A Simple Schiff Base as Fluorescent Probe for Detection of Al3+ in Aqueous Media and its Application in Cells Imaging.

A novel fluorescence probe for the detection of Al3+ was developed based on methionine protected gold nanoclusters (Met-AuNCs). A fluorescent Schiff base (an aldimine) is formed between the aldehyde group of salicylaldehyde (SA) and the amino groups of Met on the AuNCs, and developed for selective detection of Al3+ in aqueous solution. Al3+ can strongly bind with the Schiff base ligands, accompanied by the blue-shift and an obvious fluorescence emission enhancement at 455 nm. The limits of detection (LODs) of the probe are 2 pmol L-1 for Al3+. Moreover, the probe can successfully be used in fluorescence imaging of Al3+ in living cells (SHSY5Y cells), suggesting that the simple fluorescent probe has great potential use in biological imaging.

Ultrasensitive colorimetric strategy for Hg2+ detection based on T-Hg2+-T configuration and target recycling amplification.

A novelty aptasensor for ultrasensitive detection of Hg2+ is developed, exploiting the combination of plasmonic properties of gold nanoparticles (AuNPs) and exonuclease III (Exo III)-assisted target recycling for signal amplification. In the presence of Hg2+, a DNA duplex can be formed due to the strong coordination of Hg2+ and T bases of single-stranded DNA (ssDNA) probe. Exo III digests the DNA duplex from the 3' to 5' direction, resulting in the releasing of Hg2+. Then, the released Hg2+ binds with another ssDNA probe through T-Hg2+-T coordination. After Exo III-assisted Hg2+ cycles, numerous ssDNA probes are exhausted, which promotes poly(diallyldimethylammonium chloride) (PDDA)-induced AuNP aggregation, leading to an obvious color change and aggregation-induced plasmon red shift of AuNPs (from 520 to 610 nm). Therefore, this biosensor is ultrasensitive, which is applicable to the detection of trace level of Hg2+ with a linear range from 5 pM to 0.6 nM and an ultralow detection limit of 0.2 pM. Furthermore, it enables visual detection of Hg2+ as low as 50 pM by the naked eye. More importantly, the assay can be applied to the reliable determination of spiked Hg2+ in sea water samples with good recovery.

Highly sensitive and selective detection and intracellular imaging of glutathione using MnO2 nanosheets assisted enhanced fluorescence of gold nanoclusters.

Glutathione (GSH) plays a critical role in biological defense system and is associated with numerous human pathologies. However, it still remains a challenge for fluorescent detection of GSH over cysteine (Cys) and homocysteine (Hcy) because of their similar structures. In this work, MnO2 nanosheets can efficiently quench the fluorescence of gold nanoclusters (Met-AuNCs) prepared by blending methionine and HAuCl4 owing to their superior absorption capability. However, GSH can reduce MnO2 nanosheets into Mn2+ which leads to the fluorescence recovery of Met-AuNCs. More intriguingly, GSH can dramatically and selectively enhance the fluorescence intensity of Met-AuNCs. Hence, a low background, ultrasensitive fluorescent detection of GSH was obtained with a detection limit of 68 nM. Moreover, the assay has been successfully used for GSH detection in human serum samples and cellular imaging with high selectivity over Cys and Hcy.

Colorimetric determination of DNA using an aptamer and plasmonic nanoplatform.

A facile plasmonic nanoplatform was developed for rapid and sensitive determination of nucleic acid. Hg2+-regulated molecular beacon (MB, hairpin) containing rich thymine (T) bases at both ends is used as the probe. A hairpin structure can be formed in the MB probe due to the strong binding of Hg2+ to T. However, in the presence of target DNA, the hairpin structure is opened owing to target DNA-specific hybridization with the aptamer. Simultaneously, the opened MB interacts with poly(diallyldimethylammonium chloride) (PDDA) and hinders PDDA-induced aggregation of AuNPs, accompanied by a color change from blue to red and a decrease in absorption ratio (A620/A520). Hence, a good linear relationship was observed between the decreased absorption ratio (A620/A520) and DNA concentration ranging from 0.02 to 2 nmol/L with a low detection limit of 4.42 pmol/L. Moreover, this nanoplatform has been successfully utilized to discriminate between perfect target and mismatch sequences. More importantly, the bioassay is simple, versatile, rapid, and cost-effective compared with other common methods, which holds great promise for clinical diagnosis and biomedical application. Graphical abstract.

