AIE fluorescent nanozyme-based dual-mode biosensor for analysis of the bioactive component hypoxanthine in meat products.

The development of facile, low-cost reliable, and precise onsite assays for the bioactive component hypoxanthine (Hx) in meat products is significant for safeguarding food safety and public health. Herein, we proposed a smartphone-assissted aggregation-induced emission (AIE) fluorogen tetraphenylethene (TPE)-incorporated amorphous Fe-doped phosphotungstates (Fe-Phos@TPE) nanozyme-based ratiometric fluorescence-colorimetric dual-mode biosensor for achieving the onsite visual detection of Hx. When the Hx existed, xanthine oxidase (XOD) catalyzed Hx into H2O2 to be further catalyzed into •OH by the prominent peroxidase activity of Fe-Phos@TPE at pH = 6.5, resulting in the oxidization of nonfluorescent o-phenylenediamine (OPD, naked-eye colorless) to be yellow fluorescent emissive 2,3-diaminophenazine (DAP, naked-eye dark yellow) at 550 nm as well as the intrinsic blue fluorescence of Fe-Phos@TPE at 440 nm to be decreased via inner-filter effect (IFE) action, thereby realizing a multi-enzyme cascade catalytic reaction at near-neutral pH to overcome the traditional acidity dependence-induced time-consuming and low sensitivity troublesome.

ß-Glucuronidase-triggered reaction for fluorometric and colorimetric dual-mode assay based on the in situ formation of silicon nanoparticles.

BACKGROUND: ß-Glucuronidase (GUS) is considered as a promising biomarker for primary cancer. Thus, the reliable detection of GUS has great practical significance in the discovery and diagnosis of cancer. Compared with traditional organic probes, silicon nanoparticles (Si NPs) have emerged as robust optical nanomaterials due to their facile preparation, superior photobleaching resistance and excellent biocompatibility. However, most nanomaterials-based methods only output a single signal which is easily influenced by external factors in complex systems. Hence, developing nanomaterial-based multi-signal optical assays for highly sensitive GUS determination is still urgently desired. RESULTS: In this study, we developed a simple and efficient one-step method for the in situ preparation of yellow color and yellow-green fluorescent Si NPs. This was achieved by combining 3-[2-(2-aminoethylamino) ethylamino] propyl-trimethoxysilane with p-aminophenol (AP) in an aqueous solution. The obtained Si NPs showed yellow-green fluorescence at 535 nm when excited at 380 nm, while also exhibiting an absorption peak at a wavelength of 490 nm. Taking inspiration from the easy synthesis step regulated by AP, which is generated through the hydrolysis of 4-aminophenyl ß-D-glucuronide catalyzed by GUS, we constructed a direct fluorometric and colorimetric dual-mode method to measure GUS activity. The developed fluorometric and colorimetric sensing platform showed high sensitivity and accuracy with detection limits for GUS determination as low as 0.0093 and 0.081 U/L, respectively. SIGNIFICANCE: This study provides a facile dual-mode fluorometric and colorimetric approach for determination of GUS activity based on novel Si NPs for the first time. This designed sensing approach was successfully employed for the quantification of GUS in human serum samples and screening of GUS inhibitors, indicating the feasibility and potential applications in clinical cancer diagnosis and anti-cancer drug discovery.

Development of a fluorometric and colorimetric dual-mode sensing platform for acid phosphatase assay based on Fe3+ functionalized CuInS2/ZnS quantum dots.

BACKGROUND: The spectral dual-mode response towards analyte has been attracted much attention, benefiting from the higher detection accuracy of such strategy in comparison to single signal readout. However, the currently reported dual-mode sensors for acid phosphatase (ACP) activity are still limited, and most of them more or less exist some deficiencies, such as complicated construction procedure, high-cost, poor biocompatibility, aggregation-caused quenching and limited emission capacity. RESULTS: Herein, we employed Fe3+ functionalized CuInS2/ZnS quantum dots (CIS/ZnS QDs) as nanosensor to develop a novel fluorometric and colorimetric dual-mode assay for ACP activity, combing with ACP-triggered hydrolysis of ascorbic acid 2-phosphate (AAP) into ascorbic acid (AA). The Fe3+ binding to CIS/ZnS QDs can be reduced into Fe2+ during the determination, resulting in the dramatically weakened photoinduced electron transfer (PET) effect and the disappearance of competition absorption. Thus, a highly sensitive ACP assay in the range of 0.22-12.5 U L-1 through fluorescence "turn-on" mode has been achieved with a detection of limit (LOD) of 0.064 U L-1. Meanwhile, the ACP activity can also be quantified by spectrophotometry based on the chromogenic reaction of the formed Fe2+ with 1,10-phenanthroline (Phen). Moreover, the designed nanosensor with good biocompatibility was successfully applied to image and monitor the ACP levels in living cells. SIGNIFICANCE: We believe that the proposed method has remarkable advantages and potential application for ACP assay in terms of the high accuracy, simplicity, low cost, as well as its adequate sensitivity.

