Oligonucleotide-induced regulation of the oxidase-mimicking activity of octahedral Mn3O4 nanoparticles for colorimetric detection of heavy metals.

A colorimetric assay for the determination of heavy metal ions is presented that is based on the regulation of the oxidase-mimicking activity of Mn3O4 nanoparticles (NPs) by oligonucleotides. The chromogenic agent tetramethylbenzidine (TMB) is oxidized by the catalytic action of Mn3O4 NPs to generate products that have a yellow color in acidic solution, with a peak at 450 nm. It is found that random oligonucleotides are absorbed on the regular surface of the Mn3O4 NPs and temporarily inhibit the oxidation of TMB. This leads to a decrease in absorbance and a light-green coloration of the solution. The results show that the purine bases in oligonucleotides play a key role in their regulation of the activity of the NPs. The regulatory effect is assumed to be of the noncompetitive type. In the presence of heavy metal ions like Hg(II) or Cd(II), the inhibition is canceled due to the binding of heavy metal ions to thymine bases, and the color of the solution changes from light green to yellow. The increase in absorbance at 450 nm is related to the amount of heavy metal ions present. The method allows Hg(II) and Cd(II) to be determined visually in concentrations as low as 20 µg·L-1. The detection limit of the colorimetric assay is 3.8 and 2.4 µg·L-1 of Hg(II) and Cd(II), respectively. The assay displays good selectivity over other heavy metal ions. The method was successfully validated by analyzing several water samples. Graphical abstract Schematic representation of the colorimetric assay for Hg(II) and Cd(II) based on the intrinsic oxidase-mimicking activity of Mn3O4 nanoparticles that is regulated by oligonucleotides.

Ratiometric fluorescent test pen filled with a mixing ink of carbon dots and CdTe quantum dots for portable assay of silver ion on paper.

A ratiometric fluorescent test pen filled with a mixing ink of blue carbon dots (CDs) and red CdTe quantum dots (CdTe QDs) is introduced for portable assay of silver ion (Ag(I)) on paper. The mixing ink was tuned with ratiometric fluorescent intensity of 1:5, and then filled into a vacant commercial fluorescent pen core. Writing/painting a random word/figure on a blank paper can make the most portable nanoprobe determining Ag(I) by visualization. Ag(I) can adsorb onto the surface of CdTe QDs, which leads to the formation of surficial Ag2Te layer by an ion-exchanging reaction. This enables the red fluorescence of CdTe QDs (with excitation/emission maxima at 360/628 nm) to be quenched. Due to the unchanged blue fluorescence of CDs (with excitation/emission maxima at 360/440 nm) as internal standard, the solution color evolves gradually from red to blue with the increase of Ag(I) concentration with a detection limit of 3.48 nM. This is at least 2 orders of magnitude lower than the limit defined by World Health Organization (WHO) in drinkable water. The fluorescent test pen has also been used for the determination of Ag(I) in wastewater. Graphical abstract Ag(I) can adsorb on the surface of CdTe QDs to quench their fluorescence, while the fluorescent intensity of CDs keep constant, accompanying color change with the increase of Ag(I) concentration. The method offers a visual assay of Ag(I) in water.

Nuclear fast red-based colorimetric sensors for sensitive and selective detection of Ag ions.

The reduction of nuclear fast red (NFR) stain by sodium tetrahydroboron was catalyzed in the presence of silver ions (Ag+ ). The fluorescence properties of reduced NFR differed from that of NFR. The product showed fluorescence emission at 480 nm with excitation at 369 nm. Furthermore, the fluorescence intensity of the mixture increased strongly in the presence of Ag+ and Britton-Robinson buffer at pH 4.78. There was a good linear relationship between increased fluorescence intensity (ΔI) and Ag+ concentration in the range 5.0 × 10-9 to 5.0 × 10-8  M. The correlation coefficient was 0.998, and the detection limit (3σ/k) was 1.5 × 10-9  M. The colour of the reaction system changed with variation in Ag+ concentration over a wide range. Based on the colour change, a visual semiquantitative detection method for recognition and sensing of Ag+ was developed for the range 1.0 × 10-8  to 5.0 × 10-4  M, with an indicator that was visible to the naked eye. Therefore, a sensitive, simple method for determination of Ag+ was developed. Optimum conditions for Ag+ detection, the effect of other ions and the analytical application of Ag+ detection of synthesized sample were investigated.

CdSe/ZnS quantum dots coated with carboxy-PEG and modified with the terbium(III) complex of guanosine 5'-monophosphate as a fluorescent nanoprobe for ratiometric determination of arsenate via its inhibition of acid phosphatase activity.

