‐Based Electrochemiluminescence Immunoassay for Selectively Determining CA125 in Greenish Waveband

AbstractSpectrum‐based and color‐selective electrochemiluminescence (ECL) is crucial for the multicolored ECL assay strategy. Herein, a spectrum‐based ECL immunoassay of high color purity was performed on a home‐made ECL‐acquiring system by spectrally imaging the ECL emission of highly passivated CdSe nanocrystals (NCs), that is, the ECL tags, which were labelled onto sandwich‐type immuno‐complexes with CA125 as the target molecule. The ECL tags could give off efficient ECL around 555 nm with a full width at half‐maximum of 39 nm, so that the spectrum‐based ECL strategy could color‐selectively determine CA125 with a linear response range from 10−4 to 1 U/mL and a limit of detection of 5×10−5 U/mL. The intrinsically improved signal‐to‐noise ratio of ECL to fluorescence might eventually enable the spectrum‐based ECL immunoassay to be a promising alternative to the NCs‐based multicolored fluorescent assay strategy.

Sensitive detection of DNA methyltransferase activity based on supercharged fluorescent protein and template-free DNA polymerization

DNA methylation, catalyzed by DNA methyltransferases (MTases), is a key component of genetic regulation, and DNA MTases have been regarded as potential targets in anticancer therapy. Herein, based on our previously developed DNA-mediated supercharged green fluorescent protein (ScGFP)/graphene oxide (GO) interaction, coupled with methylation-initiated template- free DNA polymerization, we propose a novel fluorescence assay strategy for sensitive detection of DNA MTase activity. A hairpin DNA with a methylation-sensitive site and an amino-modified 3'-terminal (DNA-1) was designed and worked as a starting molecule. In the presence of DNA MTase, methylation-sensitive restriction endonuclease, and terminal deoxynucleotidyl transferase (TdT), DNA-1 can be sequentially methylated, cleaved, and further elongated. The resulting long DNA fragments quickly bind with ScGFP and form the ScGFP/DNA nanocomplex. Such nanocomplex can effectively protect ScGFP from being adsorbed and quenched by GO. Without the methylation-initiated DNA polymerization, the fluorescence of ScGFP will be quenched by GO. Thus, the DNA MTase activity, which is proportional to the amount of DNA polymerization products, can be measured by reading the fluorescence of ScGFP/GO. The method was successfully used to detect the activity of DNA adenine methylation (Dam) MTase with a wide linear range (0.1–100 U/mL) and a low detection limit of 0.1 U/mL. In addition, the method showed high selectivity and the potential to be applied in a complex sample. Furthermore, this study was successfully extended to evaluate the inhibition effect of 5-fluorouracil on Dam MTase activity and detect TdT activity.

A fluorescence resonance energy transfer (FRET) based “Turn-On” nanofluorescence sensor using a nitrogen-doped carbon dot-hexagonal cobalt oxyhydroxide nanosheet architecture and application to α-glucosidase inhibitor screening

The medicines targeted at α-glucosidase played an important role in anti-diabetes and anti-HIV therapy. Unfortunately, the method based on fluorescent assay strategy for α-glucosidase inhibitor screening remains poorly investigated. In this study, a novel “Turn On” fluorescence sensor platform has been developed for trace α-glucosidase inhibitor screening from natural medicines. Firstly, carbon dots were prepared by one-pot synthesis and used as the signal output. Combining with the carbon dots, cobalt oxyhydroxide (CoOOH) nanoflakes were employed to build the fluorescence resonance energy transfer (FRET) based sensor platform. Secondly, L-ascorbic acid-2-O-α-D-glucopyranosyl (AAG) was innovatively introduced as α-glucosidase substrate. With hydrolysis of AAG by α-glucosidase, ascorbic acids (AA) were released that can rapidly reduce CoOOH nanoflakes to Co2+, and then FRET was stopped accompanying with the fluorescence recovery of CDs. The sensor platform was ultrasensitive to AA with a detection limit of 5nM, ensuring the sensitive monitoring of enzyme activity. Acarbose was used as the inhibitor model and its inhibition rate is proportional to the logarithm of concentration in range of 10−9–10−3M with the correlation coefficient of R2=0.996, and an ultralow limit of detection of ~1×10−9M was obtained. The inhibiting ability of seven compounds isolated from natural medicines was also evaluated. The constructed sensor platform was proven to be sensitive and selective as well as cost-effective, facile and reliable, making it promising as a candidate for trace α-glucosidase inhibitor screening.