A highly sensitive self-powered sensing method designed on DNA circuit strategy and MoS2 hollow nanorods for detection of thalassemia.

Thalassemia is one of the most common monogenic diseases, which seriously affects human growth and development, cardiovascular system, liver, etc. There is currently no effective cure for this disease, making screening for thalassemia particularly important. Herein, a self-powered portable device with high sensitivity and specificity for efficiently screening of low-level thalassemia is developed which is enabled with AuNPs/MoS2@C hollow nanorods and triple nucleic acid amplification technologies. It is noteworthy that AuNPs/MoS2@C electrode shows the advantages of high electrocatalytic activity, fast carrier migration rate and large specific surface area, which can significantly improve the stability and output signal of the platform. Using high-efficiency tetrahedral DNA as the probe, the target CD122 gene associated with thalassemia triggers a catalytic hairpin assembly reaction to achieve CD122 recycling while providing binding sites for subsequent hybridization chain reaction, greatly improving the detection accuracy and sensitivity of the device. A reliable electrochemical/colorimetric dual-mode assay for CD122 is then established, with a linear response range of 0.0001-100 pM for target concentration and open circuit voltage, and the detection limit is 78.7 aM (S/N = 3); a linear range of 0.0001-10000 pM for CD122 level and RGB Blue value, with a detection limit as low as 58.5 aM (S/N = 3). This method achieves ultra-sensitive and accurate detection of CD122, providing a new method for the rapid and accurate screening of thalassemia.

Ultrathin C3N4 nanosheets-based oxidase-like 2D fluorescence nanozyme for dual-mode detection of organophosphorus pesticides.

Engineering efficient dual-mode portable sensor with built-in cross reference correction is of great significance for onsite reliable and precise detection of organophosphorus pesticides (OPs) and evading the false-positive outputs, especially in emergency case. Currently, most nanozyme-based sensors for OPs monitoring primarily replied on the peroxidase-like activity, which involved unstable and toxic H2O2. In this scenario, a hybrid oxidase-like 2D fluorescence nanozyme (PtPdNPs@g-C3N4) was yielded by in situ growing PtPdNPs in the ultrathin two-dimensional (2D) graphitic carbon nitride (g-C3N4) nanosheet. When acetylcholinesterase (AChE) hydrolyzed acetylthiocholine (ATCh) to thiocholine (TCh), it ablated O2-• from the dissolved O2 catalyzed by PtPdNPs@g-C3N4's oxidase-like activity, hampering the oxidation of o-phenylenediamine (OPD) into 2,3-diaminophenothiazine (DAP). Consequently, with the increasing concentration of OPs which inhibited the blocking effect by inactivating AChE, the produced DAP caused an apparent color change and a dual-color ratiometric fluorescence change in the response system. Through integrating into a smartphone, a H2O2-free 2D nanozyme-based onsite colorimetric and fluorescence dual-mode visual imaging sensor for OPs was proposed with acceptable results in real samples, which holds vast promise for further development of commercial point-of-care testing platform in early warning and controlling of OPs pollution for safeguarding environmental health and food safety.

Novel and sensitive electrochemical/fluorescent dual-mode biosensing platform based on the cascaded cyclic amplification of enzyme-free DDSA and functional nucleic acids.