Fluorescent methionine-capped gold nanoclusters for ultra-sensitive determination of copper(II) and cobalt(II), and their use in a test strip.

A fluorometric assay was constructed for supersensitive determination of Cu2+ and Co2+ based on their quenching effect on the orange fluorescence of methionine-capped gold nanoclusters (Met-AuNCs). A simple one-step method was developed for the preparation of the Met-AuNCs, employing L-methionine as both a reducing and protecting reagent. Within 10 min, water soluble Met-AuNCs were obtained with an average size of 2.4 nm. Under photoexcitation at 370 nm, the Met-AuNCs possess a maximum emission at 580 nm and a quantum yield of 2.3%. The response is fast (1 min), and the selectivity for Cu2+ and Co2+ is high over other metal ions. The detection limits for Cu2+ and Co2+ are around 47 and 420 pM, respectively. The effects were used to design a test paper for visual detection of Cu2+ and Co2+. Using this test paper, 1 µM of Cu2+/Co2+ can be detect under the UV lamp (365 nm excitation). It is perceived to be a promising tool for the rapid on-site determination of Cu2+ and Co2+ in real water samples. Graphical abstract Schematic presentation of gold nanoclusters (AuNCs) synthesis with methionine as both reducing and protecting reagent, Cu2+/Co2+ detection based on the quenching of the fluorescence of AuNCs, and visual detection of Cu2+/Co2+ based on a respective test strip.

A label-free hairpin aptamer probe for colorimetric detection of adenosine triphosphate based on the anti-aggregation of gold nanoparticles.

A facile and rapid colorimetric approach was described for selective and sensitive determination of adenosine triphosphate (ATP) based on a hairpin aptamer probe and the anti-aggregation of AuNPs. Poly(diallyldimethylammonium chloride) (PDDA) can induce the aggregation of AuNPs due to the electrostatic interaction causing a red to blue color change. Upon the addition of ATP, aptamer-based hairpin probe is opened and releases flexible ssDNA ends. The released flexible ssDNA ends can interact with PDDA and prevent PDDA-induced AuNPs aggregation. Thus, a visible color change from blue to red and a decrease in the absorption ratio (A610/A520) are observed. Under the optimal conditions, the hairpin aptamer-based colorimetric assay exhibits high sensibility and selectivity for the detection of ATP with a detection limit of 1.7nM. Moreover, this assay is successfully used in the rapid determination of ATP in spiked human serum samples with good recoveries in the range of 102.88 to 104.07%.

A hairpin-type DNA probe for direct colorimetric detection of endonuclease activity and inhibition based on the deaggregation of gold nanoparticles.

A method is described for the determination of the activity of endonuclease. It based on the deaggregation of gold nanoparticles (AuNPs) aggregated by the action of poly(diallyldimethylammonium chloride) (PDDA). A single-stranded DNA (ssDNA) is released after enzymatic cleavage catalyzed by endonuclease. The released fragments bind electrostatically to PDDA and inhibit the PDDA-induced aggregation of AuNPs. This is accompanied by a color change from blue to red and a decrease in the absorption ratio (A630/A520). Under the optimal conditions, this ratio increases linearly in the 0.001 to 1 U·µL-1 EcoRI endonuclease activity range. The detection limit is of 2 × 10-4 U·µL-1 which is much better or at least comparable to previous reports. The method is deemed to have wide scope in that it may be used to study other endonuclease activity (such as BamHI) by simply changing the specific recognition site of the hairpin-like DNA probe. The assay may also be employed to screening for inhibitors of EcoRI endonuclease. Graphical abstract Schematic presentation of the colorimetric assay based on the deaggregation of AuNPs for the detection of endonuclease activity. A single-stranded sequence (ssDNA) is released by the EcoRI cleavage, which electrostatically binds to PDDA and inhibits the PDDA-induced aggregation of AuNPs accompanying with a color change from blue to red.

