Ratiometric sensing of butyrylcholinesterase activity based on the MnO2 nanosheet-modulated fluorescence of sulfur quantum dots and o-phenylenediamine.

Butyrylcholinesterase (BChE) can modulate the expression level of cholinesterase, which emerges as an important clinical diagnose index. However, the currently reported assays for BChE are suffering from the problem of interferences. A ratiometric fluorescence assay was developed based on the MnO2 nanosheet (NS)-modulated fluorescence of sulfur quantum dots (S-dots) and o-phenylenediamine (OPD). MnO2 NS can not only quench the fluorescence of blue emissive S-dots, but also enhance the yellow emissive OPD by catalyzing its oxidation reactions. Upon introducing BChE and substrate into the system, their hydrolysate can reduce MnO2 into Mn2+, leading to the fluorescence recovery of S-dots and failure of OPD oxidation. BChE activity can be quantitatively detected by recording the change of fluorescence signals in the blue and yellow regions. A linear relationship is observed between the ratio of F435/F560 and the concentration of BChE in the range 30 to 500 U/L, and a limit of detection of 17.8 U/L has been calculated. The ratiometric fluorescence assay shows an excellent selectivity to acetylcholinesterase and tolerance to various other species. The method developed  provides good detection performances in human serum medium and for screening of  inhibitors.

Probing the Structural Dynamics of the Activation Gate of KcsA Using Homo-FRET Measurements.

The allosteric coupling between activation and inactivation processes is a common feature observed in K+ channels. Particularly, in the prokaryotic KcsA channel the K+ conduction process is controlled by the inner gate, which is activated by acidic pH, and by the selectivity filter (SF) or outer gate, which can adopt non-conductive or conductive states. In a previous study, a single tryptophan mutant channel (W67 KcsA) enabled us to investigate the SF dynamics using time-resolved homo-Förster Resonance Energy Transfer (homo-FRET) measurements. Here, the conformational changes of both gates were simultaneously monitored after labelling the G116C position with tetramethylrhodamine (TMR) within a W67 KcsA background. At a high degree of protein labeling, fluorescence anisotropy measurements showed that the pH-induced KcsA gating elicited a variation in the homo-FRET efficiency among the conjugated TMR dyes (TMR homo-FRET), while the conformation of the SF was simultaneously tracked (W67 homo-FRET). The dependence of the activation pKa of the inner gate with the ion occupancy of the SF unequivocally confirmed the allosteric communication between the two gates of KcsA. This simple TMR homo-FRET based ratiometric assay can be easily extended to study the conformational dynamics associated with the gating of other ion channels and their modulation.

Ratiometric assay of mercury ion based on nitrogen-doped carbon dots with two different optical signals: second-order scattering and fluorescence.

Ratiometric assays, which can effectively surmount external interference, have attracted extensive research interests. Herein, a novel ratiometric sensing platform for Hg2+ is designed based on nitrogen-doped carbon dots (N-CDs) with two different optical signals. Under a single excitation, N-CDs have two emission peaks around 668 nm and 412 nm, which are second-order scattering and fluorescence, respectively. Upon the addition of Hg2+, the weak scattering emission at 668 nm can be increased apparently, while the strong fluorescence intensity at 412 nm is weakened. Moreover, the ratio of scattering intensity to fluorescence intensity is linearly dependent on Hg2+ concentration (0.1-10 µM and 10-30 µM, respectively), and the detection limit is 66 nM. In addition, the ratiometric sensing mechanism is investigated in detail, which is due to the combined effect of aggregation-induced fluorescence quenching and scattering enhancement. Furthermore, the developed sensing approach holds a promising application for Hg2+ detection in actual samples. Graphical abstract.

Silver nanoparticle probe for colorimetric detection of aminoglycoside antibiotics: picomolar-level sensitivity toward streptomycin in water, serum, and milk samples.

