Facile and sensitive detection of mercury ions based on fluorescent structure-switching aptamer probe and exonuclease â ¢-assisted signal amplification.

Hg2+ is highly toxic to human health and ecosystem. In this work, based on the unique fluorescent property of 2-Aminopurine (2-AP), the formation of T-Hg2+-T mismatch structure and the signal amplification of exonuclease III (Exo III) assisted target cycle, a fluorescent probe for facile and sensitive detection of Hg2+ is constructed. The hairpin-looped DNA probe is rationally designed with 2-AP embedded in the stem and thymine-rich recognition overhangs extended at the termini. The cleavage of the double stranded DNA stem with stable T-Hg2+-T pairs catalyzed by Exo III is prompted to happen upon recognition of trace Hg2+. Under the optimal reaction conditions, there is an excellent linear relationship between Hg2+ concentration and fluorescence intensity in the range of 7.5-200 nM with a detection limit of 0.38 nM. In addition, the detection results of Hg2+ in Songhua River water and fish samples are satisfactory. The fluorescent probe avoids labeling additional quenchers or quenching materials and has strong anti-interference ability. Thus, the fluorescent probe has a broad prospect in practical application.

Metabolic engineering of the malonyl-CoA pathway to efficiently produce malonate in Saccharomyces cerevisiae.

Malonate is a platform chemical that has been utilized to synthesize many valuable chemical compounds. Here, Saccharomyces cerevisiae was metabolically engineered to produce malonate through the malonyl-CoA pathway. To construct the key step of converting malonyl-CoA to malonate, a native mitochondrial 3-hydroxyisobutyryl-CoA hydrolase gene EHD3 was mutated to target the cytoplasm and obtain malonyl-CoA hydrolase activity. The malonyl-CoA hydrolase activity of Ehd3 was achieved by mutating the malonyl-CoA binding site F121 to I121 and the active site E124 to seven amino acids (S/T/H/K/R/N/Q). We identified that the strain with E124S mutation had the highest malonate titer with 13.6 mg/L. Genomic integration of the mutant EHD3 and ACC1** to delta sequence sites was further explored to increase their reliable expression. Accordingly, a screening method with the work flow of fluorescence detection, shake-tube fermentation, and shake-flask fermentation was constructed to screen high copy delta sequences efficiently. The malonate titer was improved to 73.55 mg/L after screening the ∼1500 integrative strains, which was increased 4.4-folds than that of the episomal strain. We further engineered the strain by regulating the expression of key enzyme in the malonyl-CoA pathway to improve the precursor supply and inhibiting its competing pathways, and the final engineered strain LMA-16 produced 187.25 mg/L in the flask, 14-fold compared with the initial episomal expression strain. Finally, the combined efforts increased the malonate titer to 1.62 g/L in fed-batch fermentation.

A Nanobody-on-Quantum Dot Displacement Assay for Rapid and Sensitive Quantification of the Epidermal Growth Factor Receptor (EGFR).

Biosensing approaches that combine small, engineered antibodies (nanobodies) with nanoparticles are often complicated. Here, we show that nanobodies with different C-terminal tags can be efficiently attached to a range of the most widely used biocompatible semiconductor quantum dots (QDs). Direct implementation into simplified assay formats was demonstrated by designing a rapid and wash-free mix-and-measure immunoassay for the epidermal growth factor receptor (EGFR). Terbium complex (Tb)-labeled hexahistidine-tagged nanobodies were specifically displaced from QD surfaces via EGFR-nanobody binding, leading to an EGFR concentration-dependent decrease of the Tb-to-QD Förster resonance energy transfer (FRET) signal. The detection limit of 80±20 pM (16±4 ng mL-1 ) was 3-fold lower than the clinical cut-off concentration for soluble EGFR and up to 10-fold lower compared to conventional sandwich FRET assays that required a pair of different nanobodies.

Rapid and Wash-Free Time-Gated FRET Histamine Assays Using Antibodies and Aptamers.

Histamine (HA) is an indicator of food freshness and quality. However, high concentrations of HA can cause food poisoning. Simple, rapid, sensitive, and specific quantification can enable efficient screening of HA in food and beverages. However, conventional assays are complicated and time-consuming, as they require multiple incubation, washing, and separation steps. Here, we demonstrate that time-gated Förster resonance energy transfer (TG-FRET) between terbium (Tb) complexes and organic dyes can be implemented in both immunosensors and aptasensors for simple HA quantification using a rapid, single-step, mix-and-measure assay format. Both biosensors could quantify HA at concentrations relevant in food poisoning with limits of detection of 0.19 µg/mL and 0.03 µg/mL, respectively. Excellent specificity was documented against the structurally similar food components tryptamine and l-histidine. Direct applicability of the TG-FRET assays was demonstrated by quantifying HA in spiked fish and wine samples with both excellent concentration recovery and agreement with conventional multistep enzyme-linked immunosorbent assays (ELISAs). Our results show that the simplicity and rapidity of TG-FRET assays do not compromise sensitivity, specificity, and reliability, and both immunosensors and aptasensors have a strong potential for their implementation in advanced food safety screening.

