Dual-readout Assay Research Articles

Development of MnO2 nanosheets nanozyme-based colorimetric and photothermal dual-signal platform for monitoring alkaline phosphatase activity

Herein, a dual-signal biosensing platform for monitoring alkaline phosphatase (ALP) activity was developed. This platform combined the oxidase-mimicking activity of MnO2 nanosheets (NS) with the colorimetric and photothermal properties of oxidized 3,3′,5,5′-tetramethylbenzidine (oxTMB). MnO2 NS effectively catalyzed the oxidation of TMB to oxTMB, resulting in a color change from colorless to blue and a significant light absorption band at 652 nm. Meanwhile, oxTMB demonstrated pronounced photothermal properties, with the solution exhibiting a notable increase in temperature upon irradiation with an 808 nm laser. In the presence of ALP, 2-Phospho-L-ascorbic acid trisodium salt (AAP) was converted into L-ascorbic acid (L-AA), which then reacted with MnO2 NS. Consequently, MnO2 NS was inhibited from oxidizing TMB to oxTMB. Based on this principle, a dual-readout assay that integrated both colorimetric and photothermal signals for ALP detection was developed. The detection limits were 0.08 mU/mL for the colorimetric method and 0.12 mU/mL for the photothermal method. Furthermore, the platform was successfully applied to human serum samples, demonstrating its effectiveness in detecting ALP in complex biological matrices. The combination of colorimetric and photothermal responses enhanced both the accuracy and reliability of ALP detection, making this platform a promising tool for biomedical applications.

Fluorescent and Colorimetric Dual-Readout Immunochromatographic Assay for the Detection of Phenamacril Residues in Agricultural Products.

The fungicide phenamacril has been employed to manage Fusarium and mycotoxins in crops, leading to persistent residues in the environment and plants. Detecting phenamacril is pivotal for ensuring environmental and food safety. In this study, haptens and artificial antigens were synthesized to produce antiphenamacril monoclonal antibodies (mAbs). Additionally, gold nanoparticles coated with a polydopamine shell were synthesized and conjugated with mAbs, inducing fluorescence quenching in quantum dots. Moreover, a dual-readout immunochromatographic assay that combines the positive signal from fluorescence with the negative signal from colorimetry was developed to enable sensitive and precise detection of phenamacril within 10 min, achieving detection limits of 5 ng/mL. The method's reliability was affirmed by using spiked wheat flour samples, achieving a limit of quantitation of 0.05 mg/kg. This analytical platform demonstrates high sensitivity, outstanding accuracy, and robust tolerance to matrix effects, making it suitable for the rapid, onsite, quantitative screening of phenamacril residues.

Colorimetric and dynamic light scattering dual-readout assay for formaldehyde detection based on the hybridization chain reaction and gold nanoparticles

A novel colorimetric and dynamic light scattering dual-readout assay for FA detection was developed based on the HCR by using AuNPs.

Fluorometric and colorimetric quantitative analysis platform for acid phosphatase by cerium ions-directed AIE and oxidase-like activity.

A facile and sensitive fluorescent and colorimetric dual-readout assay for detection of acid phosphatase (ACP) was developed via Ce(III) ions-directed aggregation-induced emission (AIE) of glutathione-protected gold nanoclusters (GSH-AuNCs) and oxidase-mimicking activity of Ce(IV) ions. Free Ce(IV) ions exhibited a strong oxidase-mimetic activity, catalytically oxidizing colorless 3,3',5,5'-tetramethylbenzidine (TMB) into its blue product oxTMB in the presence of dissolved O2, thus triggering a remarkable color reaction detectedvisually. ACP can hydrolyze L-ascorbic acid-2-phosphate (AAP) with the production of ascorbic acid (AA). The AA is able to reduce Ce(IV) ions to Ce(III) ions, thus quenching the oxidase-mimetic activity of Ce(IV) ions. Meanwhile, Ce(III) ions induce AIE of GSH-AuNCs, resulting in the enhancement of thefluorescence signal of GSH-AuNCs. Both the fluorescent and colorimetric dual-mode analysis platforms exhibit a sensitive response to ACP, providing detection limits as low as 0.101 U/L and 0.200 U/L, respectively. Besides, this fabricated dual-mode detection platform holds the potential for analysis of ACP in human serum samples and screening inhibitors for ACP. With good performance and practicability, this study shows promising application in the convenient and reliable determination of ACP activity.

