Noninvasive and Spatiotemporal Control of DNAzyme-Based Imaging of Metal Ions In Vivo Using High-Intensity Focused Ultrasound.

Detecting metal ions in vivo with a high spatiotemporal resolution is critical to understanding the roles of the metal ions in both healthy and disease states. Although spatiotemporal controls of metal-ion sensors using light have been demonstrated, the lack of penetration depth in tissue and in vivo has limited their application. To overcome this limitation, we herein report the use of high-intensity focused ultrasound (HIFU) to remotely deliver on-demand, spatiotemporally resolved thermal energy to activate the DNAzyme sensors at the targeted region both in vitro and in vivo. A Zn2+-selective DNAzyme probe is inactivated by a protector strand to block the formation of catalytic enzyme structure, which can then be activated by an HIFU-induced increase in the local temperature. With this design, Zn2+-specific fluorescent resonance energy transfer (FRET) imaging has been demonstrated by the new DNAzyme-HIFU probes in both HeLa cells and mice. The current method can be applied to monitor many other metal ions for in vivo imaging and medical diagnosis using metal-specific DNAzymes that have either been obtained or can be selected using in vitro selection.

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.

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'.

A colorimetric zinc(II) assay based on the use of hairpin DNAzyme recycling and a hemin/G-quadruplex lighted DNA nanoladder.

A method is described for sensitive colorimetric determination of zinc(II) ions that uses (a) a Zn(II)-responsive hairpin DNAzyme that assists target recycling, (b) hybridization chain reaction, and (c) hemin/G-quadruplex DNA nanoladder. The Zn(II)-responsive split of the hairpin DNAzyme (HD) acts as the recognition and transformation probe. Upon addition of Zn (II) and enzyme strand, a duplex is formed in the loop region of hairpin. The caged initiator sequence is subsequently liberated from the HD by the Zn(II)-selective split of the substrate strand. This cleavage induces an enzyme strand recycling for the next round of cleavage. As a result, the initiator DNA is accumulated and cross-opened H1 and H2 to start a hybridization chain reaction (HCR). The caged G-quadruplex is released after the HCR to recruit hemin to form the hemin/G-quadruplex that is inserted into the DNA nanoladder. Once formed in the DNA nanoladder, these act as catalytic labels for the ABTS, resulting a green color change. This cascade amplification strategy allows 10 nM to 100 µM of Zn(II) to be linearly quantified by colorimetry at 415 nm with a detection limit of 3.5 nM. The recoveries ranged from 97.7 to 108.3% were obtained, confirming high reliability of the method for Zn2+ analysis in lake water samples. Graphical abstractA colorimetric assay for Zn2+ using hairpin DNAzyme (HD) assistant cycle and hemin/G-quadruplex lighted nanoladders is designed. A Zn2+-responsive split of HD is designed as the recognition and transformation probe. The hemin/G-Quadruplex inserted nanoladder act as reporter for signal readout.

Colorimetric detection and typing of E. coli lipopolysaccharides based on a dual aptamer-functionalized gold nanoparticle probe.

A rapid method for identification and typing of lipopolysaccharides (LPS) was developed by utilizing the different binding affinities between two kinds of gold nanoparticles (AuNPs) functionalized with two aptamers. Aptamers against ethanolamine and E. coli O111:B4 LPS were used to functionalize the AuNPs. The AuNPs functionalized with ethanolamine aptamer can bind to ethanolamine and are termed general probe (G-probe). The G-probe can recognize any type of LPS because ethanolamine is a component of every type of LPS. This causes a sandwich-mediated aggregation of the AuNPs and a color change from red to blue. The AuNPs functionalized with aptamer against the LPS of E. coli O111:B4 specifically bind to O111:B4 LPS and are termed specific probe (S-probe). By using these two probes, a logic typing method was developed. It can detect LPS in concentrations between 2.5 and 20 µg·mL-1 and with a 1 µg·mL-1 detection limit. In the authors' perception, the use of a dual aptamer-based colorimetric method has a large potential in terms of selective detection of microorganisms. Graphical abstract Two aptamer functionalized AuNP probes, G-probe and S-probe, were prepared for LPS typing and detecting. E. coli O111:B4 LPS was easily distinguished from O55:B5 LPS according to the signal output configurations (On & On Vs On & Off) of a general probe (G-probe) and a specific probe (S-probe).

