Magnetic aptamer copper nanoclusters fluorescent biosensor for the visual detection of zearalenone based on docking-aided rational tailoring.

To address the food safety issues caused by toxins, we established a fluorescent copper nanocluster biosensor based on magnetic aptamer for the visual and quantitative detection of ZEN. Specifically, we utilized the docking-aided rational tailoring (DART) strategy to analyze intermolecular force and interaction sites between zearalenone (ZEN) and the aptamer, and optimize the long-chain aptamer step by step to enhance the binding affinity by 3.4 times. The magnetic bead-modified aptamer underwent conformational changes when competing with complementary sequences to bind with ZEN. Then, the released complementary sequences will be amplified in template-free mode with the presence of the terminal deoxynucleotidyl transferase (TdT), and generating T-rich sequences as the core sequences for the luminescence of copper nanoclusters. The luminescence could be visualized and quantitatively detected through ultraviolet irradiation. The proposed label-free aptasensor exhibited high sensitivity and specificity, with a low limit of detection (LOD) of 0.1 ng/mL.

A turn-on signal biosensor for cadmium(II) based on DNAzyme and stem-loop qPCR.

Cadmium is a heavy metal that is exceedingly hazardous to humans and can enter the body through tainted food or drink, causing severe harm. It is critical to develop a technology for detecting cadmium in food and water that is sensitive and accurate. One such approach, which employs nucleases, is uncommon. A cadmium(II) turn-on biosensor was successfully created in this work using repetitive cleavage of certain specific nucleases for signal conversion and sophisticated stem-loop qPCR (quantitative polymerase chain reaction) for quick signal amplification and output. The method has strong selectivity and sensitivity for precise quantification, with a detection limit of 6 nmol L-1, i.e. 0.948 g L-1, which is far lower than the 5.0 g L-1 set by the United States Environmental Protection Agency, and it also operates well in retail rice samples.

Coordination-Driven One-Step Rapid Self-Assembly Synthesis of Dual-Functional Ag@Pt Nanozyme.

Realizing high-precise and adjustable regulation of engineering nanozyme is important in nanotechnology. Here, Ag@Pt nanozymes with excellent peroxidase-like and antibacterial effects are designed and synthesized by nucleic acid and metal ions coordination-driven one-step rapid self-assembly. The adjustable NA-Ag@Pt nanozyme is synthesized within 4 min using single-stranded nucleic acid as templates, and peroxidase-like enhancing FNA-Ag@Pt nanozyme is received by regulating functional nucleic acids (FNA) based on NA-Ag@Pt nanozyme. Both Ag@Pt nanozymes that are developed not only has simple and general synthesis approaches, but also can produce artificial precise adjustment and possess dual-functional. Moreover, when lead ion-specific aptamers as FNA are introduced to NA-Ag@Pt nanozyme, the Pb2+ aptasensor is successfully constructed by increasing electron conversion efficiency and improving the specificity of nanozyme. In addition, both nanozyme has good antibacterial properties, with ~100% and ~85% antibacterial efficiency against Escherichia coli and Staphylococcus aureus, respectively. This work provides a synthesis method of novelty dual-functional Ag@Pt nanozymes and successful application in metal ions detection and antibacterial agents.

Cavity hairpin ThT-light nucleic acid switches: the construction of label- and enzyme-free sensing and imaging platforms.

Thioflavin T (ThT) is a classical fluorescent dye gaining prominence in current research regarding nucleic acid conformations (NACs). However, most NACs with the ability to excite ThT fluorescent are unique or form in demanding conditions, limiting the extensiveness and depth of ThT application in sensing and imaging. Therefore, this study proposed CGG-AAA mismatched cavity hairpin ThT-light nucleic acid switches (CHTLNAS) with excellent fluorescence excitation over 500-fold higher than spontaneous, 17∼20-fold higher than ssDNA and 2.5∼5-fold higher than complementary duplex. Based on the excellent fluorescence excitation, convenient conformation formation, good sequence programmability, and flexible allosteric ability (known as the Worm-crack pod mechanism mediated by the target), it achieved the label- and enzyme-free detection of tetracycline (TET) and berberine (BB) at the pM level within 10 min. Moreover, it was found enable to realize the sensitive tracking of intracellular carriers at the nM level of ThT entry concentration, and prolongated its cell nuclear-entry time of ThT over 8 h, overcoming the non-specific high background signal interference of ThT in the nuclear region, and expanding the diversified application of ThT in cell biology research. Therefore, CHTLNAS is a more universal, practical tool than G-quadruplex or other kinds of NACs for ThT development and utilization in sensing and imaging platforms.