BACKGROUND: The low cost of aminoglycoside (AMG) antibiotics facilitates their excessive use in animal husbandry and the agriculture sector. This scenario has led to the occurrence of residues in the food chain. After several years of AMG use in antibacterial therapy, resistance to streptomycin has begun to appear. Most of the detection methods developed for AMG antibiotics lacks specificity. A broad target specific nanoprobe would be ideal for detecting the entire class of AMGs. A rapid and sensitive method for the detection of AMGs is urgently needed. RESULTS: Gallic acid-coated silver nanoparticles (AgNPs) were demonstrated as a nanoprobe for the colorimetric detection of AMGs (yellow to orange / red). A linear dynamic range of 50-650 pmol L-1 was achieved readily by ratiometric spectrophotometry (A560 /A400 ) with a limit of detection (LOD) as low as 36 pmol L-1 . The amine-groups of the AMGs function as molecular linkers, so that electrostatic coupling interactions between neighboring particles drive the formation of AgNP aggregates. The assay can also be applied for the determination of streptomycin residues in serum and milk samples. CONCLUSION: This study revealed the potential of an AgNP probe for the rapid and cost-effective detection of low-molecular-weight target analytes, such as the AMGs. A ligand-induced aggregation of AgNPs coated with gallic acid was reported to be a rapid and sensitive assay for AMGs. Analysis of streptomycin was demonstrated with excellent picomolar-level sensitivity. Thus, the validated method can find practical applications in the ultrasensitive detection of AMGs in complex and diagnostic settings. © 2019 Society of Chemical Industry.

Polylysine modified conjugated polymer nanoparticles loaded with the singlet oxygen probe 1,3-diphenylisobenzofuran and the photosensitizer indocyanine green for use in fluorometric sensing and in photodynamic therapy.

Conjugated polymer hybrid nanoparticles (NPs) loaded with both indocyanine green (ICG) and 1,3-diphenylisobenzofuran (DPBF) are described. The NPs are dually functional in that ICG acts as the photosensitizer, and DPBF as a probe for singlet oxygen (1O2 probe). The nanoparticle core consists of the energy donating host poly(9,9-dioctylfluorenyl-2,7-diyl)-co-(2,5-p-xylene) (PFP). The polymer is doped with the energy acceptor DPBF. Ratiometric fluorometric detection of singlet oxygen is accomplished by measurement of fluorescence at wavelengths of 415 and 458 nm. In addition, the shell of the positively charged polymeric nanoparticles was modified, via electrostatic interaction, with negatively charged PDT drugs ICG. The integrated nanoparticles of type ICG-DPBF-PFP display effective photodynamic performance under 808-nm laser irradiation. The 1O2 sensing behaviors of samples are evaluated based on the ratiometric fluorescent responses produced by DPBF and PFP. 1O2 can be fluorimetically sensed with a detection limit of 28 µM. The multifunctional nanoprobes exhibit effortless cellular uptake, superior photodynamic activity and a rapid ratiometric response to 1O2. Graphical abstractSchematic of a dual-functional nanoplatform for photodynamic therapy (PDT) and singlet oxygen (1O2) feedback. It offers a new strategy for self-monitoring photodynamic ablation. FRET: fluorescence resonance energy transfer. Indocyanine green is attached in the shell of nanoparticles, and 1,3-diphenylisobenzofuran is doped into the energy donating host conjugated polymer.

A ratiometric electrochemiluminescent immunoassay for calcitonin by using N-(aminobutyl)-N-(ethylisoluminol) and graphite-like carbon nitride.

A ratiometric electrochemiluminescent (ECL) assay is described for the determination of the calcium(II) regulator calcitonin (CT). The method is making use of (a) graphite-like carbon nitride (g-C3N4) as the cathodic luminophore, (b) N-(aminobutyl)-N-(ethylisoluminol) (ABEI) as the anodic luminophore, and (c) peroxodisulfate and dissolved oxygen as coreactants. The luminous potential of g-C3N4 and ABEI can be well distinguished because of their different luminescent properties. Energy transfer between g-C3N4 and ABEI is not observed, and the coreactants peroxodisulate and oxygen do not interfere with each other. Au nanoparticles were functionalized with g-C3N4 and placed on the electrode to serve as a matrix for immobilization of primary antibody (Ab1). In the presence of CT, it will bind to the electrode. Then secondary antibody (Ab2) modified with polyaniline (PANI) and ABEI is incubated onto the electrode. With the increase in the concentration of CT, the blue ECL of g-C3N4 is quenched by PANI, while the blue luminescence of ABEI is enhanced. This enables ratiometric detection of calcitonin by ratioing the internsities at 460 and 475 nm. Response is linear in the 0.1~40 pg·mL-1 CT concentration range, and the limit of detection is 23 fg·mL-1. The method breaks the limitation of common ECL ratiometric strategy, namely, two luminophores often share the common coreactant. Graphical abstractSchematic representation of an immunoassay where polyaniline (PANI) in a BSA-Ab2-ABEI-Au@PANI composite quenches the cathodic signal of a graphitic carbon nitride (Au-g-C3N4) modified with gold nanoparticles (Au), while N-(aminobutyl)-N-(ethylisolumino) (ABEI) in the BSA-Ab2-ABEI-Au@PANI composit produces an anodic signal that enables quantitation of calcitonin.