Dual-readout assay for determination of Ag+ and Cu2+ based on in situ fluorogenic and chromogenic reaction between dopamine and naphthoresorcin

Silver ions (Ag+) and copper ions (Cu2+) are toxic environmental pollutants, and their excess accumulation can cause adverse effects on human health. Thus, the development of a simple, convenient, economic and sensitive sensor for Ag+ and Cu2+ is an urgent need. Herein, we find that the fluorogenic and chromogenic reaction between dopamine (DA) and naphthoresorcin (NR) could be effectively facilitated by Ag+ or Cu2+, with significant increase of fluorescent and colorimetric signals. Accordingly, we report a novel “turn-on” dual-mode sensor for directly fluorescent and colorimetric detection of Ag+ and Cu2+. Mechanistic investigation reveals that Ag+ serves as an oxidant while Cu2+ acts as a catalyst to facilitate DA and NR reaction. By incorporating EDTA and NaCl, Ag+ and Cu2+ could be selectively measured at concentrations as low as 157.1 nM and 11.9 nM, respectively. Applicability of this sensor to detect Ag+ and Cu2+ in real water samples has also been demonstrated through a mix-and-detect procedure, and the detection results obtained by dual-readout modes agree well with correspond to those from inductively coupled plasma–mass spectroscopy (ICP-MS). This dual-mode detection method provides the advantages of simplicity, easy-operation and low experimental cost without any sophisticated synthesis, modification or labeling process, which is promising for inspection of Ag+ and Cu2+ in environmental water samples.

A novel functionalized CdTe@MOFs based fluorometric and colorimetric biosensor for dual-readout assay of creatinine

Creatinine (Cre) is a crucial biomarker in the clinical diagnosis and therapy of chronic kidney disease (CKD) and muscle diseases. Therefore, designing and developing highly sensitive, multi-signal assays for the detection of Cre is highly significant. Herein, by in situ encapsulating CdTe QDs and post-synthetically modifying pyridine-2-carboxaldehyde (2-PC), a novel ratiometric fluorescence and colorimetric dual-signal sensor CdTe@UiO-66-PC-Cu was constructed for the detection of Cre with low detection limits (LOD) 2.48 nM and 1.78 μM, respectively. Satisfactory recovery of 93.0–105.0% (RSD < 4.8%, n = 3) was also acquired for determination of Cre in actual samples. In addition, by integrating smartphone-based portable colorimetric sensing, a novel hydrogel-based colorimetric paper analytical device (c-PAD) platform was fabricated for the visual colorimetric assay of Cre in whole blood without any separation process. The dual-signal sensing system of ratiometric fluorescence and colorimetry can observably improve the sensitivity and accuracy of Cre detection. Meanwhile, this portable smartphone-integrated colorimetric sensing platform features low cost, operational simplicity, and broad adaptability, providing an ideal way to achieve point-of-care testing (POCT) of other small molecule biomarkers in human whole blood.

Highly sensitive and selective colorimetric and fluorescent dual-readout assay for the analysis of spermine, spermidine and cysteine

Fluorescent cuprous iodide (CuI)-based coordination polymers (CuI-CPs) were widely used in the sensor field for high selectivity and sensitivity. Here, a new CuI-CP fluorescent probe was fabricated for the detection of spermine, spermidine and cysteine with dual-mode, which was capable of both convenient colorimetric analysis and ratiometric fluorescent detection. The fluorescence of CuI-CP solution was quenched and the solution changed from white to purple upon addition of spermine or spermidine, while the solution changed from white to yellow for cysteine. This probe exhibited selective, sensitive, rapid and linear response to spermine, spermidine and cysteine with a limit of detection of 0.57, 1.06 and 1.18 μM, respectively. Furthermore, cellulose acetate was used as the matrix to fabricate intelligent fluorescent film for real-time and visual monitoring the freshness of pork samples based on distinct fluorescence changes under UV light. This work realized a rapid, efficient, and profitable method for visually evaluating food quality.