An aptamer-based colorimetric Pt(II) assay based on the use of gold nanoparticles and a cationic polymer.

A colorimetric method is described for the determination of Pt(II). It is based on the use of gold nanoparticles (AuNPs) which are known to aggregate in the presence of a cationic polymer such as poly(diallyldimethylammonium chloride) (PDDA). If, however, a mismatched aptamer (AA) electrostatically binds to PDDA, aggregation is prevented. Upon the addition of Pt(II), it will bind to the aptamer and induce the formation of a hairpin structure. Hence, interaction between aptamer and PDDA is suppressed and PDDA will induce the aggregation of the AuNPs. This is accompanied by a color change from red to blue. The effect can be observed with bare eyes and quantified by colorimetry via measurement of the ratio of absorbances at 610 nm and 520 nm. Response is linear in the 0.24-2 µM Pt(II) concentration range, and the detection limit is 58 nM. The assay is completed within 15 min and selective for Pt(II) even in the presence of other metal ions. It was successfully applied to the rapid determination of Pt(II) in spiked soil samples. Graphical abstract Schematic representation of the method for detection of Pt(II) based on the use of a cationic polymer and gold nanoparticles. In the presence of Pt(II), aptamer interacts with the Pt(II) and prevents the interaction between aptamer and cationic polymer. Hence, cationic polymer induce the aggregation of the AuNPs and lead to the color change from red to blue.

Quantum dots for a high-throughput Pfu polymerase based multi-round polymerase chain reaction (PCR).

Multi-round PCR is an important technique for obtaining enough target DNA from rare DNA resources, and is commonly used in many fields including forensic science, ancient DNA analysis and cancer research. However, multi-round PCR is often aborted, largely due to the accumulation of non-specific amplification during repeated amplifications. Here, we developed a Pfu polymerase based multi-round PCR technique assisted by quantum dots (QDs). Different PCR assays, DNA polymerases (Pfu and Taq), DNA sizes and GC amounts were compared in this study. In the presence of QDs, PCR specificity could be retained even in the ninth-round amplification. Moreover, the longer and more complex the targets were, the earlier the abortion happened in multi-round PCR. However, no obvious enhancement of specificity was found in multi-round PCR using Taq DNA polymerase. Significantly, the fidelity of Pfu polymerase based multi-round PCR was not sacrificed in the presence of QDs. Besides, pre-incubation at 50 °C for an hour had no impact on multi-round PCR performance, which further authenticated the hot start effect of QDs modulated in multi-round PCR. The findings of this study demonstrated that a cost-effective and promising multi-round PCR technique for large-scale and high-throughput sample analysis could be established with high specificity, sensibility and accuracy.

Recyclable colorimetric sensor of Cr3+ and Pb2+ ions simultaneously using a zwitterionic amino acid modified gold nanoparticles.

In this work, a rapid, simple and sensitive colorimetric sensor for simultaneous (or respective) detection of Cr3+ and Pb2+ using tyrosine functionalized gold nanoparticles (AuNPsTyr) has been developed. Tyrosine, a natural and zwitterionic amino acid, could be as a reducing and capping agent to synthesise AuNPs and allow for the simultaneous and selective detection of Cr3+ and Pb2+. Upon the addition of Cr3+ or Pb2+ (a combination of them), the color of AuNPsTyr solution changes from red to blue grey and the characteristic surface plasmon resonance (SPR) band is red-shifted to 580nm due to the aggregation of AuNPs. Interestingly, the aggregated AuNPsTyr can be regnerated and recycled by removing Pb2+ and Cr3+. Even after 3 rounds, AuNPsTyr show almost the same A580nm/A520nm value for the assays of Pb2+ and Cr3+, indicating the good recyclability of the colorimetric sensor. The responding time (within 1min) and sensitivity of the colorimetric sensor are largely improved after the addition of 0.1M NaCl. Moreover, the AuNPsTyr aggregated by Cr3+ or Pb2+ (a combination of them) show excellent selectivity compared to other metal ions (Cr3+, Pb2+, Fe2+,Cu2+,Zn2+,Cr6+,Ni2+,Co2+,Hg2+,Mn2+,Mg2+,Ca2+,Cd2+). More importantly, the developed sensor manifests good stability at room temperature for 3months, which has been successfully used to determine Cr3+ and Pb2+ in the real water samples with a high sensitivity.