A ratiometric fluorometric method is described for the determination of arsenate via its inhibitory effect on the activity of the enzyme acid phosphatase. A nanoprobe was designed that consists of CdSe/ZnS quantum dots (QDs) coated with the terbium(III) complex of guanosine monophosphate (Tb-GMP). The nanoprobe was synthesized from carboxylated QDs, Tb(III) and GMP via binding of Tb(III) by both the carboxy and the phosphate groups. The nanoprobe, under single-wavelength excitation (at 280 nm), displays dual (red and green) emission, with peaks at around 652 nm from the QDs, and at 547 nm from the Tb-GMP coordination polymers. It is shown to be a viable nanoprobe for fluorometric determination of As(V) detection through it inhibitory action on the activity of acid phosphatase (ACP). The enzyme destroys the Tb-GMP structure via hydrolysis of GMP, and hence the fluorescence of the Tb-GMP complex is quenched. In contrast, the fluorescence of the CdSe/ZnS QDs remains inert to ACP. It therefore can serve as an internal reference signal. In the presence of arsenate (an analog of phosphate), the activity of ACP is inhibited due to competitive binding. Thus, hydrolysis of GMP is prevented. These findings were used to design a ratiometric fluorometric method for the quantification of As(V). The ratio of fluorescences at 547 and 652 nm increases linearly in the 0.5 to 200 ppb As(V) concentation range, and the limit of detection is 0.39 ppb. Under a UV lamp, the probe shows distinguishable color from green to red on increasing the concentration of As(V). Graphical abstract Schematic illustration of CdSe/ZnS quantum dot coated with carboxy-PEG and modified with the terbium(III)-GMP complex as a fluorescent nanoprobe for ratiometric determination of arsenate via its inhibition of ACP activity.

High-throughput clone screening followed by protein expression cross-check: A visual assay platform.

In high-throughput biotechnology and structural biology, molecular cloning is an essential prerequisite for attaining high yields of recombinant protein. However, a rapid, cost-effective, easy clone screening protocol is still required to identify colonies with desired insert along with a cross check method to certify the expression of the desired protein as the end product. We report an easy, fast, sensitive and cheap visual clone screening and protein expression cross check protocol employing gold nanoparticle based plasmonic detection phenomenon. This is a non-gel, non-PCR based visual detection technique, which can be used as simultaneous high throughput clone screening followed by the determination of expression of desired protein.

Automated, high-throughput, in vivo analysis of visual function using the zebrafish.

BACKGROUND: Modern genomics has enabled the identification of an unprecedented number of genetic variants, which in many cases are extremely rare, associated with blinding disorders. A significant challenge will be determining the pathophysiology of each new variant. The Zebrafish is an excellent model for the study of inherited diseases of the eye. By 5 days post-fertilization (dpf), they have quantifiable behavioral responses to visual stimuli. However, visual behavior assays can take several hours to perform or can only be assessed one fish at a time. RESULTS: To increase the throughput for vision assays, we used the Viewpoint Zebrabox to automate the visual startle response and created software, Visual Interrogation of Zebrafish Manipulations (VIZN), to automate data analysis. This process allows 96 Zebrafish larvae to be tested and resultant data to be analyzed in less than 35 minutes. We validated this system by disrupting function of a gene necessary for photoreceptor differentiation and observing decreased response to visual stimuli. CONCLUSIONS: This automated method along with VIZN allows rapid, high-throughput, in vivo testing of Zebrafish's ability to respond to light/dark stimuli. This allows the rapid analysis of novel genes involved in visual function by morpholino, CRISPRS, or small-molecule drug screens. Developmental Dynamics 245:605-613, 2016. © 2016 Wiley Periodicals, Inc.

Visual assessment of parasitic burden in infected macrophage by plasmonic detection of leishmania specific marker RNA.

Visceral leishmaniasis is a neglected tropical disease and may prove fatal if not diagnosed and treated early. The amastigotes of Leishmania donovani nest in the macrophage of human host and thus, determination of parasitic burden in the infected macrophages has been the most crucial step in diagnosis, dose determination and medical management of relapse cases of this fatal disease. Microscopic count following Giemsa staining and other morphological analysis are the classical ways vastly used in the resource stringent endemic areas. The current method introduced a high throughput, rapid, cheap, non-gel, non-PCR and nonculture based visual detection platform employing salt triggered aggregation of gold nanoparticle in presence of extracted total RNA from infected macrophages and leishmania specific oligo-nucleotide probe to determine the parasite burden in macrophages. Amastigote's small subunit ribosomal RNA (SSU rRNA, PMID 1565128) was used as the leishmania specific marker and its abundance in the total RNA extracts of infected macrophages were determined by this visual colorimetric assay.

A smartphone-based ratiometric fluoroprobe based on blue-red dual-emission signals of thiochrome and copper nanoclusters for sensitive assay of metam-sodium in cucumbers.

