Dual-readout Assay Research Articles

A dual-signal readout sensor for highly sensitive detection of iodide ions in urine based on catalase-like reaction of iodide ions and N-doped C-dots

In this work, a novel colorimetric and ratiometric fluorescence detection method for iodide ions (I−) was developed. In the presence of I−, the color of N-doped C-dots/H2O2/OPD system aqueous solution varied from colorless to yellow due to 2,3-diaminophenazine (OPDox) produced from I−-catalyzed H2O2-o-phenylenediamine (OPD) reaction. At the same time, the OPDox had an excellent fluorescence (FL) property. Under the excitation wavelength at 390nm, a novel FL emission ascribed to OPDox was clearly observed at 565nm. In addition, the FL at 565nm gradually increased, while the FL of emission peak at 450nm attributed to N-doped C-dots gradually decreased with the increasing concentration of I− due to the FL resonance energy transfer (FRET) between N-doped C-dots and OPDox. Therefore, a dual-signal optical sensor was developed for detection of I−. Under the optimized conditions, the linear response to I− was in the range of 0.09–50μM and the limit of detection was 0.06μM. Moreover, this proposed assay was successfully applied for the detection of I− in urine. These results indicate that the colorimetric and ratiometric fluorescence dual-readout assay method has great potential for applications in physiological and pathological diagnosis.

One-Pot Hydrothermal Synthesis of Carbon Dots with Efficient Up- and Down-Converted Photoluminescence for the Sensitive Detection of Morin in a Dual-Readout Assay

Blue luminescent carbon dots (CDs) with a high photoluminescence (PL) quantum yield (48.3 ± 5.3%) were prepared by the one-pot hydrothermal reaction of citric acid with poly(ethylenimine) (PEI). The CDs display bright PL, narrow emission spectra, pH-dependent PL intensity, high photostability, and up-converted luminescence. The CDs exhibit a quenching of both down- and up-conversion PL in the presence of morin and thus serve as useful probes for morin detection. Both down- and up-conversion measurements allow the quantification of concentrations from 0 to 300 μmol/L with a detection limit of 0.6 μmol/L, and this dual-mode detection increases the reliability of the measurement. The proposed method of determination is simple, sensitive, and cost-effective, with potential applications in clinical and biochemical assays.

Upconversion ratiometric fluorescence and colorimetric dual-readout assay for uric acid

A new upconversion colorimetric and ratiometric fluorescence detection method for uric acid (UA) has been designed. Yb3+, Er3+ and Tm3+ co-doped NaYF4 nanoparticles (UCNPs) was synthesized. The co-doped NaYF4 nanoparticles, emit upconversion fluorescence with four typical emission peaks centered at 490nm, 557nm, 670nm and 705nm under the 980nm near-infrared (NIR) irradiation. The ZnFe2O4 magnetic nanoparticles (MNPs) possessing excellent peroxidase-like activity was prepared and used to catalyze oxidation the coupling of N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt (TOPS) and 4-amino-antipyrine (4-AAP) in the presence of H2O2 to form purple products (compound 1) which has a characteristic absorption peak located at 550nm. The upconversion fluorescence at 557nm was quenched by the compound 1 while the upconversion emission at 705nm was essentially unchanged, the fluorescence ratio ((I557/I705)0/(I557/I705)) is positively proportional to UA concentration in existence of uricase. More importantly, colorimetric signal can be easily observed and applied to directly distinguish the concentration of UA by the naked eye. Under the optimized conditions, the linear range of colorimetric and ratiometric fluorescence sensing towards UA was 0.01–1mM, the detection limits were as low as 5.79μM and 2.86μM (S/N=3), respectively. The proposed method has been successfully applied to the analysis of UA in human serum. These results indicate that the colorimetric and ratiometric fluorescence dual-readout assay method has great potential for applications in physiological and pathological diagnosis.

Fluorescence turn-on and colorimetric dual readout assay of glutathione over cysteine based on the fluorescence inner-filter effect of oxidized TMB on TMPyP

Quantitative fluorescence turn-on and colorimetric detection of glutathione (GSH) with rapid speed, low cost have attained much attention. Herein, we developed a sensitive fluorescence turn-on and colorimetric sensor for GSH based on the inner-filter effect (IFE), which is the first time to select oxTMB and TMPyP as the IFE absorber and fluorophore pair, respectively. The absorption band of oxTMB matches well with the emission band of TMPyP in the IFE-based fluorescent assay. In the absence of GSH, the absorption peak of oxTMB at 652nm significantly overlaps with the emission of TMPyP, resulting in the efficient IFE and inhibition of the fluorescence of TMPyP. In the presence of GSH, the absorption intensity at 652nm decreases, generating the recovery of the fluorescence of TMPyP. Therefore, this approach is demonstrated to be a novel candidate for detection of GSH, with high sensitivity and selectivity. The linear dynamic range for the concentrations of GSH is between 0.1μM to 20μM along with a limit of detection (LOD) of about 30nM (calculated LOD as 3σ/slope). Finally, this novel sensor was successfully applied for GSH detection in fetal calf serum, and satisfactory recovery was achieved.