Programmable Synthetic Upstream Activating Sequence Library for Fine-Tuning Gene Expression Levels in Saccharomyces cerevisiae.

A wide dynamic range of promoters is necessary for fine-tuning transcription levels. However, weak intensity and narrow dynamic range limit transcriptional regulation via constitutive promoters. The upstream activation sequence (UAS) located upstream of the core promoter is a crucial region that could obviously enhance promoter strength. Herein, we created a random mutagenesis library consisting of 330 different variants based on the UAS of the TDH3 promoter with an ∼37-fold dynamic range by error-prone polymerase chain reaction (PCR) and obtained strong intensity mutant UAS, which was ∼12-fold greater than the wild-type UASTDH3. Analysis of the mutant library revealed 15 strength-enhancing sites and their corresponding bases of the UASTDH3 regions, which provided the impetus for a synthetic library. The resulting 32 768 mutant UAS library was constructed by permutation and combination of the bases of the 15 enhancing sites. To characterize the library, a strength prediction model was built by correlating DNA structural features and UAS strength, which provided a model between UAS sequence and intensity. Following characterization, the UAS library was applied to precisely regulate gene expression in the production of ß-carotene, proving that the UAS library would be a useful tool for gene tuning in metabolic engineering. In summary, we designed, constructed, and characterized a UAS library that facilitated precise tuning of transcription levels of target proteins.

A signal-on fluorescent aptasensor by sensitized Tb3+ luminescence for detection of melamine in milk.

A fluorescent aptasensor based on sensitized terbium(III) luminescence was constructed to detect melamine in milk. Tb3+ as the fluorescence probe can be sensitized by a guanine-rich single-stranded DNA sequence, so the complementary sequence of the polythymidine aptamer (cDNA) was modified with six consecutive guanine bases (G6). In the absence of melamine, melamine aptamer combined with cDNA to form a double helix structure, and G6 hybridized with the extended cytosine bases in the aptamer, resulting in low fluorescence intensity of Tb3+. In the presence of melamine, cDNA was released due to the specific recognition of melamine to the aptamer, resulting in stronger sensitized fluorescence intensity of Tb3+. Under the optimum conditions, the linear concentration of melamine in the milk ranged from 1.0 µg/mL to 10.0 µg/mL. This aptasensor can be used for the accurate and rapid detection of melamine in milk with a detection limit of 0.02 µg/mL, and has the advantages of high sensitivity, high efficiency, simple operation and low cost.

Mechanistic analysis of cadmium toxicity in Saccharomyces cerevisiae.

As a potentially toxic heavy metal, Cadmium (Cd) can cause endoplasmic reticulum and oxidative stress, and thus lead to cell death. To explore the mechanisms of Cd toxicity, we investigated the UPRE-lacZ expression, the intracellular reactive oxygen species (ROS) and cell death in the 151 Cd-sensitive mutants of Saccharomyces cerevisiae in response to Cd stress. We identified 101 genes regulating UPRE-lacZ expression were involved in preventing ROS production and/or cell death from increasing to high levels, while mutants for 72 genes caused both elevated ROS production and cell death, indicating the Cd-induced ROS production and cell death are mediated by UPR. Genes involved in cell wall integrity (CWI) pathway, vacuolar protein sorting (VPS) and vacuolar transport, calcium/calcineurin pathway and PHO pathways were all required for the Cd-induced UPR, intracellular ROS and cell death. To conclude, this study highlights the importance of Cd-induced UPR, intracellular ROS levels and cell death that may play crucial roles in Cd-induced toxicity.

Effect of magnesium ions on glucaric acid production in the engineered Saccharomyces cerevisiae.

Glucaric acid has been successfully produced in Escherichia coli and fungus. Here, we first analyzed the effects of different metal ions on glucaric acid production in the engineered Saccharomyces cerevisiae Bga-3 strain harboring the glucaric acid synthesis pathway. We found that magnesium ions could promote the growth rate of yeast cells, and thus, increase the glucaric acid production by elevating the glucose and myo-inositol utilization of Bga-3 strain. RNA-Seq transcriptome analysis results showed that the upregulation of genes involved in the gluconeogenesis pathway, as well as the downregulation of genes associated with the glycolysis pathway and pentose phosphate pathway in response to MgCl2 were all benefit for the enhancement of the glucose-6-phosphate flux, which was the precursor for myo-inositol and glucaric acid. In addition, we found that MgCl2 could also increase the activity of MIOX4, which was also crucial for glucaric acid synthesis. At last, a final glucaric acid titer of 10.6 g/L, the highest reported titer, was achieved in the fed-batch fermentation using a 5-L bioreactor by adding 100 mM MgCl2. Our findings will provide a new way of promoting the production of other chemicals in the engineered yeast cells.