On-site quantitation of xanthine in fish and serum using a smartphone-based spectrophotometer integrated with a dual-readout nanosensing assay

Rapid and quantitative biochemical analysis at points-of-need is imperative for food safety inspection. This work reports on: 1) a stand-alone smartphone-based “two-in-one” spectrophotometer (the SAFS) installed with a self-developed application (the SAFS-App) which can precisely collect both absorption spectra and fluorescence spectra in a reproducible manner within 5 s; and 2) a straightforward protocol for xanthine detection using fluorescent carbon nanodots and silver nanoparticles. The assay performed with the SAFS demonstrates high specificity towards xanthine, and a linear range of 1–60 μM with LODs of 0.38 and 0.58 μM for colorimetric and fluorometric readouts, respectively. The reliability and robustness of the SAFS are validated by on-site quantitation of xanthine in fish and serum samples, with comparable accuracy to HPLC method. More importantly, the SAFS presents itself as an appealing device which is accessible to everyone through the Internet of Things and can be tailored for diverse point-of-care testing applications.

Cell-free transcription-translation system: a dual read-out assay to characterize riboswitch function.

Cell-free protein synthesis assays have become a valuable tool to understand transcriptional and translational processes. Here, we established a fluorescence-based coupled in vitro transcription-translation assay as a read-out system to simultaneously quantify mRNA and protein levels. We utilized the well-established quantification of the expression of shifted green fluorescent protein (sGFP) as a read-out of protein levels. In addition, we determined mRNA quantities using a fluorogenic Mango-(IV) RNA aptamer that becomes fluorescent upon binding to the fluorophore thiazole orange (TO). We utilized a Mango-(IV) RNA aptamer system comprising four subsequent Mango-(IV) RNA aptamer elements with improved sensitivity by building Mango arrays. The design of this reporter assay resulted in a sensitive read-out with a high signal-to-noise ratio, allowing us to monitor transcription and translation time courses in cell-free assays with continuous monitoring of fluorescence changes as well as snapshots of the reaction. Furthermore, we applied this dual read-out assay to investigate the function of thiamine-sensing riboswitches thiM and thiC from Escherichia coliand the adenine-sensing riboswitch ASW from Vibrio vulnificusand pbuE from Bacillus subtilis, which represent transcriptional and translational on- and off-riboswitches, respectively. This approach enabled a microplate-based application, a valuable addition to the toolbox for high-throughput screening of riboswitch function.

Ultrasensitive colorimetric and fluorescent dual-readout assay for D-penicillamine based on the chloride boosted oxidative ability of copper ions

Dual-readout protocols with two signal transduction channels can significantly improve the detection precision and diversity. In contrast to the previous tedious and time-consuming preparation of two response materials, a convenient and commercially available small molecule o-Phenylenediamine (OPD)-based dual-mode system was developed for the ultrasensitive detection of D-penicillamine (D-PA). Fortunately and interestingly, it is demonstrated that chloride ions (Cl−) could largely enhance the oxidative ability of copper ions (Cu2+) and improve the reaction rate and efficiency of the Cu2+-OPD system, leading to the boosted colorimetric and fluorescent signals and shorter reaction time. Meanwhile, D-PA could strongly bind with Cu2+ to form a stable Cu2+-D-PA complex, thereby inhibiting the aforementioned oxidation process and exhibiting decrease of absorbance values and fluorescence intensity. Inspired by the excellent analytical performance, an “INH” molecular logic gate was devised by employing Cl− and D-PA as the inputs, and the dual-mode strategy has been successfully employed to probe D-PA in tablets and human serum samples. Such cost-effective yet efficacious analytical tool not only improve the sensing performance of the Cu2+-OPD reaction system, but also enhance the detection sensitivity for D-PA and promote the application and development of OPD-based reactions.

The self-nitrogen-doped carbon quantum dots derived from Morus alba L. leaves for the rapid determination of tetracycline