A Dual Readout Assay Based on Fluorescence Polarization and Time-Resolved Fluorescence Resonance Energy Transfer to Screen for RSK1 Inhibitors.

A dual readout assay based on fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET) exhibits many advantages over single assay technology in terms of screening quality and efficiency. In this study, we developed a dual readout assay combining FP and TR-FRET to identify ribosomal S6 kinase 1 (RSK1) inhibitors. This dual readout assay can monitor both FP and TR-FRET signals from a single RSK1 kinase reaction by using the immobilized metal affinity for phosphochemical (IMAP)-based assay. The Z' value and signal to background (S/B) ratio were 0.85 and 4.0 using FP, and 0.79 and 10.6 using TR-FRET, which led to performance of a pilot library screening against the drug repositioning set consisting of 2320 compounds with a reasonable reproducibility. From this screening, we identified 16 compounds showing greater than 50% inhibition against RSK1 for both FP and TR-FRET; 6 compounds with greater than 50% inhibition only for FP; and 4 compounds with greater than 50% inhibition only for TR-FRET. In a cell-based functional assay to validate the hit compounds, 10 compounds identified only in a single assay had little effect on the RSK-mediated phosphorylation of liver kinase B1, whereas 5 compounds showing greater than 80% inhibition for both FP and TR-FRET reduced the phosphorylation of liver kinase B1. These results demonstrate that the dual readout assay can be used to identify hit compounds by subsequently monitoring both FP and TR-FRET signals from one RSK1 reaction.

A highly sensitive dual-readout assay based on gold nanoclusters for folic acid detection

We describe a sensitive fluorometric and colorimetric dual-readout probe for folic acid (FA). It is based on the use of the gold nanoclusters (AuNCs) and cysteamine–modified gold nanoparticles (cyst-AuNPs). The bovine serum albumin stabilized AuNCs exhibit strong fluorescence emission at 652 nm. Upon addition of cyst-AuNPs, the fluorescence intensity of the AuNCs showed dramatic decrease due to the surface plasmon enhanced energy transfer process. This is due to an FA-induced aggregation of the cyst-AuNPs which shifts the absorption peaks from 530 to 670 nm. Thus, the surface plasmon enhanced energy transfer between cyst-AuNPs and AuNCs is weakened and the fluorescence intensity of AuNCs is recovered. The fluorescence intensity of the AuNCs/cyst-AuNPs system is proportional to the concentration of FA in the range from 0.11 to 2.27 μmol L−1. The dual-readout probe reported here was successfully applied to the determination of FA in spiked serum samples and folic acid tablets.

A highly sensitive dual-readout assay based on poly(A) and gold nanoparticles for palmatine hydrochloride

This report presents a highly sensitive, poly(A)-stabilized gold nanoparticle-based assay with dual readouts (resonance light scattering and colorimetric) for detecting palmatine hydrochloride (PaH) in real samples. The detection mechanism is based on the fact that palmatine hydrochloride has strong affinity to poly(A), which can stabilize gold nanoparticles at high ionic strength, and cause the aggregation of poly(A)-stabilized AuNPs, resulting in the enhanced resonance light scattering (RLS). At the same time, the color change of poly(A)-stabilized AuNPs solution is from red to blue via purple. Thus a highly sensitive RLS assay for PaH has been developed with a linear range of 0.023–2.5μg/mL. The limit of detection (LOD, 3σ) is 2.3ng/mL. In this work, the reaction mechanism of this system was investigated by scanning electron microscope (SEM), dark-field light scattering images (DLSI), dynamiclight scattering (DLS) and circular dichroism (CD). This proposed method was also applied successfully for the determination of PaH in pharmaceutical preparations and urine samples with RSD⩽4.0%. The results are in good agreement with those from the official method.

Dual-Readout Fluorescent Assay of Protein Kinase Activity by Use of TiO2-Coated Magnetic Microspheres