Docking-aided rational tailoring of a fluorescence- and affinity-enhancing aptamer for a label-free ratiometric malachite green point-of-care aptasensor.

Although nucleic acid aptasensors are increasingly applied in the detection of environmentally hazardous biomolecules, several formidable challenges remain with this technique because of their vulnerability, high cost and suboptimal sensitivity. Here, a docking-aided rational tailoring (DART) strategy was established at three levels and in two dimensions for the refinement of malachite green (MG) DNA aptamers. Guided by in silico molecular docking, coarse and fine tailoring were conducted at three levels each, to significantly enhance fluorescence activation intensity and binding affinity in two dimensions. Empowered by the results of the rational tailoring, a mechanistic view of the MG DNA aptamer-target interaction was thoroughly analyzed via four types of interactions. To meet the demand for point-of-care testing (POCT), a label-free and ratiometric fluorescent aptasensor was developed leveraging the tailored MG aptamer, based on the binding site competition-equilibrium effect via the introduction of a reference dye. This sensitive, specific, low-cost and rapid aptasensor subsequently demonstrated outstanding detection performance, achieving an ideal signal response range of 5 nmol·L-1 - 6 µmol·L-1 and a low limit of detection (LOD) of 1.49 nmol·L-1. The DART strategy and systematic exploration of the MG DNA luminescent aptamers herein will provide a valuable reference in the field of aptamer tailoring, biosensing and bioimaging. The proposed label-free ratiometric aptasensor also provides a highly generalizable strategy for hazardous biomolecular detection.

Nano-gold-enhanced LAMP method for qualitative visual detection of Salmonella in milk.

Since Salmonella can cause foodborne disease and public health safety issues and requires a robust, rapid, on-site detection method, a novel visual qualitative method with nano-gold-enhanced loop-mediated isothermal amplification (LAMP) reaction was established for detecting Salmonella in an integrated tube. During the experiment, nano-gold were used to enhance LAMP amplification, improving amplification efficiency and shortening the reaction time to within 30 min. Visual qualitative detection is achieved via negative staining, involving the addition of CuSO4 to the final products of the LAMP reaction. Ring-like white accumulation occurs in the absence of Salmonella targets but not when they are present. After completing the LAMP reaction, the integration tube was shaken gently for 1 min to observe the liquid phase system changes, realizing the closed tube detection of Salmonella. The process resolved the challenge presented by cross-contamination, false positives, and nonspecific amplification during the LAMP reaction. This method was used to detect Salmonella in milk, further highlighting its prospects in the field of rapid food safety detection.

Visualization of copper nanoclusters for SARS-CoV-2 Delta variant detection based on rational primers design.

Here, based on the design of rational primers and copper nanoclusters (CuNCs), we present a method for the accurate detection of the SARS-CoV-2 Delta variant, which is capable of distinguishing the Delta variant with its single nucleotide polymorphism from the 'wild type' coronavirus (NC_045512.2), and realizing visualization signal out. Specifically, we show that dual priming oligonucleotide (DPO) primers and AT primers can be used to distinguish between wild types and mutations of this virus by polymerase chain reaction (PCR) analysis and that visualization can be achieved via the red fluorescence of CuNCs in ultraviolet radiation. Among the results, it was found that the R-1-down (DPO)-6I and F-1-30 AT, with the single nucleotide deletion site designed at the 3' end of the downstream primer, showed the best specificity towards the Delta variant. Moreover, the use of AT primers increased the AT contents of the PCR products, thus meeting the template requirements generated by the CuNCs. It was also found that the AT primers could assist with improving detection specificity. Finally, we demonstrate that the visualization of the CuNCs-based detection assay exhibited a linear relationship in 0.5 pg µL-1-50 ng µL-1, with a limit of quantitation (LOQ) of 0.5 pg µL-1.

Fusion of binary split allosteric aptasensor for the ultra-sensitive and super-rapid detection of malachite green.

The complicated labeling procedure and high cost of split aptasensors have hitherto limited their application in the detection of hazardous substances. Herein we report the first examples of label-free aptasensors based on the fusion of a binary split G-quadruplex and malachite green (MG) aptamer, transducing recognition events into fluorescent signals through the allosteric regulation of the aptamer to achieve selective and sensitive detection. Specifically, RNA MGA was successfully converted into DNA MGA with comparable affinity and improved stability, thereby overcoming the limitations of poor stability and high expense. We subsequently split the DNA MGA and attached them to a G-rich DNA sequence at the 5' and 3' ends, to construct the binary split allosteric aptasensor. The performance of the binary split aptasensor for MG detection was significantly improved based on proposed allosteric regulation strategy, and the reconfiguration capability of the aptamers upon binding with targets was proven, providing the binary split aptasensor with superior sensitivity and selectivity. This sensing method has a wide dynamic detection range of 5 nmol·L-1 to 500 µmol·L-1, with a low limit of detection (LOD) of 4.17 nmol·L-1, and achieves the ultra-sensitive and super-rapid detection of MG. This newly proposed aptasensor is equipped with the advantages of being label-free, time saving and economical. More importantly, this successful construction of a fused aptasensor expands the principles of split aptasensor design and provides a universal platform for the detection of environmental contaminants.