Gold nanoparticles stabilized with four kinds of amino acid-derived carbon dots for colorimetric and visual discrimination of proteins and microorganisms.

A colorimetric array is described for the sensitive discrimination of proteins and microorganisms. Carbon dots (CDs) were prepared from citric acid and one of the amino acids glycine, lysine, serine or aspartic acid. They act as stabilizers for gold nanoparticles (AuNPs). The interactions between target protein and the CD/AuNPs induce a different aggregation behavior. This provides the basis for colorimetric discrimination of protein species and results in color changes from red/purple to purple/blue. Specific response patterns are analyzed by linear discriminant analysis. Twelve kinds of proteins with different pI and molecular weight were visually discriminated at nanomolar concentration levels. Alternatively, discrimination can be performed by measurement of the ration of absorbance at 525 nm and 620 nm. The discrimination sensitivity is as low as 2 nM. The method can differentiate between BSA and HSA. Twelve proteins were successfully distinguished in (spiked) urine samples. The discrimination accuracy is 100% at the 500 nM protein concentration level. In addition, different strains of microorganisms (E. coli O157:H7, E.coli ER2738, P. aeruginosa CICC10204; P. aeruginosa CICC21954; B.subtilis CICC10071; B.subtilis CICC10275) can be discriminated successfully via this array. Graphical abstract A CD/AuNPs-based colorimetric array sensor is proposed for the discrimination of protein, offering a discrimination sensitivity low down to 2 nM. The accurate differentiations of microorganisms originated from same species are achieved.

Bare eye detection of Hg(II) ions based on enzyme inhibition and using mercaptoethanol as a reagent to improve selectivity.

The authors describe a colorimetric method for the determination of Hg2+ ions based on the inhibition of the activity of the enzyme urease. The pH value of solution increases when urease hydrolyzes urea, which can be visualized by adding a pH indicator such as Phenol Red (PhR). Mercaptoethanol as a typical thiol is added to the system to improve selectivity because it binds metal ions and then - unlike the Hg2+ mercaptoethanol complex - does not inhibit urease. Hence, the color of the pH indicator PhR turns from yellow to pink as the solution becomes alkaline. The Hg2+ mercaptoethanol complex, in contrast, strongly inhibits urease and the color of the solution remains yellow. The findings were used to design a photometric assay based on the measurement of the ratio of absorptions of PhR at 558 nm and 430 nm. It has a linear response over the 25 to 40 nM Hg2+ concentration range and a 5 nM detection limit. This is well below the guideline values of Hg2+ specified by the US Environmental Protection Agency and the World Health Organization for drinking water (10 nM and 30 nM, respectively). The method was employed to the determination of Hg2+ in water samples spiked with 10 nM levels of Hg2+ where color changes still can be observed visually. Graphical Abstract Schematic presentation of a colorimetric method for the ultrasensitive detection of Hg2+ based on the inhibition of urease activity. Mercaptoethanol is used to improve the selectivity. Even at Hg2+ concentrations as low as 5 nM, the color change still can be easily observed by bare eyes.

Colorimetric detection of L-histidine based on the target-triggered self-cleavage of swing-structured DNA duplex-induced aggregation of gold nanoparticles.

A rapid, highly sensitive and selective colorimetric assay is presented for visually detecting L-histidine. It is based on L-histidine-triggered self-cleavage of DNA duplex-induced gold nanoparticle (AuNP) aggregation. The citrate-capped AuNPs easily aggregate in a high concentration of salt environment. However, in the presence of L-histidine aptamers (DNA1 and DNA2), the partial strands of DNA1 and DNA2 hybridize to form a DNA duplex with a swing structure. The swing-like DNA duplexes are adsorbed on the surface of AuNPs to improve the stability of AuNPs, and the AuNPs also are better dispersed in high-salt media. When L-histidine is added to the solutions, it catalyzes the self-cleavage of DNA1 to form many single-stranded DNA (ssDNA) fragments. These ssDNA segments are adsorbed on the AuNPs and weaken the stability of AuNPs. Hence, the AuNPs aggregate in high-salt environment, and this results in a red-to-blue color change. Under the optimized conditions, L-histidine can be determined with a limit of detection of 3.6 nM. In addition, the sensor was successfully applied to the determination of L-histidine in spiked serum samples. Graphical abstract Schematic of a rapid and homogeneous colorimetric L-histidine assay. It combines L-histidine-triggered self-cleavage of the swing-like DNA duplexes and self-cleavage of DNA-induced AuNP aggregation.

Simple dual-readout immunosensor based on phosphate-triggered and potassium permanganate for visual detection of fenitrothion.

