Co-Immobilization of GOD & HRP on Y-Shaped DNA Scaffold and the Regulation of Inter-Enzyme Distance.

In multienzymes cascade reaction, the inter-enzyme spacing is supposed to be a factor affecting the cascade activity. Here, a simple and efficient Y-shaped DNA scaffold is assembled using two partially complementary DNA single strands on magnetic microspheres, which is used to coimmobilize glucose oxidase (GOD) and horseradish peroxidase (HRP). As a result, on poly(vinyl acetate) magnetic microspheres (PVAC), GOD/HRP-DNA@PVAC multienzyme system is obtained, which can locate GOD and HRP accurately and control the inter-enzyme distance precisely. The distance between GOD and HRP is regulated by changing the length of DNA strand. It showed that the cascade activity is significantly distance-dependent. Moreover, the inter-enzyme spacing is not the closer the better, and too short distance would generate steric hindrance between enzymes. The cascade activity reached the maximum value of 967 U mg-1 at 13.6 nm, which is 3.5 times higher than that of free enzymes. This is ascribed to the formation of substrate channeling.

Engineering of catalytic hairpin-rigidified Y-shaped DNA-functionalized nanomachine to rapidly detect mRNA.

The catalytic hairpin-rigidified Y-shaped DNA through layer-by-layer assembly has been fixed on the surface of copper sulfide nanoparticles for the detection of survivin mRNA. The distance between the CHA probes fixed on the Y-shaped DNA is significantly shortened. The results show that the fluorescence of this nanomachine reached the maximum value in 50 min (excitation wavelength at 488 nm and emission wavelength 526 nm), and its reaction rate is more than 5-fold faster than that of the free-CHA control system. In addition, the nanomachine showed high sensitivity (LOD of 3.5 pM) and high specificity for the survivin mRNA detection. Given its fast response time and excellent detection performance, we envision that the catalytic hairpin-rigidified Y-shaped DNA-functionalized nanomachine will offer potential applications in disease diagnostics and clinical applications.

Surface-assisted DNA self-assembly: An enzyme-free strategy towards formation of branched DNA lattice.

DNA based self-assembled nanostructures and DNA origami has proven useful for organizing nanomaterials with firm precision. However, for advanced applications like nanoelectronics and photonics, large-scale organization of self-assembled branched DNA (bDNA) into periodic lattices is desired. In this communication for the first time we report a facile method of self-assembly of Y-shaped bDNA nanostructures on the cationic surface of Aluminum (Al) foil to prepare periodic two dimensional (2D) bDNA lattice. Particularly those Y-shaped bDNA structures having smaller overhangs and unable to self-assemble in solution, they are easily assembled on the surface of Al foil in the absence of ligase. Field emission scanning electron microscopy (FESEM) analysis shows homogenous distribution of two-dimensional bDNA lattices across the Al foil. When the assembled bDNA structures were recovered from the Al foil and electrophoresed in nPAGE only higher order polymeric bDNA structures were observed without a trace of monomeric structures which confirms the stability and high yield of the bDNA lattices. Therefore, this enzyme-free economic and efficient strategy for developing bDNA lattices can be utilized in assembling various nanomaterials for functional molecular components towards development of DNA based self-assembled nanodevices.

A dual-signaling surface-enhanced Raman spectroscopy ratiometric strategy for ultrasensitive Hg2+ detection based on Au@Ag/COF composites.

Heavy metal ion pollution poses significant risks to human health and ecological systems, and its monitoring is important. A sensitive and accurate surface-enhanced Raman spectroscopy (SERS) detection assay for Hg2+ was developed using Au@Ag/COF substrates and Y-shaped DNA labeled with two Raman reporters. The Au@Ag NPs in the COF produced robust and uniform E-fields, improving their detection reproducibility. The Y-shaped DNA design increased sensitivity with a low detection limit of 5.0 × 10-16 M by bringing the Raman reporter closer to the substrate surface. Additionally, the use of two Raman reporters allowed for a ratiometric method, improving detection accuracy by detecting both "signal-off" and "signal-on" signals. This selective sensor exhibited excellent recovery in river water, tap water, and milk samples, showcasing its robust biosensing capability for the detection of Hg2+ and its potential for sensing other heavy-metal ions in food and environmental applications.