Herein, we present a novel electrochemical (EC)/fluorescent (FL) dual-mode biosensor for sensitive and accurate detection of target nucleic acids, which was based on the functional nucleic acids-involved enzyme-free dynamic DNA self-assembly of catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) for cascaded cyclic amplification. Originally, the CHA reaction of three well-designed hairpin probes were initiated by target sequence, forming abundant Mg2+-dependent three-way DNAzyme junctions (MTWDJ) which could recognize and cleave the methylene blue-labeled substrate hairpin (MB-Hs) to generate the MB-labeled fragments s1 (MB-s1) and the HCR initiator s2. Then, s2 triggered the HCR of four hairpins to produce long DNA nanowires which contained numerous G-quadruplex sequences and the same Mg2+-dependent DNAzyme (MNAzyme) sequences as MTWDJ. Therefore, the HCR copolymer could not only emerge the fluorescent signals through combining thioflavin T with G-quadruplex, but also generate MB-s1 and s2 via MNAzyme cleavage of MB-Hs to continue initiating the HCR. Meanwhile, MB-s1, the cleavage product of MTWDJ and MNAzyme, was captured on the DNA tetrahedron nanostructure modified electrode surface to bring electrochemical signals. Benefiting from integrating the efficient cyclic cleavage of MTWDJ and MNAzyme, the concatenated CHA and HCR amplification circuit, and the dual-mode detection, the sensitivity and accuracy of this biosensor were significantly improved. Under the optimal conditions, the proposed EC/FL dual-mode sensing strategy exhibited a superior analytical performance toward target nucleic acids, showing the promising application in bioanalysis and early disease diagnosis.

Dual mode assay of glutathione with Tb-doped g-C3N4/MnO2 nanoconjugates as fluorescence probe and Mn as elemental target.

Glutathione (GSH) plays important roles in various physiological processes, thus highly sensitive assay of GSH and timely warning of its variation at trace level in complex biological matrixes is of great significance. However, this is challenging due to the coexisting reductive biomolecules and dynamic change of GSH levels in responding to various stimuli which remain largely unexploited. Herein, we report a dual mode protocol for the assay of GSH based on nanoconjugate g-C3N4:Tb/MnO2 between MnO2 nanosheets and terbium-doped g-C3N4 (g-C3N4:Tb) nanosheets. MnO2 moiety effectively quenches the emission at 546 nm from Tb3+ in the nanoconjugate, which is restored under the reduction of MnO2 by GSH to ensure fluorescence turn-on assay of GSH. Meanwhile, the generated Mn2+ facilitates inductively coupled plasma mass spectrometry (ICP-MS) detection to endow indirect highly sensitive assay of GSH. Fluorescence mode derived a limit of detection (LOD) of 0.17 µmol L-1 within a linear range of 0.5-160 µmol L-1, while ICP-MS resulted in a superior LOD of 0.016 µmol L-1 within 0.05-160 µmol L-1. Both detection modes provide excellent selectivity to GSH. The dual mode platform was validated by GSH assay in cell lysates. It was further demonstrated by monitoring the variation of dynamic change of GSH level under CuSO4 or cisplatin induced GSH consumption.

Cysteamine-Gold Coated Carboxylated Fluorescent Nanoparticle Mediated Point-of-Care Dual-Modality Detection of the H5N1 Pathogenic Virus.

Globally, point-of-care testing (POCT) is the most preferable on-site technique for disease detection and includes a rapid diagnostic test (RDT) and fluorescent immunochromatographic strip test (FICT). The testing kits are generally insufficient in terms of signal enhancement, which is a major drawback of this approach. Sensitive and timely on-site POCT methods with high signal enhancement are therefore essential for the accurate diagnosis of infectious diseases. Herein, we prepare cysteamine-gold coated carboxylated europium chelated nanoparticle (Cys Au-EuNPs)-mediated POCT for the detection of the H5N1 avian influenza virus (AIV). Commercial nanoparticles were used for comparison. The spectral characteristics, surface morphologies, functional groups, surface charge and stability of the Cys AuNPs, EuNPs, and Cys Au-EuNPs were confirmed by UV-visible spectrophotometry, fluorescence spectrometry, transmission electron microscope with Selected area electron diffraction (TEM-SAED), Fourier-transform infrared spectroscopy (FTIR) and zeta potential analysis. The particle size distribution revealed an average size of ~130 ± 0.66 nm for the Cys Au-EuNPs. The Cys Au-EuNP-mediated RDT (colorimetric analysis) and FICT kit revealed a limit of detection (LOD) of 10 HAU/mL and 2.5 HAU/mL, respectively, for H5N1 under different titer conditions. The obtained LOD is eight-fold that of commercial nanoparticle conjugates. The photo luminance (PL) stability of ~3% the Cys Au-EuNPs conjugates that was obtained under UV light irradiation differs considerably from that of the commercial nanoparticle conjugates. Overall, the developed Cys Au-EuNPs-mediated dual-mode POCT kit can be used as an effective nanocomposite for the development of on-site monitoring systems for infectious disease surveillance.