Spatial imaging of glycoRNA in single cells with ARPLA.

Little is known about the biological roles of glycosylated RNAs (glycoRNAs), a recently discovered class of glycosylated molecules, because of a lack of visualization methods. We report sialic acid aptamer and RNA in situ hybridization-mediated proximity ligation assay (ARPLA) to visualize glycoRNAs in single cells with high sensitivity and selectivity. The signal output of ARPLA occurs only when dual recognition of a glycan and an RNA triggers in situ ligation, followed by rolling circle amplification of a complementary DNA, which generates a fluorescent signal by binding fluorophore-labeled oligonucleotides. Using ARPLA, we detect spatial distributions of glycoRNAs on the cell surface and their colocalization with lipid rafts as well as the intracellular trafficking of glycoRNAs through SNARE protein-mediated secretory exocytosis. Studies in breast cell lines suggest that surface glycoRNA is inversely associated with tumor malignancy and metastasis. Investigation of the relationship between glycoRNAs and monocyte-endothelial cell interactions suggests that glycoRNAs may mediate cell-cell interactions during the immune response.

Simultaneous Fe2+/Fe3+ imaging shows Fe3+ over Fe2+ enrichment in Alzheimer's disease mouse brain.

Visualizing redox-active metal ions, such as Fe2+ and Fe3+ ions, are essential for understanding their roles in biological processes and human diseases. Despite the development of imaging probes and techniques, imaging both Fe2+ and Fe3+ simultaneously in living cells with high selectivity and sensitivity has not been reported. Here, we selected and developed DNAzyme-based fluorescent turn-on sensors that are selective for either Fe2+ or Fe3+, revealing a decreased Fe3+/Fe2+ ratio during ferroptosis and an increased Fe3+/Fe2+ ratio in Alzheimer's disease mouse brain. The elevated Fe3+/Fe2+ ratio was mainly observed in amyloid plaque regions, suggesting a correlation between amyloid plaques and the accumulation of Fe3+ and/or conversion of Fe2+ to Fe3+. Our sensors can provide deep insights into the biological roles of labile iron redox cycling.

Insights into nucleic acid-based self-assembling nanocarriers for targeted drug delivery and controlled drug release.

Over the past few decades, rapid advances of nucleic acid nanotechnology always drive the development of nanoassemblies with programmable design, powerful functionality, excellent biocompatibility and outstanding biosafety. Nowadays, nucleic acid-based self-assembling nanocarriers (NASNs) play an increasingly greater role in the research and development in biomedical studies, particularly in drug delivery, release and targeting. In this review, NASNs are systematically summarized the strategies cooperated with their broad applications in drug delivery. We first discuss the self-assembling methods of nanocarriers comprised of DNA, RNA and composite materials, and summarize various categories of targeting media, including aptamers, small molecule ligands and proteins. Furthermore, drug release strategies by smart-responding multiple kinds of stimuli are explained, and various applications of NASNs in drug delivery are discussed, including protein drugs, nucleic acid drugs, small molecule drugs and nanodrugs. Lastly, we propose limitations and potential of NASNs in the future development, and expect that NASNs enable facilitate the development of new-generation drug vectors to assist in solving the growing demands on disease diagnosis and therapy or other biomedicine-related applications in the real world.

Correction: Mercury nanoladders: a new method for DNA amplification, signal identification and their application in the detection of Hg(ii) ions.