Detection of long mRNA sequences by a Y-shaped DNA probe with three target-binding segments.

Tumor-related mRNA detection is significant and interesting. The current mRNA detection method has the challenge of quantifying long mRNA sequences. Herein, a Y-shaped DNA probe with three target-binding segments was developed to detect tumor-related mRNA. This Y-shaped DNA probe (Y-probe) was assembled by six single DNA strands. Among these DNA strands, two DNA strands contained the split G-quadruplex sequence, and two DNA strands were modified with a pair of fluorophore and quencher, which were used to produce the detectable signal. In the presence of a long target mRNA sequence, target mRNA was hybridized with the three target-binding segments of the Y-probe, resulting in the increased fluorescence of G-quadruplex specific dye Thioflavin T and the decreased fluorescence of fluorophore, which could achieve the ratio detection of target mRNA. The Y-probe exhibited a low detection limit of 17.53 nM. Moreover, this probe showed high accuracy due to the benefits of three target-binding segments.

Programmable Y-Shaped Probes with Proximity Bivalent Recognition for Rapid Electrochemiluminescence Response of Acute Myocardial Infarction.

Herein, an exogenous luminophore-free and disposable electrochemiluminescence (ECL) biosensor was established for rapid response of acute myocardial infarction (AMI) using programmable Y-shaped probes (Y-probes) with proximity bivalent recognition. Specifically, the indium tin oxide thin film coated glass electrode (ITO) was modified with urchin-like porous TiO2 microspheres (pTiO2 MSs), which could achieve strong and stable ECL in S2O82- solution due to the dual promoting effect of the coreaction accelerator pTiO2 MSs, exhibiting 2.7-fold higher ECL intensity in comparison with that of bare ITO. Moreover, the Y-probes as bivalent recognition elements containing two kinds of cardiac troponin I (cTnI, a biomarker of AMI) aptamers and a linker labeled with ferrocene (L-Fc) were designed to export a "signal off" mode. When the target cTnI was in the proximity of the Y-probes, the L-Fc was separated from the electrode surface due to the proximity recognition of cTnI and its aptamers, achieving the highly effective recovery of ECL, which allowed for a much more rapid detection of cTnI than the sandwich-type immunoassay. As a proof of concept, an exogenous luminophore-free and disposable ECL platform for rapid and sensitive monitoring of cTnI was obtained and displayed a desired linear range from 100 fg mL-1 to 100 ng mL-1 with a limit of detection (LOD) of 30.1 fg mL-1, which can be ingeniously expanded as a portable home tester with ECL biosensors developments.

Y-shaped DNA-Mediated hybrid nanoflowers as efficient gene carriers for fluorescence imaging of tumor-related mRNA in living cells.

Organic-inorganic hybrid nanomaterial has gained much attention due to its excellent performances in bioanalysis and biomedicine. However, the preparation of DNA-inorganic hybrid nanomaterial with suitable size for cell uptake remains a huge challenge. Herein, a moderate biomineralization strategy for synthesis of Y-DNA@Cu3(PO4)2 (Y-DNA@CuP) hybrid nanoflowers is reported. Y-DNA with a loop structure is used as both the biomineralization template and the recognition unit for thymidine kinase 1 (TK1) mRNA. The Y-DNA probe can linearly response to TK1 mRNA target sequence in a range from 2 nM to 150 nM with the limit of detection as low as 0.56 nM. Interestingly, the presence of Y-DNA significantly decreases the size of Cu3(PO4)2 (CuP) particles, which allows them suitable for intracellular applications as gene nanocarriers. Once inside the cells, the hybrid nanoflowers dissolve and release the Y-DNA probes. Then, the intracellular TK1 mRNA hybridizes with the loop region of Y-DNA, which dissociates the Cy3-labeled loop strand and turns on the red fluorescence. Through the real-time imaging of the intracellular TK1 mRNA, the assessment of tumor cells before and after the treatment of drugs including ß-estradiol and tamoxifen is achieved.