Morus alba L. (Mulberry), one of industrial crops, have been widely cultivated in China, mulberry leaves (Folium Mori), one of the main products of Morus alba L., is high yield and rich in nitrogen element, high-value-utilization of mulberry leaves is very important for agricultural economic development. Carbon quantum dots have been attracting extensive attention because of their excellent properties including tunable fluorescence, bio-compatibility, non-toxicity, high water-solubility, and ease of functionalization. Heteroatoms doping is an effective way to improve quantum yield and fluorescence performance of carbon quantum dots. In this work, the self-nitrogen-doped carbon quantum dots was prepared from mulberry leaves, a nitrogen-rich, economic value-high and eco-friendly crops products, via a facile one-step hydrothermal method. The as-prepared self-nitrogen-doped carbon quantum dots exhibited certain stability, solubility and fluorescence intensity (quantum yield is 9.3%). With the introduction of tetracycline, the efficiency fluorescence quenching was observed, and an obvious color change can also be identified through naked eye. The fluorescence quenching mechanism is induced by internal filtering effect and static quenching. Based on this phenomenon, a rapid, naked-eye, and dual-readout assay for tetracycline detection was constructed and prepared as a fluorescent test paper. The linear range and detection limit of tetracycline were 0.158–40 μM and 158 nM, respectively. Other types of antibiotics, comment ions and chemicals show little influence on tetracycline detection. Furthermore, the constructed dual-readout assay was employed to detect tetracycline in river water with recovery range of 98.6–102.2%, relative standard deviation less than 2.54%. The constructed fluorescence probe show potential application to tetracycline detection in the real water sample.

An enzyme-activatable dual-readout probe for sensitive β-galactosidase sensing and Escherichia coli analysis

Rapid and accurate sensing of β-galactosidase (β-gal) activity is particularly critical for the early detection of many diseases and has become a topic of interest in recent years. However, most traditional probes for β-gal sensing often suffer from the disadvantages of narrow dynamic range, low reaction efficiency and are only employed with either colorimetric or fluorescence sensing. Furthermore, β-galactosidase sensing based assay for efficient detection and antibiotic resistance analysis of Escherichia coli (E.coli) is not available. Here, an enzyme-induced probe assay was reported for dual sensitive fluorescence and colorimetric measurement of β-gal activity, and was further employed for detection of Escherichia coli and their antibiotic resistance analysis. The DCM-βgal probe was virtually non-emissive in aqueous solution, while it could be activated by β-gal to produce bright emission. Under optimized conditions, DCM-βgal displayed high sensitivity, selectivity and rapid response to β-gal with a low detection limit of 1.5 × 10-3 Uml-1. Importantly, this assay was successfully applied to sensitive detection of E. coli cells with a fast detection process within 5h and a low detection concentration of 1 × 103CFUml-1. Furthermore, the enzyme-activatable assay was also successfully applied for high throughput E. coli antibiotic resistance analysis. The DCM-βgal strategy is applied for the first time on the detection of E. coli cells and their antibiotic resistance analysis. It is provided with the advantages of high selectively, a simple operation, low cost and rapid detection. The detection platform can also be extended to analyze the level of β-gal in other types of cells or biological samples. Overall, the simple, effective and dual-readout assay holds promise for efficient sensing of β-gal activity and provides a potential tool for E. coli detection and their antibiotic resistance analysis.

Green-synthesized two-dimensional nanocarriers with photothermal capability for portable dual-readout immunochromatographic assay of clenbuterol

Immunochromatographic test strips (ITSs) have gained great practicability in point-of-care testing (POCT). Developing novel labels of ITSs with an additional signal to carry out quantitative analysis favors the development of sensitive POCT. Herein, tannic acid (TA) assisted stripping WS2 nanosheets (TA-WS2 NSs) based on colorimetric/photothermal ITS (TWITS) for clenbuterol (CLE) monitoring. After WS2 NSs loaded TA, it retained excellent photothermal performance and an intact surface structure for monoclonal antibody (mAb) labeling, at the same time, the stability of WS2 NSs and the activity of mAb were improved effectively. Satisfactory, the visual limit of detection (vLOD) of the photothermal signal was 0.3 ng/mL, which was enhanced 7-fold at least compared with traditional gold-based nanoparticles ITS (GNPs ITS). Importantly, this work can offer a new referenced method for other small molecule pollutants.