Multicolor-based visual immunosensor is a promising tool for rapid analysis without the use of bulky instruments. Herein, an anti-fenitrothion nanobody-alkaline phosphatase fusion protein (VHHjd8-ALP) was employed to develop a multicolor visual immunosensor (MVIS) and a ratiometric fluorescence MVIS (RFMVIS, respectively). After one-step competitive immunoassay, the VHHjd8-ALP bound to microplate catalyzed phenyl phosphate disodium salt (ArP) into phenol. Under high alkaline condition (pH 12), the phenol reduced KMnO4 to intermediate (K2MnO4) and further to MnO2 in alkaline condition (pH 12), accompanied by a visible color transition of purple-green-yellow, which can be used for semiquantitative visual analysis or qualitative detection by measuring RGB value. RFMVIS was proposed on the basis of MVIS to further improve sensitivity. The CdTe quantum dot and fluorescein were used as signal probes to develop the fluorescent immunosensor. The CdTe dots with red emission (644 nm) was quenched by oxidation of KMnO4, whereas the fluorescein with green emission (520 nm) remained constant, accompanied by a fluorescent color transition of green-yellow-red. By measuring the ratio of the fluorescence intensity (I644/I520), the ratiometric fluorescence immunosensor was developed for qualitative analysis. The two visual immunosensors were sensitive and simple, and they showed good accuracy and practicability in the recovery test, thus are ideal tools for rapid screening.

Activity-Based Self-Enriched SERS Sensor for Blood Metabolite Monitoring.

The monitoring of metabolites in biofluids provides critical clues for disease diagnosis and evaluation. Yet, the quantitative detection of metabolites remains challenging for surface-enhanced Raman spectroscopy (SERS) due to poor reproducibility in preparation and manipulation of SERS nanoprobes. Herein, we develop an activity-based, slippery liquid-infused porous surface SERS (abSLIPSERS) sensor for facile quantification of metabolites with unmodified naked metal nanoparticles (NPs) by integrating biocatalysis-boronate oxidation cascades with SLIPS-driven self-concentration and delivering. Upon mixing the target metabolite with a specific oxidase, a H2O2-sensitive phenylboronate probe, and the naked Au NPs, H2O2 produced from the biocatalytic reaction oxidizes the phenylboronate probe to phenol, resulting in a ratiometric SERS response. Meanwhile, the SLIPS enables the complete enrichment of molecules and NPs within an evaporating liquid droplet, delivering the probes to the SERS-active sites for Raman amplification. Compared with conventional SERS biosensors, abSLIPSERS avoids multistep synthesis and biofunctionalization of nanoprobes, which significantly simplifies the detection workflow and improves the reproducibility. The abSLIPSERS sensor also shows tunable dynamic range beyond 4 orders of magnitude and allows quantifying any other metabolites with specific enzymes. We demonstrate abSLIPSERS sensing of lactate, glucose, and choline in human serum for exploring energy metabolism in lung cancer. This study opens up a new opportunity for future point-of-care testing of circulating metabolites by SERS and will help to facilitate the translation of SERS bioanalysis to clinical settings.

Room-temperature phosphorescence and fluorescence nanocomposites as a ratiometric chemosensor for high-contrast and selective detection of 2,4,6-trinitrotoluene.

Reports on using complementary colours for high-contrast ratiometric assays are limited to date. In this work, graphitized carbon nitride (g-C3N4) nanosheets and mercaptoethylamine (MEA) capped Mn-doped ZnS QDs were fabricated by liquid exfoliation of bulk g-C3N4, and by a coprecipitation and postmodification strategies, respectively. Mn-doped ZnS quantum dots were deposited onto g-C3N4 nanosheets through an electrostatic self-assembly to form new nanocomposites (denoted as Mn-ZnS QDs@g-C3N4). Mn-ZnS QDs@g-C3N4 can emit a pair of complementary colour light, namely, orange room-temperature phosphorescence (RTP) at 582 nm and blue fluorescence at 450 nm. After 2,4,6-trinitrotoluene (TNT) dosing into Mn-ZnS QDs@g-C3N4 aqueous solution, and pairing with MEA to generate TNT anions capable of quenching the emission of Mn-doped ZnS QDs, the fluorescence colours of the solution changed from orange to blue across white, exhibiting unusual high-contrast fluorescence images. The developed ratiometric chemosensor showed very good linearity in the range of 0-12 µM TNT with a limit of detection of 0.56 µM and an RSD of 6.4 % (n = 5). Also, the ratiometric probe had an excellent selectivity for TNT over other nitroaromatic compounds, which was applied in the ratiometric test paper to image TNT in water, and TNT sensing under phosphorescence mode to efficiently avoid background interference. A high-contrast dual-emission platform for selective ratiometric detection of TNT was therefore established.