It is of great importance to develop the highly efficient fluorescence strategy for rapid/sensitive detection of metam-sodium (MES) in evaluating its residual safety, especially in fresh vegetables. Herein, we prepared an organic fluorophore (thiochrome, TC) and glutathione-capped copper nanoclusters (GSH-CuNCs), and their combination (TC/GSH-CuNCs) was sucessfully employed as a ratiometric fluoroprobe by means of the blue-red dual emission. The fluorescence intensities (FIs) of TC decreased upon the addition of GSH-CuNCs via the fluorescence resonance energy transfer (FRET) process. When fortified at the constant levels of GSH-CuNCs and TC, MES substantially reduced the FIs of GSH-CuNCs, while this was not the case in the FIs of TC except for the prominent red-shift of ∼30 nm. Compared to the previous fluoroprobes, the TC/GSH-CuNCs based fluoroprobe supplied wider linear range of 0.2-500 µM, lower detection limit (60 nM), and satisfactory fortification recoveries (80-107%) for MES in the cucumber samples. Based on the fluorescence quenching phenomenon, a smartphone application was used to output RGB values of the captured images for the colored solution. The smartphone-based ratiometric sensor could be utilized for the visual fluorescent quantitation of MES by virtue of the R/B values in cucumbers, which gave linear range (1-200 µM) and LOD (0.3 µM). By means of blue-red dual-emission fluorescence, the smartphone-based fluoroprobe provides a cost-effective, portable and reliable avenue for the on-site, rapid and sensitive assay of MES's residues in complex vegetable samples.

A novel raiometric fluorescence probe based on silicon quantum dots and copper nanoclusters for visual assay of l-cysteine in milks.

l-cysteine (l-Cys) plays an important role in many physiological processes. The previously reported methodologies for l-Cys detection have many drawbacks, and thus the development of specific/sensitive approaches is crucial for its direct/quick assay in food matrices. Herein, silicon quantum dots (SiQDs) and glutathione-stabilized copper nanoclusters (CuNCs) were successfully synthesized and integrated as a ratiometric fluorescent probe for assay l-Cys assay in milks. The fluorescence of SiQDs was quenched by CuNCs through fluorescence resonance energy-transfer process. Upon addition of l-Cys, the 440-nm fluorescence of SiQDs changed insignificantly, while the 650-nm fluorescence of CuNCs decreased significantly. Ratiometric fluorescence signal was linear in the l-Cys concentration range of 0.25 µM to 2.5 mM with a detection limit of 75 nM and visual color changes from red to blue. The probe can realize rapid and portable detection without the participation of intermediate ions, and has good selectivity and accuracy for l-cysteine in milk samples.

Functional Role of the RNA-Binding Protein Rbm24a and Its Target sox2 in Microphthalmia.

Congenital eye defects represent a large class of disorders affecting roughly 21 million children worldwide. Microphthalmia and anophthalmia are relatively common congenital defects, with approximately 20% of human cases caused by mutations in SOX2. Recently, we identified the RNA-binding motif protein 24a (Rbm24a) which binds to and regulates sox2 in zebrafish and mice. Here we show that morpholino knockdown of rbm24a leads to microphthalmia and visual impairment. By utilizing sequential injections, we demonstrate that addition of exogenous sox2 RNA to rbm24a-deplete embryos is sufficient to suppress morphological and visual defects. This research demonstrates a critical role for understanding the post-transcriptional regulation of genes needed for development.

Label-free peptide nucleic acid biosensor for visual detection of multiple strains of influenza A virus suitable for field applications.

Accurate and rapid diagnosis of Influenza A viruses (IAVs) is challenging because of multiple strains circulating in humans and animal populations, and the emergence of new strains. In this study, we demonstrate a simple and rapid strategy for visual detection of multiple strains of IAVs (H1 to H16 subtypes) using peptide nucleic acid (PNA) as a biosensor and unmodified gold nanoparticles (AuNPs) as a reporter. The design principle of the assay is based on the color change on account of free PNA-induced aggregation of AuNPs in the presence of non-complementary viral RNA sequence and vice-versa. The assay could detect IAV RNA with a visual limit of detection of 2.3 ng. The quantification of RNA with a considerable accuracy on a simple spectrophotometer was achieved on plotting the PNA-induced colorimetric changes (absorption ratio of A640/A520) in the presence of a varying concentration of complementary RNA. As a proof-of-concept, the visual assay was validated on 419 avian clinical samples and receiver operating characteristic (ROC) curve analysis showed a high diagnostic specificity (96.46%, 95% CI = 93.8 to 98.2) and sensitivity (82.41%, 95% CI = 73.9 to 89.1) when RT-qPCR was used as reference test. Hence, the simplicity, rapidity, and universality of this strategy make it a potential candidate visual assay for clinical diagnosis and surveillance of IAVs, especially in the resource-limited settings. The proposed strategy establishes new avenues for developing a simple and rapid diagnostic system for viral infections and biomolecules.