A fluorescent and colorimetric sensor based on ionic liquids for the on-site monitoring trace gaseous SO2

SO2 could cause severe environmental pollution and health threat, so real-time and on-site monitoring of SO2 has attracted considerable attention. This work proposed a novel ionic liquid-based sensor, called trihexyl (tetradecyl) phosphonium fluorescein ionic liquid, which can accurately detect SO2 with its fluorescent and colorimetric dual-readout assay without seventeen gases interference (eg: NO, N2, CO2, O2, COS, HCl, CHCl3). GC-MS was also used to verify the validation of the detection method. First, this fluorescein-based IL sensor exhibited fluorescence green and colorimetric yellow signals. When the sensor was exposed to gaseous SO2, the green fluorescence quenched, and the colorimetric yellow color faded due to chemical bond interaction. Also, the proposed IL sensor exhibited good linearity in the SO2 concentration range of 5.0–95.0 ppm with a detection limit of 0.9 ppm (fluorescence) and 1.9 ppm (colorimetry), and recoveries of 97%∼103% with RSD less than 1.21%. Besides, the IL sensor could be easily assembled into a paper device by simple immersion, and the paper strip was exploited to realize a semiquantitative visual detection of SO2. These results indicated that the proposed fluorescence-colorimetric dual-signal chemosensor could be used as intelligent paper labels for real-time and on-site monitoring of SO2 in ambient air.

Synthesis, Molecular Modeling Study, and Quantum-Chemical-Based Investigations of Isoindoline-1,3-diones as Antimycobacterial Agents.

The condensation of phthalic anhydride afforded structurally modified isoindoline-1,3-dione derivatives with selected amino-containing compounds. The title compounds (2–30) have been characterized by thin-layer chromatography (TLC), infrared spectroscopy, 1H and 13C NMR spectroscopy, and mass spectroscopy. All of the compounds were assessed for their antimycobacterial activity toward the H37Rv strain by a dual read-out assay method. Among the synthesized compounds, compound 27 possessed a significant IC50 of 18 μM, making it the most potent compound of the series. The InhA inhibitory (IC50) activity of compound 27 was 8.65 μM in comparison to Triclosan (1.32 μM). Computational studies like density functional theory (DFT) study, molecular docking, and dynamic simulation studies illustrated the reactivity and stability of the synthesized compounds as InhA inhibitors. A quantum-mechanics-based DFT study was carried out to investigate the molecular and electronic properties, reactivities, and nature of bonding present in the synthesized compounds and theoretical vibrational (IR) and isotropic value (1H and 13C NMR) calculations.

Fluorescent and colorimetric detection of Norfloxacin with a bifunctional ligand and enzymatic signal amplification system

We report a dual signal read-out enzyme-linked immunosorbent assay (ELISA) strategy for the sensitive detection of Norfloxacin (Nor) using a bifunctional ligand and enzymatic signal amplification system. In the proposed method, the immune response of an anti-Nor antibody against Nor-Biotin bifunctional ligands triggers the immobilization of streptavidin–alkaline phosphatase and the alkaline-phosphatase could continuous triggers the hydrolysis of L-ascorbic acid-phosphoric acid to produce abundant L-ascorbic acid (AA). Colorimetric signal is generated from the aggregation of azide and alkyne-based colloidal gold generated by a click-chemistry reaction with Cu+ catalysis (reduced by the AA), which changes its color from red to blue. A fluorescence signal is generated by the opening of the spirolactam ring caused by unreacted Cu2+ binding to the Cu2+ probe. Our method was successfully illustrated by detecting Nor in the range of3.18 × 10−2 to 6.88 × 103 pg/mL.The efficient detection of Nor was further confirmed using Nor added to commercially available milk, with a recovery in the range of 98.65%–104.01%. This work provides a promising method to detect Nor and other veterinary drug residues to guarantee food safety.

Enzyme-free ratiometric fluorescence and colorimetric dual read-out assay for glyphosate with ultrathin g-C3N4 nanosheets

Herein, a sensing platform for the rapid quantitative detection of glyphosate with high sensitivity and selectivity was constructed by enzyme-free ratiometric fluorescence and colorimetric dual read-out assay based on ultrathin g-C3N4 nanosheets (UCN NS).Fluorescent diaminophenylazine (DAP) with fluorescence was from the oxidation of o-phenylenediamine (OPD) in the presence of Cu2+. DAP could inspire a significant fluorescence quenching of nanosheets due to photo-induced electron transfer (PET), therefore exhibiting a ratiometric fluorescence response. In the presence of glyphosate, the strong chelation of Cu2+ by glyphosate inhibited the oxidation of OPD, which reduced the fluorescence intensity of DAP, while the fluorescence of the ultrathin nanosheets was restored. Moreover, the oxidation of OPD (colorless) to DAP (yellow) was a distinctly visible color change process, which provides well-suited conditions for colorimetric analysis. At the optimum experimental conditions, the detection concentration range of glyphosate was 0.001–0.48 μg/mL, and the limit of fluorometric detection was 0.49 ng/mL (S/N = 3). This assay was simple and easy to use for the detection of glyphosate in food safety and the environment with satisfactory results.