Construction of a magnetic-fluorescent-plasmonic nanosensor for the determination of MMP-2 activity based on SERS-fluorescence dual-mode signals.

Matrix metalloproteinase 2 (MMP-2) is a crucial biomarker of tumor growth, invasion and metastasis. In the present study, a core-satellite magnetic-fluorescent-plasmonic nanosensor (FMNS@Au) was constructed through biological self-assembly to generate localized SERS "hot spots" and an efficient FRET system for the sensitive determination of MMP-2 activity in a SERS-fluorescence dual-mode assay. In this hybrid nanosensor, a biotin-labeled peptide containing a specific MMP-2 substrate (PLGVR) was employed as a bridge for the assembly of gold nanoparticles (AuNPs) and avidin functionalized fluorescent-magnetic nanospheres (FMNS). The modified RB on FMNS served as a Raman reporter and a donor of FRET, while the AuNPs assembled on FMNS acted as SERS substrates and acceptors of FRET. In the presence of MMP-2, the SERS "hot spot" effect was weakened and the FRET system was disrupted through enzymatic cleavage of PLGVR, resulting in a reduction of SERS signal and the recovery of fluorescence emission. Importantly, this combination of SERS and fluorescence assay methods in the dual-mode nanosensor broadened the detection range for MMP-2 to 1-200 ng mL-1, with a limit of detection of 0.35 ng mL-1 and a limit of quantitation of 1.17 ng mL-1. In addition, our novel nanosensor affords semi-quantitative sensing of MMP-2 by naked-eye observation and accurate detection of MMP-2 through dual-mode analysis. The practicality of FMNS@Au was validated by determination of MMP-2 activity in cell secretions and human serum samples. The designed FMNS@Au nanosensor holds great potential for clinical diagnosis of protease-related diseases.

A novel sensing platform for the determination of alkaline phosphatase based on SERS-fluorescent dual-mode signals.

Alkaline phosphatase (ALP), as an important biomarker, is closely associated with various diseases. Multi-mode sensing platforms can combine the advantages of different technologies and solve their inherent or practical limitations. Herein, we developed a sensing platform for the determination of alkaline phosphatase (ALP) in human serum based on SERS-fluorescent dual-mode assay. Based on the fact that ALP can trigger the in-situ reaction between o-phenylenediamine (OPD) and ascorbic acid (AA), we connected gold nanoparticles (AuNPs) to 3,4-diaminobenzene-thiol (OPD(SH)) through an Au-S covalent bond to synthesize a nanoprobe (OPD(S)-AuNPs). The nanoprobe provides a unique interactive ammonium group for the diol group of AA, which was then used to generate an N-heterocyclic compound that can exhibit good SERS and fluorescence signals without adding SERS reporter and fluorophores or quantum dots (QDs). When being excited at different wavelengths as 360 nm and 785 nm, the fluorescence and SERS signals can be separately generated, which can avoid the disturbance from each other. The response of the fluorescence system was linear from 1.0 to 20 mU mL-1 (R2 = 0.994) with a detection limit of 0.3 mU mL-1, while that of the SERS system was linear from 0.5 to 10 mU mL-1 (R2 = 0.998) with a detection limit of 0.2 mU mL-1. The sensing platform developed was further employed in ALP inhibitor evaluation.

Silicon quantum dots based dual-mode fluorometric and colorimetric sensing of D-penicillamine.