A simple, highly sensitive, and dual-readout fluorescent assay is developed for the detection of protein kinase activity based on the specific recognition utility of TiO2-coated Fe3O4/SiO2 magnetic microspheres (TMSPs) for kinase-induced phosphopeptides. When the fluorophore-labeled substrate peptides are phosphorylated by the kinase reaction, they can bind specifically to the TiO2 layer of TMSPs by means of phosphate groups, resulting in fluorophore enrichment on the TMSP surfaces. The accumulated fluorophores on the TMSPs are proportional to the kinase activity, and the fluorescence signal readout could be run through either direct fluorescent imaging of the TMSPs or measurement of the fluorescence intensity by simply detaching the fluorescent phosphopeptides into the solution. The TMSPs exhibit extremely high selectivity for capturing phosphorylated peptides over the nonphosphorylated ones, resulting in an ultrahigh fluorescence signal-to-background ratio of 42, which is the highest fluorescence change thus far in fluorescent assays for detection of protein kinase activities. Therefore, the proposed fluorescent assay presents high sensitivity, low detection limit of 0.1 milliunit/μL, and wide dynamic range from 0.5 milliunit/μL to 0.5 unit/μL with protein kinase A (PKA) as a model target. Moreover, the TMSP-based fluorescent assay can simultaneously quantify multiple kinase activities with their specific peptides labeled with different dyes. This new strategy is also successfully applied to monitoring drug-triggered PKA activation in cell lysates. Therefore, the TMSP-based fluorescent assay is very promising in high-throughput screening of kinase inhibitors and in highly sensitive detection of kinase activity, and thus it is a valuable tool for development of targeted therapy, clinical diagnosis, and studies of fundamental life science.

Abstract 2469: Identification of novel Mcl-1 inhibitors using integrated screening approach: combining high throughput and virtual screening.

Abstract Myeloid cell leukemia-1 (Mcl-1) is a potent anti-apoptotic protein, a member of the prosurvival Bcl-2 family, and its role is emerging as a critical survival factor in a broad range of human cancers. Mcl-1 represents a very attractive molecular target for development of a new class of cancer therapy and there is still need for developing BH3 mimetics that can efficiently and selectively target Mcl-1 protein. We employed integrated screening approach through combining high throughput and virtual screening, to identify novel chemical classes as Mcl-1 inhibitors. For this purpose we developed a dual-readout HTS assay that combines two assay technologies, FP and FRET, into one system using the Mcl-1 and labeled Noxa BH3 derived peptide and optimized in a 1,536-well ultra-HTS format. This assay was used for screening a library of 102,255 compounds. As two assay platforms were utilized for the same target simultaneously, hit information was enriched identifying 1214 compounds. To further improve the output and the quality of the identified inhibitors, as well as to incorporate the structure-based knowledge of the interactions between Mcl-1 and number of BH3 peptides, we have integrated in silico target-based screening for selection of the most promising hits for further validation. The complex structure between Mcl-1 and Noxa BH3 peptide (PDB ID: 2NLA) was used for the induced fit docking of 1214 hits. Followed by the scoring and ranking of the identified hits, 62 compounds were selected for further evaluation in a series of complementary biochemical, biophysical and functional assays. Several in vitro binding assays with different platform, FP, SPR, and (HSQC) NMR spectroscopy, were used to determine the binding affinity of the compounds. The binding data revealed that we have identified inhibitors with different selectivity profiles against five members of the Bcl-2 family: Mcl-1, Bcl-2, Bcl-xL, Bcl-w and A1. 15N HSQC spectra conclusively showed that newly identified compounds interact with the BH3 domain in Mcl-1 protein and affect many residues in the BH3 binding groove. Using pull down assay it was demonstrated that the identified compounds were able in a dose dependent manner to disrupt interactions between endogenous Mcl-1 protein and biotin labeled Noxa BH3 peptide. Functional studies showed that compounds can antagonize Mcl-1 function and induce cytochrome c and Smac release from the isolated mitochondria. By using murine embryonic fibroblasts (MEFs), wild type and deficient in both Bax and Bak (double knock out), it was further demonstrated that the cytotoxic activity and induction of apoptosis, depend on Bax and/or Bak, suggesting that the compounds function as BH3 mimetics. Collectively, these findings provide several different chemical classes for further chemical modifications and optimization toward developing a new class of anticancer drugs as Mcl-1 inhibitors. Citation Format: Ahmed Mady, Chenzhong Liao, Fardokht Abulwerdi, Chenxi Shen, Yuhong Du, Jeanne Stuckey, Haian Fu, Zaneta Nikolovska-Coleska. Identification of novel Mcl-1 inhibitors using integrated screening approach: combining high throughput and virtual screening. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2469. doi:10.1158/1538-7445.AM2013-2469

A dual read-out assay to evaluate the potency of compounds active against Mycobacterium tuberculosis.