Dual-emitting BP-CdTe QDs coupled with dual-function moderator TiO2 NSs for electrochemiluminescence ratio bioassay.

The limitation of the selection of dual-emitting luminophor and dual-function moderator makes the construction of single-luminophor-based electrochemiluminescence (ECL) ratio strategy extremely challenging. This work developed black phosphorus nanosheet loaded with CdTe quantum dots (BP-CdTe QDs) as dual-emitting luminophor, and TiO2 nanosheets (NSs) as dual-function moderator to construct single-luminophor-based ECL ratio biosensor for amyloid-ß protein (Aß) detection. The BP-CdTe QDs modified glassy carbon electrode (GCE) firstly captured the anti-Aß1, and then the target Aß. Finally, the secondary antibody composites containing N,N-diethylethylenediamine (DEDA) and TiO2 NSs were captured on the electrode surface. DEDA in the secondary antibody composites and S2O82- added in the detection solution served as the anodic and cathodic co-reactants, respectively, and TiO2 NSs as co-reaction accelerator can simultaneously enhance two emissions. The increase of target concentration made the two signals show different increasing trend, thus achieving a sensitive ratio detection of target Aß with the detection limit of 21 fg/mL. The dual-emitting BP-CdTe QDs coupled with dual-function moderator TiO2 NSs provide an attractive single-luminophor-based ECL ratio platform for bioassay, which can not only be applied to the detection of Aß, but also to the detection of other disease biomarkers.

Fluorescence ratiometric assay for discriminating GSH and Cys based on the composites of UiO-66-NH2 and Cu nanoclusters.

The discriminative detection of glutathione (GSH) from cysteine (Cys) remains a challenge because of their similarity in structure and chemical properties. This study reported a strategy for selective and sensitive detection of GSH based on the GSH-promoted blue fluorescence of UiO-66-NH2 and aggregation-enhanced emission (AEE) feature of orange emissive Cu nanoclusters (NCs). A relatively weak blue fluorescence of UiO-66-NH2 was converted to strong after reacting with GSH due to the rotation-restricted emission enhancement mechanism. In addition, the GSH-activated UiO-66-NH2 was further used as a template and reducing reagent for synthesizing orange-red AEE active Cu NCs composites (UiO-66-NH2@Cu NCs). A ratiometric fluorescence response was observed after forming UiO-66-NH2@Cu NCs, helping discriminate GSH over Cys. In addition, UiO-66-NH2@Cu NCs were further utilized for the detection of GSH in clinical samples. The present findings provide an efficient strategy to discriminate GSH over Cys and open a new door for utilizing and functionalizing metal-organic frameworks (MOFs) for various applications.

A microchip electrophoresis-based assay for ratiometric detection of kanamycin by R-shape probe and exonuclease-assisted signal amplification.

Excessive intake of kanamycin (KANA) can cause some serious drug-resistant diseases, so it is urgent to develop some accurate and rapid analytical methods for monitor KANA residues in foodstuffs with complex matrix. Recently, many ratiometric assays were reported to be capable of overcoming matrix interference. Herein, a ratiometric and homogeneous assay for KANA detection based on microchip electrophoresis (MCE) was developed. First, by one single strand DNA (S-DNA) and one hairpin DNA (H-DNA), a novel R shape DNA probe (R-DNA) was prepared. After the probe was incubated with KANA, the S-DNA-KANA complex was formed, and H-DNA was released. Moreover, in the presence of exonuclease I (Exo-I), S-DNA-KANA complex would be digested to release the captured KANA for triggering target recycling and signal amplification. With the reaction going on, the fluorescence intensity of H-DNA (IH) increased and that of R-DNA (IR) decreased. They can be separated at different voltage intensities and converted to fluorescent signals for signal readout by MCE. The signal ratio of IH/IR was found to be linear toward target from 0.5 pg mL-1 to 10 ng mL-1, and the limit of detection was 150 fg mL-1. Moreover, it was successfully employed for KANA detection in milk and fish samples with consistent results of enzyme linked immune sorbent assay (ELISA). The R-DNA probe can quantitatively convert the amount of target to the intensity of DNA without label by MCE, and achieved exonuclease assisted signal amplification in homogenous solution. It was valuable to detect antibiotics residues in foodstuff with complex matrix. This approach broadened the application field of MCE to detect antibiotics without derivatization, which provided a promising platform for rapid screening of antibiotic residues in food.