Identification and characterization of a ferritin gene involved in the immune defense response of scallop Chlamys farreri.

Scallop Chlamys farreri is an important aquaculture species in northern China. However, its mass mortality caused by several pathogens can result in great economic loss and negative impacts to the sustainable development of the scallop industry. Thus, improving the overall understanding of immune response mechanisms involved in host-pathogen interactions is necessary. Ferritins are conserved molecules in organisms that are involved in diverse biological processes, such as mediating host-pathogen responses. In this study, we report a novel ferritin gene from C. farreri (denoted as CfFER). The full length of CfFER is 848 bp and contains a 5'-UTR of 113 bp, a 3'-UTR of 219 bp, and a complete open reading frame (ORF) of 516 bp. The ORF encodes a polypeptide of 171 amino acid residues with a molecular weight of approximately 19.95 kDa and an isoelectric point of 5.07. The CfFER protein exhibited typical ferritin structures, namely, a ferroxidase diiron center, a ferrihydrite nucleation center, and an iron-binding response signature. Phylogenetic analysis revealed that CfFER was closely related to other mollusk ferritin proteins. Expression of CfFER in different tissues was analyzed by quantitative real-time PCR, and results showed that CfFER was ubiquitously expressed in all examined tissues. The highest and lowest expression levels of CfFER were measured in the muscle and hemocyte, respectively. The relative mRNA expression of CfFER in response to bacterial (Vibrio anguillarum) and viral (acute viral necrobiotic virus) challenges sharply increased by ca. 5-fold about12 h post-infection (hpi) and then normalized at 48 hpi. Western blot analysis with polyclonal antibodies generated from the recombinant product of CfFER also demonstrated the presence of ferritin protein in hemocytes. These findings strongly suggest that CfFER is involved in the immune response of C. farreri and protection against pathogen challenge.

Development of a high-throughput real time PCR based on a hot-start alternative for Pfu mediated by quantum dots.

Hot start (HS) PCR is an excellent alternative for high-throughput real time PCR due to its ability to prevent nonspecific amplification at low temperature. Development of a cost-effective and simple HS PCR technique to guarantee high-throughput PCR specificity and consistency still remains a great challenge. In this study, we systematically investigated the HS characteristics of QDs triggered in real time PCR with EvaGreen and SYBR Green I dyes by the analysis of amplification curves, standard curves and melting curves. Two different kinds of DNA polymerases, Pfu and Taq, were employed. Here we showed that high specificity and efficiency of real time PCR were obtained in a plasmid DNA and an error-prone two-round PCR assay using QD-based HS PCR, even after an hour preincubation at 50 °C before real time PCR. Moreover, the results obtained by QD-based HS PCR were comparable to a commercial Taq antibody DNA polymerase. However, no obvious HS effect of QDs was found in real time PCR using Taq DNA polymerase. The findings of this study demonstrated that a cost-effective high-throughput real time PCR based on QD triggered HS PCR could be established with high consistency, sensitivity and accuracy.

A hot start alternative for high-fidelity DNA polymerase amplification mediated by quantum dots.