Supramolecular interaction of labetalol with cucurbit[7]uril for its sensitive fluorescence detection.

This work studied the host-guest interaction between cucurbit[7]uril (CB[7]) and labetalol in acidic aqueous solution and proposed a simple competitive method for fluorescence detection of labetalol. The binding constant of labetalol-CB[7] was (1.83 ± 0.22) × 10(6) M(-1), which was greater than those of palmatine-CB[7], berberine-CB[7], and coptisine-CB[7] complexes. The fluorescence intensity of palmatine-CB[7], berberine-CB[7], and coptisine-CB[7] complexes decreased linearly with increasing concentration of labetalol ranging from 0.014 to 2.06, 0.014 to 1.15, and 0.034 to 1.23 μM, respectively. Based on the competitive interaction, the proposed detection method for labetalol showed limits of detection of 4.9 nM, 4.9 nM, and 12.0 nM, respectively, and was successfully applied for the determination of labetalol in human urine samples with good precision and recoveries from 95.4% to 102.5%. Moreover, it could be employed to monitor the time-dependent concentration of labetalol in urine from a healthy volunteer after oral medication. The superstructure-based competitive mode provided a promising fluorescence assay strategy for various potential applications.

Using copper ions to amplify ROS-mediated fluorescence for continuous online monitoring of extracellular glucose in living rat brain

In this study we developed a facile and sensitive method for continuous monitoring of extracellular glucose concentration in living rat brain through microdialysis (MD) sampling in conjunction with (i) online sample derivatization using glucose oxidase to generate H2O2, which converted a reactive oxygen species-responsive fluorescent dye, 2′,7′-dichlorodihydrofluorescein (DCFH), into fluorescent species, and (ii) a novel non-immobilized enzyme-based fluorescence assay strategy, featuring copper ion (Cu2+)-facilitated amplification of the fluorescence intensity. After evaluating the experimental conditions for glucose oxidation and fluorescence generation, the introduction of Cu2+ ions to this system resulted in an additional 51-fold amplification of the net fluorescence intensity. By sequentially loading brain microdialysate into the dual sample collection loops, the sampling frequency was 7.5h−1. Based on a 40-μL sample volume, the system's detection limit reached as low as 0.18mM, sufficiently accurate to determine the extracellular glucose concentrations in living rat brains. To demonstrate the proposed system's practical performance and applicability, we conducted (i) spike analyses of biomolecule-rich fetal bovine serum sample, confirming that the analytical reliability was similar to that of a commercial glucose kit, and (ii) in vivo dynamic monitoring of the extracellular glucose concentrations in living rat brains after inducing neural depolarization by perfusing a high-K+ medium from the MD probe.

Target-mediated consecutive endonuclease reactions for specific and sensitive homogeneous fluorescence assay of O6-methylguanine-DNA methyltransferase