Correction for 'Mercury nanoladders: a new method for DNA amplification, signal identification and their application in the detection of Hg(ii) ions' by Yuxiang Feng et al., Chem. Commun., 2018, 54, 8036-8039.

Detachable nanoladders: A new method for signal identification and their application in the detection of ochratoxin A (OTA).

A highly sensitive fluorescence turn-off biosensor for the detection of ochratoxin A (OTA) was developed based on graphene oxide (GO) and an aptamer-induced detachable nanoladders. In this assay, two types of ssDNA (H1 and H2) were involved in the assembly of the DNA nanoladders, in which H1 was labeled with fluorophore, and H2 was the OTA binding aptamer. Self-assembly of the DNA nanoladders with the addition of GO weakened its adsorption and the fluorescence intensity remained strong. In the presence of OTA, the aptamer was specifically recognized and an aptamer-OTA complex was formed, leading to the detached of DNA nanoladders. With the addition of GO, the released H1 was adsorbed on the GO surface, thus efficiently quenching the fluorescence signal (turning off). The detection limit of OTA in this assay was 4.59 nM. To improve the sensitivity of this strategy, we creatively developed an alternative strategy to replace the sturdy nanoladders with frail nanoladders. More precisely, the sequences of H1 had mismatched bases, which, when hybridized with H2 could be used to create long non-perfect complementary nanoladders. For the mismatched bases-based frail nanoladders, it was easier for OTA to bind its aptamer sequence, thus enabling a more thorough and faster detachment of the nanoladders, along with a greater degree of fluorescence quenching. The detection limit for OTA was estimated to be 0.1 nM. The biosensors we developed were sensitive, specific, enzyme-free, cost-effective and can be applied in red wine samples spiked with known concentration of OTA.

An electrochemical biosensor based on nucleic acids enzyme and nanochannels for detecting copper (II) ion.

An electrochemical biosensor based on Cu2+-dependent cleavage DNAzyme (cDNAzyme), Exponential Amplification Reaction (EXPAR), and single-strand triggered DNA hybridization chain reaction (HCR) was developed for detecting the copper (II) ions. This method capitalizes on the specific recognition of cDNAzyme, the single strand accumulated isothermal amplification of EXPAR, and the enzyme-free isothermal DNA assembly of HCR. In the presence of Cu2+, the catalytic chain of cDNAzyme split the substrate chain to produce single-stranded DNA (Oligo X). With the help of the EXPAR, the trace Oligo X was amplified and converted to the initiator DNA (Oligo Y). After adding the initiator DNA (Oligo Y) into nanochannel, massive DNA superstructures grew from the capture probe (CP) that were pre-immobilized on the nanochannel wall, resulting in a sharp decrease of the effective diameter of the nanochannel. As a result, the transmembrane ionic current significantly decreased due to the effective closing of the nanochannel. Finally, the copper (II) ion was detected by monitoring the changes of transmembrane ionic current. The developed electrochemical biosensor also displayed high selectivity for Cu2+, which could be useful for monitoring Cu2+ above ten picomole.

Mercury nanoladders: a new method for DNA amplification, signal identification and their application in the detection of Hg(ii) ions.

A biosensor based on Hg(ii) nanoladders integrated with graphene oxide (GO) for Hg(ii) detection was developed. Nanoladders were essentially DNA sandwich structures supported by T-Hg(ii)-T and were used for Hg(ii) trapping and signal translation. GO was used to differentiate the fluorescence generated by Hg(ii) nanoladders from a background signal.

A rapid and visual aptasensor for Lipopolysaccharides detection based on the bulb-like triplex turn-on switch coupled with HCR-HRP nanostructures.