Label-free fluorescence "turn-on" strategy for mercury (II) detection based on the T-Hg2+-T configuration and the DNA-sensitized luminescence of terbium (III).

A novel label-free fluorescence "turn-on" strategy was developed for the sensitive detection of Hg2+ based on the thymine-Hg2+-thymine (T-Hg2+-T) coordination and the fact that single-stranded DNA (ssDNA) greatly enhances the fluorescence of terbium (III) (Tb3+), but double-stranded DNA (dsDNA) does not. In the absence of Hg2+, the mercury-specific DNA (MSD) hybridized with the corresponding complementary strand (cDNA) to form a double helix structure in solution based on Watson-Crick base pairings, which cannot enhance the fluorescence of Tb3+. In the presence of Hg2+, MSD preferentially bound with Hg2+ to form the T-Hg2+-T complex due to the strong affinity of Hg(II) for the T bases of DNA, thus avoiding the hybridization of the cDNA to MSD. The free cDNA can greatly enhance the emission of Tb3+, leading to a sharp increase in fluorescence intensity. Under the optimized conditions, the increased fluorescence intensity was proportional to the concentration of Hg2+ in the range from 10 to 600 nM, and this method can detect concentrations of Hg2+ as low as 0.24 nM. Moreover, satisfactory results were obtained for the detection of Hg2+ in river water and fish samples, and the results were consistent with those from the atomic fluorescence spectroscopy (AFS). Thus, the fluorescence characteristics of Tb3+ were here used in a "turn on" approach to detect Hg2+, which represents a new opportunity for Hg2+ analysis in the field of environmental monitoring and food safety.

Facile detection of melamine by a FAM-aptamer-G-quadruplex construct.

The development of a novel method for melamine detection that uses a FAM-aptamer-G-quadruplex construct due to the efficient quenching ability of an aptamer-linked G-quadruplex is reported herein. The construct, which is labeled with the fluorescent dye 6-carboxyfluorescein (FAM), consists of two parts: a melamine-binding aptamer and a G-rich sequence that can form a G-quadruplex structure. Because of the specific recognition of melamine by the T-rich aptamer, this aptamer folds into a hairpin structure in the presence of melamine, which draws the G-quadruplex closer to the FAM fluorophore, leading to the quenching of the fluorescence of FAM. Thus, a highly sensitive and selective fluorescence strategy for assaying melamine was established. Under optimal conditions, the fluorescence quenching is proportional to the concentration of melamine within the range 10-90 nM, and the method has a detection limit of 6.32 nM. Further application of the method to plastic cup samples suggested that it permitted recoveries of between 97.15% ± 1.02 and 101.92% ± 2.07. The detected amounts of melamine spiked into the plastic cup samples and the corresponding amounts measured by HPLC were in good accordance, indicating that this fluorescent method is reliable and practical. Owing to its high sensitivity, excellent selectivity, and convenient procedure, this strategy represents a promising alternative method of melamine screening. Graphical abstract.

Development of Structure-Switching Aptamers for Kanamycin Detection Based on Fluorescence Resonance Energy Transfer.

The structure-switching aptamers are designed for the simple and rapid detection of kanamycin based on the signal transduction principle of fluorescence resonance energy transfer (FRET). The structure switch is composed of kanamycin-binding aptamers and the complementary strands, respectively labeled with fluorophore and quencher, denoted as FDNA and QDNA. In the absence of kanamycin, FDNA and QDNA form the double helix structure through the complementary pairing of bases. The fluorophore and the quencher are brought into close proximity, which results in the fluorescence quenching because of the FRET mechanism. In the presence of kanamycin, the FDNA specifically bind to the target due to the high affinity of aptamers, and the QDNA are dissociated. The specific recognition between aptamers and kanamycin will obstruct the formation of structure switch and reduce the efficiency of FRET between FDNA and QDNA, thus leading to the fluorescence enhancement. Therefore, based on the structure-switching aptamers, a simple fluorescent assay for rapid detection of kanamycin was developed. Under optimal conditions, there was a good linear relationship between kanamycin concentration and the fluorescence signal recovery. The linear range of this method in milk samples was 100-600 nM with the detection limit of 13.52 nM (3σ), which is well below the maximum residue limit (MRL) of kanamycin in milk. This method shows excellent selectivity for kanamycin over the other common antibiotics. The structure-switching aptamers have been successfully applied to the detection of kanamycin spiked in milk samples with the satisfying recoveries between 101.3 and 109.1%, which is well-consistent with the results from LC-MS/MS. Due to the outstanding advantages of facile operation, rapid detection, high sensitivity, excellent specificity, and low cost, the application and extension of this strategy for rapid determination of antibiotics in food samples may greatly improve the efficiency in food safety and quality supervision.