Quantum dots (QDs) are of great interest due to their unique chemical and physical properties. Recently, a hot start (HS) polymerase chain reaction (PCR) amplification performance based on QDs with a high-fidelity Pfu DNA polymerase has been reported. However, whether QDs can trigger HS effects with other high-fidelity or conventional DNA polymerases is yet to be understood. In the present study, we studied the QD-triggered HS effects with four high-fidelity and three conventional DNA polymerases, and the HS effect comparisons among them were also made. It was found that QDs could trigger a distinct HS PCR amplification performance with all the four tested high-fidelity DNA polymerases, and specific target DNA could be well amplified even if the PCR mixture was pre-incubated for 2 h at 50°C. On the contrary, the HS effects were not prominent with all the three conventional Taq DNA polymerases. Specifically, the fidelity of Pfu is not sacrificed in the presence of QDs, even after a 1 h pre-incubation at 50°C before PCR. Furthermore, the electrophoresis results preliminarily demonstrated that QDs prefer to adsorb high-fidelity polymerases rather than conventional ones, which might result in the QD-triggered HS effects on PCR performance by using high-fidelity DNA polymerases.

Characterization and separation of semiconductor quantum dots and their conjugates by capillary electrophoresis.

Semiconductor quantum dots (QDs) are very important luminescent nanomaterials with a wide range of potential applications. Currently, QDs as labeling probes are broadly used in bioassays, including immunoassay, DNA hybridization, and bioimaging, due to their excellent physical and chemical properties, such as broad excitation spectra, narrow and size-dependent emission profiles, long fluorescence life time, and good photostability. The characterization of QDs and their conjugates is crucial for their wide bioapplications. CE has become a powerful tool for the separation and characterization of QDs and their conjugates. In this review, some CE separation models of QDs are first introduced, mainly including CZE, CGE, MEKC, and ITP. And then, some key applications, such as the measurements of size, surface charge, and concentration of QDs and the characterization of QDs conjugates (e.g. QD-protein, QD-DNA, QD-small molecule), are also described. Finally, future perspectives are discussed.

CdTe quantum dots enhance feasibility of EvaGreen-based real-time PCR with decent amplification fidelity.

Quantitative real-time PCR (qPCR), as an important quantitative technique for nucleic acids, has been widely used in many fields including clinical diagnosis, molecular biology, and cancer research. However, non-specific amplification products are still a frequent problem in qPCR. In this study, we investigated the effects of QDs on real-time amplification based on either SYBR Green I or EvaGreen. It was found that QDs could raise the amplification sensitivity and thus enhance the efficiency using SYBR Green I detection system. In the case of EvaGreen detection systems, addition of QDs also led to a better correlation coefficient than without QDs. EvaGreen-based system gave sharper peaks for melting curves than SYBR Green I. The experiments indicated that the polymerase activity could be partially blocked by QDs at the pre-PCR temperatures, resulting in the improvement of PCR specificity. These results indicated that CdTe QDs could be used as a descent qPCR enhancer. Good amplification fidelity in QDs-facilitated qPCR was also a plus that has not been reported elsewhere.

Studies on interaction of CdTe quantum dots with bovine serum albumin using fluorescence correlation spectroscopy.

Luminescent quantum dots (QDs) have widely used in some biological and biomedical fields due to their unique and fascinating optical properties, meanwhile the interaction of QDs with biomolecules recently attract increasing attention. In this paper, we employed fluorescence correlation spectroscopy (FCS) to investigate the nonspecific interaction between CdTe QDs and bovine serum albumin (BSA) as a model, and evaluate their stoichiometric ratio and association constant. Our results documented that BSA was able to bind to CdTe QDs and form the QD-BSA complex by a 1:1 stoichiometric ratio. The association constant evaluated is 1.06+/-0.14x10(7) M(-1) in 0.01 M phosphate buffer (pH=7.4). Furthermore, we found that QD-BSA complex dissociated with increase of ion strength, and we speculated that the interaction of CdTe QDs with BSA was mainly attributed to electrostatic attraction. Our preliminary results demonstrate that fluorescence correlation spectroscopy is an effective tool for investigation of the interaction between quantum dots (or nanoparticles) and biomolecules.

Fabrication of PDMS/glass microchips by twofold replication of PDMS and its application in genetic analysis.