Functional Nucleic Acid Nanomaterials: Development, Properties, and Applications.

Functional nucleic acid (FNA) nanotechnology is an interdisciplinary field between nucleic acid biochemistry and nanotechnology that focuses on the study of interactions between FNAs and nanomaterials and explores the particular advantages and applications of FNA nanomaterials. With the goal of building the next-generation biomaterials that combine the advantages of FNAs and nanomaterials, the interactions between FNAs and nanomaterials as well as FNA self-assembly technologies have established themselves as hot research areas, where the target recognition, response, and self-assembly ability, combined with the plasmon properties, stability, stimuli-response, and delivery potential of various nanomaterials can give rise to a variety of novel fascinating applications. As research on the structural and functional group features of FNAs and nanomaterials rapidly develops, many laboratories have reported numerous methods to construct FNA nanomaterials. In this Review, we first introduce some widely used FNAs and nanomaterials along with their classification, structure, and application features. Then we discuss the most successful methods employing FNAs and nanomaterials as elements for creating advanced FNA nanomaterials. Finally, we review the extensive applications of FNA nanomaterials in bioimaging, biosensing, biomedicine, and other important fields, with their own advantages and drawbacks, and provide our perspective about the issues and developing trends in FNA nanotechnology.

Universal linker Polymerase Chain Reaction-triggered Strand Displacement Amplification visual biosensor for ultra-sensitive detection of Salmonella.

Salmonella is a principal causal agent of pathogenic outbreaks via food. A universal, highly sensitive and visual Salmonella detection method was proposed in this paper, based on a universal linker PCR (UL-PCR)-triggered Strand Displacement Amplification (SDA). In this research, the UL-PCR achieved the primary amplification. The universal linker primer was ingeniously designed and composed of two parts, one of which was the binding sequence of the target, and the other was the universal linker. Complementary sequences of the G-quadruplex and the nicking endonuclease recognition sequence were included in the universal linker. Therefore, the G-quadruplexes and nicking sites were successfully introduced into the UL-PCR products, providing a basis for further SDA triggering. SDA achieved the secondary signal amplification and generated a large amount of label-free DNAzymes. Following SDA, DNAzymes catalyzed 3,3',5,5'-tetramethylaniline (TMB) into colored compounds visible to the naked eye. We obtained the best experimental conditions by univariate analysis. Under optimal conditions, this proposed universal label-free method could detect Salmonella genome at level as low as 22 copies mL-1, with an excellent linear range between 102 copies mL-1 and 107 copies mL-1. And the limit of quantification (LOQ) was 102 copies mL-1. This strategy shows promise for broad applications.

Food-grade expression of nattokinase in Lactobacillus delbrueckii subsp. bulgaricus and its thrombolytic activity in vitro.

OBJECTIVES: To produce nattokinase in a food-grade expression system and evaluate its thrombolytic activity in vitro. RESULTS: No nattokinase activity from reconstituted strains was observed in simulated gastric juice, but the enzyme was stable in intestinal fluid, the relative activity of which was found to be 60% after 4 h. Due to the nattokinase being produced intracellularly by recombinant bacterial strains, the persistence of the bacteria in gastric juice ensured transmission of the nattokinase into intestinal juice. Because of subsequent disintegration of the bacteria, the highest nattokinase activity was observed after 3 h at approximately 32%, following its carriage within the recombinant strains to the intestinal fluid. CONCLUSIONS: This study demonstrated that nattokinase from recombinant strains exhibited good thrombolytic activity in vitro and may be used by the dairy fermentation industry for the development of novel thrombolytic functional foods.

Fluorescent detection of Cu (II) ions based on DNAzymatic cascaded cyclic amplification.