A label-free aptasensor for the detection of tetracycline based on the luminescence of SYBR Green I.

A novel fluorescent method based on tetracycline-binding aptamers and the luminescence of SYBR Green I (SGI) was established for the sensitive and selective detection of tetracycline. Under natural conditions, the aptamers of tetracycline show the G-quadruplex spatial structures while SGI is nearly nonfluorescent in aqueous solution. After mixture with the G-quadruplex structured aptamers, SGI can recognize and intercalate into the aptamers, resulting in a strong fluorescence emission. When the target tetracycline was added into the solution, the specific recognition and high-affinity binding of aptamers with tetracycline will induce the conformational changes of aptamers from G-quadruplex structures to hairpin structures. Thereafter, SGI will be released from the aptamer molecules, leading to the fluorescence decline. The quantitative detection of tetracycline can be achieved by measuring the fluorescence change of the system. Under the optimum conditions, the linear range of tetracycline in the milk was from 5 to 25 µg/mL, and the detection limit was as low as 0.10 µg/mL. The recoveries of the spiked milk samples were in the range of 98.98%-104.67% with the relative standard deviations (RSDs) of 0.16%-0.67%, and the results were in agreement with those from HPLC. Therefore, the biosensor based on the specific recognition of aptamers and the fluorescence properties of SGI can detect the tetracycline in milk accurately, rapidly and specifically.

Aptamers-Based Sensing Strategy for 17ß-Estradiol Through Fluorescence Resonance Energy Transfer Between Oppositely Charged CdTe Quantum Dots and Gold Nanoparticles.

A novel aptamers-based fluorescence resonance energy transfer (FRET) assay was developed employing the oppositely charged thioglycolic acid-capped CdTe quantum dots (TGA-CdTe QDs) and cysteamine-stabilized gold nanoparticles (cysteamine-AuNPs). The fluorescence of TGA-CdTe QDs was significantly quenched with addition of cysteamine-AuNPs via the FRET. The FRET process can be modulated by 17ß-estradiol in that the specific recognition between 17ß-estradiol and aptamers could show different effects on the aptamers-mediated aggregation of cysteamine-AuNPs, and correspondingly adjust the FRET process. The feasibility of the method has been demonstrated by carrying out the detection of 17ß-estradiol with the wide linear range from 0.5·ng mL-1 to 150 ng·mL-1 and the low detection limit of 0.057 ng·mL-1. The established assay exhibited favorable selectivity towards 17ß-estradiol over other endocrine disrupting compounds and probably coexisting chemicals in real samples. Furthermore, the assay has been successfully applied to detect 17ß-estradiol in real tap water samples and feeds samples with good performance. The results were in full consistence with those from HPLC method, indicating the reliability of the detection system. The aptamers-based FRET assay is expected to offer a new opportunity for the rapid analysis of 17ß-estradiol in real samples.

Aptamer contained triple-helix molecular switch for rapid fluorescent sensing of acetamiprid.

In this study, an aptamer-based fluorescent sensing platform using triple-helix molecular switch (THMS) was developed for the pesticide screening represented by acetamiprid. The THMS was composed of two tailored DNA probes: a label-free central target specific aptamer sequence flanked by two arm segments acting as a recognition probe; a hairpin-shaped structure oligonucleotide serving as a signal transduction probe (STP), labeled with a fluorophore and a quencher at the 3' and 5'-end, respectively. In the absence of acetamiprid, complementary bindings of two arm segments of the aptamers with the loop sequence of STP enforce the formation of THMS with the "open" configuration of STP, and the fluorescence of THMS is on. In the presence of target acetamiprid, the aptamer-target binding results in the formation of a structured aptamer/target complex, which disassembles the THMS and releases the STP. The free STP is folded to a stem loop structure, and the fluorescence is quenched. The quenched fluorescence intensity was proportional to the concentration of acetamiprid in the range from 100 to 1200nM, with the limit of detection (LOD) as low as 9.12nM. In addition, this THMS-based method has been successfully used to test and quantify acetamiprid in Chinese cabbage with satisfactory recoveries, and the results were in full agreement with those from LC-MS. The aptamer-based THMS presents distinct advantages, including high stability, remarkable sensitivity, and preservation of the affinity and specificity of the original aptamer. Most importantly, this strategy is convenient and generalizable by virtue of altering the aptamer sequence without changing the triple-helix structure. So, it is expected that this aptamer-based fluorescent assay could be extensively applied in the field of food safety inspection.