O6-Methylguanine-DNA methyltransferase (MGMT) is one of the most important DNA-repair enzymes. Herein, a simple, sensitive and selective homogeneous fluorescence assay strategy is developed for the detection of MGMT on the basis of target-mediated two consecutive endonuclease reactions. The activity assay of MGMT is firstly accomplished using a hairpin-structured DNA substrate to offer a specific recognition site on the substrate DNA for restriction endonuclease PvuII, and thus to initiate the first endonuclease reaction. The product which activates the second endonuclease reaction allows an efficient amplification approach to create an abundance of fluorescence signal reporters. The first endonuclease reaction offers the method high specificity and the second one furnishes the assay improved sensitivity. The results reveal that the MGMT assay strategy shows dynamic responses in the concentration range from 1 to 120ngmL−1 with a detection limit of 0.5ngmL−1. By simply altering the alkylated bases, this strategy can also be extended for the detection of other alkyltransferases. Therefore, the developed strategy might provide an intrinsically convenient, sensitive and specific platform for alkyltransferase activate assay and related biochemical studies due to its label-free, homogeneous, and fluorescence-based detection format.

A Ligation Triggered Label-Free Fluorescent Assay for Adenosine-Triphosphate Based on Nicking Endonuclease Signal Amplification and Ligand Responsive G-Quadruplex Formation

A novel label-free fluorescent assay strategy was developed for sensitive and selective detection of adenosine-triphosphate (ATP) based on nicking endonuclease signal amplification (NESA) and N-Methyl mesoporphyrin (NMM)-responsive G-quadruplex formation. In this assay, the nick in the specially designed DNA duplex can be sealed by T4 DNA ligase in the presence of ATP. The following replication and strand displacement yield the duplex that contains the full recognition site for nicking endonuclease. Nicking enzyme's cutting the replicated DNA duplex results in a new replication site for polymerase and the continuous displacement of the nicked G-rich strand. The nicked strand can fold into G-quadruplex structure in the presence of K+ and selectively binds with NMM, yielding a significant fluorescence signal. Under the optimal conditions, the fluorescent assay strategy showed a dynamic response to ATP in the concentration range from 10 nM to 1000 nM and a detection limit of 0.8 nM, which compared favorably with most of the previous ATP assay methods. In addition, the proposed strategy exhibited superior selectivity toward ATP over other nucleosides and its analogues. The fluorescent assay was also applied in detecting ATP in serum with satisfactory results.

A sensitive, homogeneous fluorescence assay for detection of thymine DNA glycosylase activity based on exonuclease-mediated amplification

A novel homogeneous fluorescence assay strategy for highly sensitive detection of thymine DNA glycosylase (TDG) enzyme activity based on the exonuclease-mediated signal amplification reaction was reported.

Endonucleolytic Inhibition Assay of DNA/Fok I Transducer as a Sensitive Platform for Homogeneous Fluorescence Detection of Small Molecule–Protein Interactions

This paper reported a novel homogeneous fluorescence assay strategy for probing small molecule-protein interactions based on endonucleolytic inhibition of a DNA/Fok I transducer. The transducer could cyclically cleave fluorescence-quenched probes to yield activated fluorescence signal, while protein binding to the small molecule label would prevent Fok I from approaching and cleaving the fluorescence-quenched probes. Because of the efficient signal amplification from the cyclic cleavage operation, the developed strategy could offer high sensitivity for detecting small molecule-protein interactions. This strategy was demonstrated using folate and its high-affinity or low-affinity binding proteins. The results revealed that the developed strategy was highly sensitive for detecting either high- or low-affinity small molecule-protein interactions with improved selectivity against nonspecific protein adsorption. This strategy could also be extended for assays of candidate small-molecule ligands using a competitive assay format. Moreover, this strategy only required labeling the small molecule on a DNA heteroduplex, circumventing protein modifications that might be harmful for activity. In view of these advantages, this new method could have potential to become a universal, sensitive, and selective platform for quantitative assays of small molecule-protein interactions.

Hybridization-triggered isothermal signal amplification coupled with MutS for label-free and sensitive fluorescent assay of SNPs

Combining the specific recognization of MutS protein for mismatched DNA sequences with the target-driven molecular switch that acts as both the template and primer of the polymerization reaction, a new label-free and sensitive fluorescent assay strategy for specific single-stranded DNA sequences or SNPs is proposed.