D-penicillamine (D-PA) plays an important role in medical and clinical treatment of some diseases. In this work, we designed a sensitive fluorometric and colorimetric dual-mode assay on the basis of fluorescent silicon quantum dots (SiQDs) and 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) to effectively detect D-PA. The sulfydryl group (-SH) of D-PA can react with DTNB to generate 5-thio-2-nitrobenzoate (TNB), which has a broad absorption peak centered at 407 nm and is capable to absorb excitation light of SiQDs and greatly quench the fluorescence of SiQDs on account of inner filter effect (IFE). Meanwhile, the color of the detection system obviously turned yellow. Consequently, the quantitative determination of D-PA can be achieved through both fluorometric and colorimetric methods. The fluorometric and colorimetric sensing platform can detect D-PA in 1-20 µM and 2-20 µM concentration range, respectively, and the limits of detection (LOD) were 0.48 µM and 0.68 µM, accordingly. Furthermore, the designed sensing platform was utilized to detect D-PA in real biological samples and the experimental results were satisfactory, suggesting the feasibility and potential applications of the sensing platform.

Aptamer and gold nanorod-based fumonisin B1 assay using both fluorometry and SERS.

An aptamer-based assay is presented for the determination of fumonisin B1 (FB1). It is bimodal in that both surface-enhanced Raman spectroscopy (SERS) and fluorometry are applied for quantitation. It makes use of platinum-coated gold nanorod (AuNR) and DNA sequences. The complementary DNA of aptamer (cDNA) against FB1 is immobilized on the surface of AuNR. The aptamer of FB1 modified with Cy5.5 are complementarily hybridized with cDNA. In the absence of FB1, the aptamer and its cDNA associate. In this situation, strong SERS and weak fluorescence signals are obtained. In the presence of FB1, the aptamer disassociates with its cDNA and binds the target. As the concentration of FB1 increases, the SERS and fluorescence signal intensities of the mixture are gradually decreased and increased, respectively. Under optimized conditions, the SERS signal at 1366 cm-1 decreases linearly in the 10-500 pg mL-1 concentration range with the calibration equation of y = 1997lgx-594 (the coefficient of determination is 0.998). The fluorescence signal at 670 nm increases linearly in the 10-250 pg mL-1 concentration range with the calibration equation of y = 500lgx-383 (the coefficient of determination is 0.991). The assay was applied to the determination of FB1 contents in spiked corn samples. The average recoveries ranged from 92 to 107%, confirming the practicality of this method. The results obtained by this assay are in good agreement with that of LC-MS/MS method. Graphical abstractSchematic illustration of a bimodal aptasensor based on surface enhanced Raman scattering (SERS) and fluorescence change for the detection of fumonisin B1 (FB1).

Competitive redox reaction of Au-NCs/MnO2 nanocomposite: Toward colorimetric and fluorometric detection of acid phosphatase as an indicator of soil cadmium contamination.

In this study, by rational regulating the competitive redox reaction of Au-NCs/MnO2 nanocomposite between the dye indigo carmine (IC) and the enzymatic product L-ascorbic acid (AA), we have established a colorimetric and fluorometric double-channel responsive assay for acid phosphatase (ACP), which could serve as an indicator of soil cadmium (Cd) contamination. Initially, the gold nanoclusters (Au-NCs) were added to the suspension of MnO2 nanosheets to form Au-NCs/MnO2 nanocomposite with enhanced oxidative degradation ability. When IC was subsequently added, the blue color of IC faded due to oxidative degradation, and the mixture showed the yellow color of Au-NCs/MnO2 nanocomposite. Meanwhile, based on the inner filter effect (IFE), the fluorescence of Au-NCs was suppressed by MnO2 nanosheets during this process. However, with the presence of AA, hydrolyzed from L-ascorbic-2-phosphate (AAP) by ACP, the MnO2 nanosheets in Au-NCs/MnO2 nanocomposite were reduced to Mn2+ immediately. As a consequence, IC remained its blue color, in the meantime, the fluorescence of Au-NCs recovered, which essentially constituted a new mechanism for ACP detection with colorimetric and fluorometric double-channel response. With the method we developed, soil ACP activity can either be directly visualized by bare eyes or detected reliably through double channels. Furthermore, the dynamic changes of ACP activity during soil Cd contamination could also be monitored; the sharp increase of ACP activity at an appropriate time point could serve as a unique alarm for cadmium (Cd) contamination in soil, which is of great importance for soil quality evaluation and ecological risk assessment.