Biocatalysis-mediated MOF-to-prussian blue transformation enabling sensitive detection of NSCLC-associated miRNAs with dual-readout signals

Sensitive and accurate miRNAs assay is critical for early diagnosis of non-small-cell lung carcinomas (NSCLC). Herein, we demonstrate a photothermal and electrochemical dual-readout assay method for miRNA detection based on a novel biocatalysis-mediated MOF-to-prussian blue (PB) transformation (BMMPT) strategy and the catalytic hairpin assembly (CHA) amplification strategy. It is found that the Fe2+-based MOF (MOF-Fe2+) can act as the Fe2+ source to react with K3[Fe(CN)6], leading to the in-situ formation of prussian blue (PB) on MOF-Fe2+. Due the inherent near-infrared (NIR) photothermal conversion ability and electrochemical signal of PB, the resulting PB@MOF-Fe2+ is employed to arouse temperature readout or electrochemical signal. The presence of target miRNA-21 triggers the CHA reaction on magnetic beads (MBs), resulting the capture of numerous glucose oxidase (GOx) tags on MBs. The GOx tags then catalyze the generation of H2O2 using glucose as substrate. The H2O2 is used to inhibit the MOF-to-PB transformation process by oxidizing Fe2+ into Fe3+, leading to the decrease in temperature and electrochemical readout aroused by PB@MOF-Fe2+. By this means, a signal-off assay mode with dual readout is established for miRNA-21. Under the optimal conditions, using temperature readout or electrochemical readout, miRNA-21 can be detected at concentrations as low as 0.3 fM and 0.32 fM, respectively. Moreover, the developed method is successfully applied to evaluate the expression level of miRNA-21 in serum of NSCLC patients. This work not only provides a practical tool for NSCLC diagnosis but also presents the new features of MOF materials as signal transduction tags.

Green and facile synthesis of iron-doped biomass carbon dots as a dual-signal colorimetric and fluorometric probe for the detection of ascorbic acid

A new method based on biomass Fe-CDs with fluorescence properties and simulated oxidase activity colorimetric and fluorometric dual-readout assay for highly effective detection of AA was established.

Ratiometric fluorescence and chromaticity dual-readout assay for β-glucuronidase activity based on luminescent lanthanide metal-organic framework

β-Glucuronidase (GCU) is a biomarker of many malignant tumors. The accurate and sensitive detection of GCU is of significance for the early diagnosis of diseases. Herein, we proposed a ratiometric fluorescence/chromaticity dual-readout assay method for GCU activity by integrating a luminescent lanthanide metal-organic framework (MOF) and fluorescence turn-on enzymolysis reaction. MOF-253 synthesized by Al3+ and 2,2′-bipyridine-5,5′-dicarboxylic acid was used as scaffold to prepare Eu3+@MOF-253 with red emission, which plays multiple roles in GCU assay including fluorescence internal standard, synergistic enzyme catalysis and chromaticity shift enhancement. 4-Methylumbelliferyl-β-glucuronide was selected as substrate and hydrolyzed by GCU to produce 4-methylumbelliferyl with blue emission, which can quench the fluorescence of Eu3+@MOF-253 due to internal filter effect. Therefore, the turn-on/off ratiometric fluorescence assay method of GCU was established, and it exhibits a wide linear range of 0.1–50 U L−1, low limit of detection (LOD) of 0.03 U L−1 and good selective response. Furthermore, a portable device with illuminometer as RGB signal readout was designed to perform the ratiometric chromaticity visual assay of GCU, which has the linear range of 0.2–40 U L−1 and the LOD of 0.06 U L−1. The dual-readout method was applied to detect GCU in human serum and shows high accuracy and precision (recovery of 92.4–114.1%, relative standard deviation < 7.9%). Both the ratiometric fluorescence and the ratiometric chromaticity assay get the same results as the classical colorimetric assay. To our knowledge, this is the first report on the ratiometric fluorescence/chromaticity dual-readout assay of GCU activity.