In this paper, we describe a simple method for fabrication of high quality poly(dimethylsiloxane) (PDMS)/glass microchip by twofold replica molding of PDMS. This technique first served to transfer the negative microchannels from the glass template to the PDMS substrate as a master, and then this PDMS master with positive microchannels was used to replicate the PDMS replica with negative microchannels. Finally, the PDMS replica was bound to a glass sheet by UV radiation. The fabricated microchips were successfully applied for the detection of C677T mutation from the human methylenetetrahydrofolate reductase gene.

Genetic mutation analysis by CE with LIF detection using inverse-flow derivatization of DNA fragments.

Inverse-flow derivatization is a novel approach to obtain fluorescent DNA derivatives in DNA analysis based on CE with LIF detection. In the present work, we want to explore the feasibility of the application of this method into the mutation detection based on constant denaturant capillary electrophoresis (CDCE) and SSCP analysis. The DNA fragments were first amplified by PCR using a pair of common primers without fluorescent label, and then the mutations were determined by CDCE or SSCP analysis based on CE-LIF with inverse-flow derivatization of DNA fragments. The experimental conditions were investigated systematically, and different labeling modes including inverse-flow derivatization, on-column derivatization and fluorescent labeled primer technique were compared. The inverse-flow derivatization was successfully used in the detection of C677T mutation in the methylenetetrahydrofolate reductase gene by CDCE or SSCP analysis. Our preliminary results demonstrate that inverse-flow derivatization is very simple, inexpensive and sensitive and well suitable for the genetic analysis in clinical diagnosis.

Capillary electrophoresis of double-stranded DNA fragments using a new fluorescence intercalating dye EvaGreen.

EvaGreen is a new DNA intercalating dye successfully used in quantitative real-time PCR. In the present work, we firstly apply EvaGreen to the analysis of dsDNA by CE with LIF detection. Comparisons of EvaGreen dye with the commonly used dyes SYBR Green I and SYBR Gold were preformed in dsDNA analysis by CE. The linear range of dsDNA using EvaGreen was slightly wider than that using SYBR Gold and SYBR Green I, and the detection limits of dsDNA were not significantly different for the three dyes. Good separations of dsDNA fragments were obtained using the three dyes. Reproducibility of migration time and the peak area of dsDNA fragments with EvaGreen were better than those for SYBR Green I and SYBR Gold. The RSD values were 0.24-0.27% for migration time and 3.45-7.59% for peak area within the same day, 1.35-1.63% for migration time and 6.72-12.05% for peak area for three days. Our data demonstrated that EvaGreen is well suited for the dsDNA analysis by CE with LIF detection.

Comparisons between capillary zone electrophoresis and real-time PCR for quantification of circulating DNA levels in human sera.

BACKGROUND: Recently, some research results showed that the circulating DNA in serum or plasma had potential for the molecular diagnosis and prognosis of certain cancers. Several methods have been employed for the quantification of circulating DNA. However, the circulating DNA levels obtained by various methods exhibited considerable differences. Additionally, these methods were labor-extensive and time-consuming, and not suitable for the quantification of circulating DNA in numerous samples due to the use of commercial DNA extraction kits for the purification of circulating DNA. We presented a new method for the quantification of circulating DNA in sera by capillary zone electrophoresis (CZE) with laser-induced fluorescence detection (LIF). METHODS: In the present work, we want to make comparison between CZE-LIF assay and real time PCR for the quantification of circulating DNA levels. Linearity, intra and inter variability of two methods were evaluated. RESULTS: The intra and inter variability of circulating DNA quantification by real-time PCR were 7.3% and 14.92%, respectively. In CZE assay the intra and inter variability were 4.19% and 6.91%, respectively. The R.S.D. values of the same coated capillary and different coated capillaries were 5.14% and 9.02%, respectively. Our data showed that the circulating DNA levels obtained by two methods had a good correlation. Moreover, we further confirmed that blood samples collection, serum preparation and other treatment procedures had a significant impact on the DNA levels in sera. CONCLUSION: Our data further illustrated that CZE-LIF is a simple, rapid and sensitive method for the quantification of circulating DNA in human sera, and well suitable for the analysis of a large number of samples in clinical diagnosis.