For previously reported aptasensor, the sensitivity and selectivity of aptamers to targets were often suppressed due to the reporter label of single-stranded molecular beacon or hindrance of the duplex DNA strand displacement. To solve the affinity declining of aptamers showed in traditional way and realize on-site rapid detection of Lipopolysaccharides (LPS), we developed an ingenious structure-switching aptasensor based on the bulb-like triplex turn-on switch (BTTS) as the effective molecular recognition and signal transduction element and streptavidin-horseradish peroxidase modified hybridization chain reaction (HCR-HRP) nanocomposites as the signal amplifier and signal report element. In the presence of LPS, the bulb-like LPS-aptamer (BLA) and LPS formed the LPS/aptamer complex, while the BTTS disassembled and liberated the dissociative bridge probes (BP) to achieve molecular recognition and signal transduction. Immobilized BP, captured by immobilized capture probes (CP), triggered hybridization chain reactions (HCR) to amplify the switching signal, and the HCR products were then modified with streptavidin-horseradish peroxidase (SA-HRP) to form HCR-HRP nanostructures to output colorimetric signals. In less than four hours, the proposed biosensor showed a detection limit of 50pg/mL of LPS quantitatively with the portable spectrophotometer and the observation limit of 20ng/mL semi-quantitatively with the naked eye, opening up new opportunities for LPS detection in future clinical diagnosis, food security and environment monitoring.

Two-Way Gold Nanoparticle Label-Free Sensing of Specific Sequence and Small Molecule Targets Using Switchable Concatemers.

A two-way colorimetric biosensor based on unmodified gold nanoparticles (GNPs) and a switchable double-stranded DNA (dsDNA) concatemer have been demonstrated. Two hairpin probes (H1 and H2) were first designed that provided the fuels to assemble the dsDNA concatemers via hybridization chain reaction (HCR). A functional hairpin (FH) was rationally designed to recognize the target sequences. All the hairpins contained a single-stranded DNA (ssDNA) loop and sticky end to prevent GNPs from salt-induced aggregation. In the presence of target sequence, the capture probe blocked in the FH recognizes the target to form a duplex DNA, which causes the release of the initiator probe by FH conformational change. This process then starts the alternate-opening of H1 and H2 through HCR, and dsDNA concatemers grow from the target sequence. As a result, unmodified GNPs undergo salt-induced aggregation because the formed dsDNA concatemers are stiffer and provide less stabilization. A light purple-to-blue color variation was observed in the bulk solution, termed the light-off sensing way. Furthermore, H1 ingeniously inserted an aptamer sequence to generate dsDNA concatemers with multiple small molecule binding sites. In the presence of small molecule targets, concatemers can be disassembled into mixtures with ssDNA sticky ends. A blue-to-purple reverse color variation was observed due to the regeneration of the ssDNA, termed the light-on way. The two-way biosensor can detect both nucleic acids and small molecule targets with one sensing device. This switchable sensing element is label-free, enzyme-free, and sophisticated-instrumentation-free. The detection limits of both targets were below nanomolar.

Highly sensitive detection of lipopolysaccharides using an aptasensor based on hybridization chain reaction.

Lipopolysaccharides (LPS), integral components of the outer membrane of all gram-negative bacteria, are closely associated with foodborne diseases such as fever, diarrhea and hypotension, and thus, the early and sensitive detection of LPS is necessary. In this study, an aptasensor assay based on hybridization chain reaction (HCR) was developed to detect LPS. Briefly, two complementary stable species of biotinylated DNA hairpins coexisted in solution until the introduction of a detection probe triggered a hybridization chain reaction cascade. The DNA conjugates specifically reacted with the LPS, which were captured by the ethanolamine aptamer attached to the reaction well surface. After optimizing the key reaction conditions, such as the reaction time of HCR, the amount of the capture probe and detection probes, the increase in the LPS concentration was readily measured by the optical density value, and a relatively low detection limit (1.73 ng/mL) was obtained, with a linear response range of 1-10(5 )ng/mL. The approach presented herein introduced the use of an aptasensor for LPS discrimination and HCR for signal amplification, offering a promising option for detecting LPS.