A fluorescent biosensor based on the cascaded cyclic amplification-lighted copper nanoparticles has been developed, optimized, and validated. In the double-modular cascaded cyclic amplification, a DNAzymatic cyclic amplification unit transforms metal ion signal to specific DNA sequences, and a linear/exponential integrated amplification unit converts as-prepared DNA codes to identical thymine (T)-rich DNA templates. T-rich scaffolds can induce the generation of red fluorescent copper nanoparticles, with fluorescence emission at 625 nm upon the excitation at 340 nm, as signal vehicles for quantitative detection of metal ions. Copper ions, selected as the model target, could be detected in a wide linear range from 10 to 104 nM depending on the increased fluorescent intensity, and the detection limit is 5.6 ± 0.52 nM (n = 3) within 40 min, which is 4 orders of magnitude lower than the limits set in drinking water. In the detection of Cu2+ in real tap and lake water, the results between inductively coupled plasma mass spectrometry (ICP-MS) and our proposed biosensor were consistent, illustrating the practicability of the fabricated method. In summary, the established fluorescent biosensor compensates the deficiency of immunoassays failing to analyze metal ions, broadens ranges of biomarkers responding to cleaved DNAzymes, provides an open platform sensing different metal ions, and meets the increasing need for the ultrasensitive detection in the field of food safety, environmental monitoring, and medical diagnosis.

ExoIII and TdT dependent isothermal amplification (ETDA) colorimetric biosensor for ultra-sensitive detection of Hg2.

As the accumulation of mercury ions has a detrimental impact on human health, the design and development of a new type of biosensor that can rapidly, sensitively and selectively detect Hg2+ in aqueous solutions are essential. In this study, we have developed an exonuclease III (ExoIII) and Terminal deoxynucleotidyl transferase (TdT) dependent isothermal amplification (ETDA) colorimetric biosensor. The template sequence is a hairpin where -NH2 is labeled at the 3'-end and both termini are T-rich sequences. In the presence of Hg2+, the template formed a blunt end, and the catalytic activity of ExoIII was activated with cleavage of the -NH2 at the 3'-end. TdT enzyme activity was initiated with the formation of a large number of G-rich nucleic acid sequences. G-rich sequences incubated with iron (III)-hemin mimicked peroxidase-like activity, catalyzing the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2. The biosensor constructed in this paper had a good linear range, 1-25 nmol/L. Its detection limit was 0.41 nmol/L (3σ), and recovery rates were between 100.5% and 103%. In conclusion, combined with the colorimetric biosensor and double enzyme cyclic amplification reaction, an ultra-sensitivity and strong specificity detection method was developed to detect Hg2+. At the same time, this method also expands the detection method of Hg2+ available in the literature.

A Universal Electrochemical Biosensor Using Nick-HCR Nanostructure as Molecular Gate of Nanochannel for Detecting Chromium(III) Ions and MicroRNA.

Solid-state nanochannels demonstrating excellent mechanical properties and chemical stability combined with programmable DNA provide an opportunity to control on-demand ion transport. However, poor functionalization of the nanochannels limits the types of detected targets, as well as its universality in the sensing field. To solve these issues, a universal nanochannel sensing platform was developed by employing a nick hybridization chain reaction (nHCR) nanostructure as a molecular gate, which could generally respond to the universal sequence Y. Metal ion-dependent DNAzyme cleavage was used to transfer the chromium(III) (Cr3+) ions into nucleic acid X, which was further amplified and converted into universal sequence Y. Upon adding sequence Y into the nHCR nanostructure-functionalized nanochannel, the disassembly of the nHCR molecular gate turned on the ionic current signal inside the nanochannel. The ON-OFF ratio displayed a linear relationship with the Cr3+ concentration in the range from 200 fM to 20 nM. In less than 66 min, the nanochannel-based biosensing platform successfully detected Cr3+ ions as low as 200 fM. In addition, the detection of microRNA with a concentration as low as 1 pM was achieved by only regulating the sequence of template X'-Y'.

The ultra-sensitive visual biosensor based on thermostatic triple step functional nucleic acid cascade amplification for detecting Zn2.

Here, we present an ultra-sensitive visual biosensor based on thermostatic triple step functional nucleic acid cascade amplification for detecting Zn2+. A Zn2+-assisted cDNAzyme assay is conducted and the Zn2+ is successfully converted into nucleic acids to achieve the first circular amplification within 1 h. The cleavage products prompted Hybridization Chain Reaction (HCR), forming multiple DNA nanowire structures with branched chains. And the Strand Displacement Amplification (SDA) were further empowered by the HCR products with the addition of the amplification enzyme and the endonuclease. In just 80 min, the second and third-order signal amplifications are reached. With the addition of Hemin and chromogenic substrates, the visual signal output is achieved in 15 min based on the large amount of G-quadruplex (G4) DNAzymes. This biosensor can detect levels as low as 1.075 pM Zn2+, showing a good linear range within 10 pM-100 nM. It shows considerable potential for the Zn2+ quantitative detection.