Tuberculosis is a serious global health problem caused by the bacterium Mycobacterium tuberculosis. There is an urgent need for discovery and development of new treatments, but this can only be accomplished through rapid and reproducible M. tuberculosis assays designed to identify potent inhibitors. We developed an automated 96-well assay utilizing a recombinant strain of M. tuberculosis expressing a far-red fluorescent reporter to determine the activity of novel compounds; this allowed us to measure growth by monitoring both optical density and fluorescence. We determined that optical density and fluorescence were correlated with cell number during logarithmic phase growth. Fluorescence was stably maintained without antibiotic selection over 5 days, during which time cells remained actively growing. We optimized parameters for the assay, with the final format being 5 days’ growth in 96-well plates in the presence of 2% w/v DMSO. We confirmed reproducibility using rifampicin and other antibiotics. The dual detection method allows for a reproducible calculation of the minimum inhibitory concentration (MIC), at the same time detecting artefacts such as fluorescence quenching or compound precipitation. We used our assay to confirm anti-tubercular activity and establish the structure activity relationship (SAR) around the imidazo[1,2-a]pyridine-3-carboxamides, a promising series of M. tuberculosis inhibitors.

Green synthesis of carbon dots with down- and up-conversion fluorescent properties for sensitive detection of hypochlorite with a dual-readout assay

High quality carbon dots (C-dots) with down- and up-conversion fluorescence have been synthesized through low-temperature carbonization using sweet pepper as the carbon source. The C-dots with a quantum yield (QY) of 19.3% exhibit superior photophysical properties, for example, narrow and symmetric emission spectra, large stock shifts, resistance to photobleaching, and excitation-dependent fluorescence behavior. The excellent C-dots serve as useful fluorescent probes for hypochlorite (ClO(-)) detection by both down- and up-conversion fluorescence. Two consecutive linear ranges allow a wide determination of ClO(-) concentrations with a low detection limit of 0.05 μmol L(-1) and 0.06 μmol L(-1) (S/N = 3) for down- and up-conversion fluorescence measurements, respectively. The proposed detection method is advantageous because it is simple, sensitive, dual-signalling model and low-cost and has potential extensive applications in environmental and biological assays.

A Highly Sensitive, Dual-Readout Assay Based on Gold Nanoparticles for Organophosphorus and Carbamate Pesticides

This report presents a highly sensitive, rhodamine B-covered gold nanoparticle (RB-AuNP) -based assay with dual readouts (colorimetric and fluorometric) for detecting organophosphorus and carbamate pesticides in complex solutions. The detection mechanism is based on the fact that these pesticides can inhibit the activity of acetylcholinesterase (AChE), thus preventing the generation of thiocholine (which turns the RB-AuNP solutions blue and unquenches the fluorescence of RB simultaneously). The color of the RB-AuNP solution remains red and the fluorescence of RB remains quenched. By use of this dual-readout assay, the lowest detectable concentrations for several kinds of pesticides including carbaryl, diazinon, malathion, and phorate were measured to be 0.1, 0.1, 0.3, and 1 μg/L, respectively, all of which are much lower than the maximum residue limits (MRL) as reported in the European Union pesticides database as well as those from the U.S. Department Agriculture (USDA). This assay allows detection of pesticides in real samples such as agricultural products and river water. The results in detecting pesticide residues collected from food samples via this method agree well with those from high-performance liquid chromatography (HPLC). This simple assay is therefore suitable for sensing pesticides in complex samples, especially in combination with other portable platforms.

A Highly Sensitive Gold‐Nanoparticle‐Based Assay for Acetylcholinesterase in Cerebrospinal Fluid of Transgenic Mice with Alzheimer's Disease

A highly sensitive, selective, and dual-readout (colorimetric and fluorometric) assay for acetylcholinesterase (AChE) based on Rhodamine B-modified gold nanoparticle is reported. Due to its good sensitivity and selectivity, the assay can be used for monitoring AChE levels in the cerebrospinal fluid of transgenic mice with Alzheimer's disease.

A Dual-Readout F2Assay That Combines Fluorescence Resonance Energy Transfer and Fluorescence Polarization for Monitoring Bimolecular Interactions

Förster (fluorescence) resonance energy transfer (FRET) and fluorescence polarization (FP) are widely used technologies for monitoring bimolecular interactions and have been extensively used in high-throughput screening (HTS) for probe and drug discovery. Despite their popularity in HTS, it has been recognized that different assay technologies may generate different hit lists for the same biochemical interaction. Due to the high cost of large-scale HTS campaigns, one has to make a critical choice to employee one assay platform for a particular HTS. Here we report the design and development of a dual-readout HTS assay that combines two assay technologies into one system using the Mcl-1 and Noxa BH3 peptide interaction as a model system. In this system, both FP and FRET signals were simultaneously monitored from one reaction, which is termed "Dual-Readout F(2) assay" with F(2) for FP and FRET. This dual-readout technology has been optimized in a 1,536-well ultra-HTS format for the discovery of Mcl-1 protein inhibitors and achieved a robust performance. This F(2) assay was further validated by screening a library of 102,255 compounds. As two assay platforms are utilized for the same target simultaneously, hit information is enriched without increasing the screening cost. This strategy can be generally extended to other FP-based assays and is expected to enrich primary HTS information and enhance the hit quality of HTS campaigns.