Cascade of deoxyribozymes for the colorimetric analysis of drug resistance in Mycobacterium tuberculosis.

A visual cascade detection system has been applied to the detection and analysis of drug-resistance profile of Mycobacterium tuberculosis complex (MTC), a causative agent of tuberculosis. The cascade system utilizes highly selective split RNA-cleaving deoxyribozyme (sDz) sensors. When activated by a complementary nucleic acid, sDz releases the peroxidase-like deoxyribozyme apoenzyme, which, in complex with a hemin cofactor, catalyzes the color change of the sample's solution. The excellent selectivity of the cascade has allowed for the detection of point mutations in the sequences of the MTC rpoB, katG, and gyrA genes, which are responsible for resistance to rifampin, isoniazid, and fluoroquinolone, respectively. When combined with isothermal nucleic acid sequence based amplification (NASBA), the assay was able to detect amplicons of 16S rRNA and katG mRNA generated from 0.1 pg and 10 pg total RNA taken for NASBA, respectively, in less than 2 h, producing a signal detectable with the naked eye. The proposed assay may become a prototype for point-of-care diagnosis of drug resistant bacteria with visual signal output.

A High-Throughput Assay for Congenital and Age-Related Eye Diseases in Zebrafish.

Debilitating visual impairment caused by cataracts or microphthalmia is estimated to affect roughly 20 million people in the United States alone. According to the National Eye Institute, by 2050 that number is expected to more than double to roughly 50 million. The identification of candidate disease-causing alleles for cataracts and microphthalmia has been accelerated with advanced sequencing technologies creating a need for verification of the pathophysiology of these genes. Zebrafish pose many advantages as a high-throughput model for human eye disease. By 5 days post-fertilization, zebrafish have quantifiable behavioral responses to visual stimuli. Their small size, many progeny, and external fertilization allows for rapid screening for vision defects. We have adapted the OptoMotor Response to assay visual impairment in zebrafish models of cataracts and microphthalmia. This research demonstrates an inexpensive, high-throughput method for analyzing candidate genes involved in visual impairment.

Visual and fluorescent assays for selective detection of beta-amyloid oligomers based on the inner filter effect of gold nanoparticles on the fluorescence of CdTe quantum dots.

Beta-amyloid (Aß) peptides are the major constituents of senile plaques in the brains of Alzheimer's disease (AD) patients. Aß monomers (AßMs) can coalesce to form small, soluble oligomers (AßOs), followed by reorganization and assembly into long, thread-like fibrils (AßFs). Recently, soluble AßOs have been regarded as reliable molecular biomarkers for the diagnosis of AD because of their high toxicity for neuronal synapse and high concentration levels in the brains of AD patients. In this work, we reported a label-free, sensitive and selective method for visual and fluorescent detection of AßOs based on the inner filter effect (IFE) of gold nanoparticles (AuNPs) on the fluorescence of CdTe quantum dots (QDs). Specifically, the fluorescence of CdTe QDs was quenched significantly by AuNPs through the IFE. PrP(95-110), an AßOs-specific binding peptide from cellular prion protein, triggered the aggregation and color change of AuNPs suspension; thus, the IFE of AuNPs on the fluorescence of CdTe QDs was weakened and the fluorescence intensity was recovered. However, in the presence of AßOs, the specific interaction of AßOs and PrP(95-110) prevented the absorption of PrP(95-110) onto the surface of AuNPs. As a result, the aggregation of AuNPs was inhibited and the fluorescence intensity of CdTe QDs was quenched again. This label-free method is specific for detection of AßOs but not for AßMs and AßFs. The detection limits were found to be 0.5nM for the visual assay and 0.2nM for the fluorescent detection. We believe that this work would be valuable for many investigations related to AD diagnosis and drug discovery.

Determination of dissolved organic matter based on UV-light induced reduction of ionic silver to metallic nanoparticles by humic and fulvic acids.

We describe a novel solution-based method for the determination of dissolved organic matter (DOM) relying on the formation of silver nanoparticles (AgNPs) via photo-stimulated reduction of silver ions by humic and fulvic acids. The method is based on natural driven formation of nanoscale materials yielding a direct relationship between DOM concentration and AgNPs formation. The aqueous dispersion of the formed AgNPs show strong and uniform UV-Vis absorption bands between 450 and 550 nm irrespectively of DOM nature and properties (i.e. humic or fulvic acids). The ensuing chromatic shift accompanying the appearance of the new absorption bands is easily conceivable by a simple spectrophotometer and the bare eye, holding great promise for the on-site, instrumental-free screening of DOM levels. Under the optimum experimental conditions the determination of DOM was successfully demonstrated to various water samples with high sensitivity (<1.0 mg L(-1)), satisfactory recoveries (87.5-123.5%) and reproducibility (5.87-6.73%).