A label-free electrochemical biosensor for microRNAs detection based on DNA nanomaterial by coupling with Y-shaped DNA structure and non-linear hybridization chain reaction.

DNA nanomaterials have been widely used in bioassays due to their promising properties for sensitive and specific detection of biomolecules. Herein, a label-free electrochemical method was developed for quantitative detection of microRNAs by integrating Y-shaped DNA (Y-DNA) structures with non-linear hybridization chain reaction (non-linear HCR). The Y-DNA structures consisting of three sequences (Y1, Y2 and Y3) serve as stable and specific probes for recognizing target miRNAs. In the presence of target miRNA, competitive hybridization occurs between miRNA and Y-DNA, resulting in the release of Y3 and the disintegration of the Y-DNA structure. The triggers, which were blocked by Y3 previously, were exposed and initiated the non-linear HCR. Remarkable electrochemical signal changes were produced after the isothermal amplification reaction. Therefore, the proposed biosensor achieved sensitive detection of microRNAs (miRNAs). Under optimal conditions, the limit of detection (LOD) was reduced to 0.3334 fM and linear range was from 1 fM to 10 pM. The special design of Y-DNA helped the biosensor obtain the ability to distinguish between single base mutations. What's more, this biosensor was capable of detecting miRNAs in clinical serum samples. We hope that this developed biosensor would provide a potential application for DNA nanomaterials in the field of microRNAs detection and inspire more interests in the development of DNA nanomaterial biosensors.

Electrochemical biosensor based on Se-doped MWCNTs-graphene and Y-shaped DNA-aided target-triggered amplification strategy.

A highly sensitive electrochemical biosensor for detection of platelet-derived growth factor-BB (PDGF-BB) is developed by using Se-doped multi-walled carbon nanotubes (MWCNTs)-graphene hybrids as electrode supporting substrate, hemin/G-quadruplex as trace labels and Y-shaped DNA-aided target recycling as signal magnifier. The aptamer-containing hairpin probes were first immobilized on the electrode. When target PDGF-BB was added, the aptamer binded PDGF-BB to trigger catalytic assembly of two other hairpins to form many G-quadruplex Y-junction DNA structures, which released PDGF-BB to again bind the intact aptamer to initiate another assembly cycle. G-quadruplex/hemin complexes were produced when hemin was added to generate substantially amplified current output. The developed assay showed a linear range toward PDGF-BB from 0.1 pM to 10 nM with a detection limit of 27 fM (S/N = 3). The method showed excellent specificity and repeatability, and could be expediently applied for sensitive detection of other molecules by simply changing the aptamers.

An electrochemical aptasensor for multiplex antibiotics detection using Y-shaped DNA-based metal ions encoded probes with NMOF substrate and CSRP target-triggered amplification strategy.

An effective electrochemical aptasensor has been developed for the detection of multiplex antibiotics using Y-shaped DNA probes. These probes-based metal ions encoded the nanoscale metal-organic frameworks (NMOF) as a substrate, and circular strand-replacement DNA polymerization (CSRP) target triggered the amplification strategy. The Y-DNA probes (Y-DNA) were assembled using an assisted DNA probe (assisted DNA labeled with magnetic gold nanoparticles) which can hybridize to the captured DNA probe (consisting of aptamer and primer recognition region), and signal tags (NMOF encapsulating signal DNAs and different metal ions such as Pb2+ or Cd2+). Notably, NMOF was employed as the developed platform with a large specific area to load abundant metal ions that can produce distinguishable signals. In the presence of targets, chloramphenicol (CAP) and oxytetracycline (OTC) as models, the conformational change of the captured DNA can disassemble the Y-DNA probes that can consequently release the signal tags in the supernatant due to the high affinity of targets towards the aptamer domain than its complementary sequences. Subsequently, the exposed sequences of captured DNA serve as the initiators for triggering the target cyclic-induced polymerization with the assistance of Bst DNA polymerase. Thus, numerous signal tags could be detected by square wave voltammetry in the supernatant after magnetic separation, thereby amplifying the electrochemical signals. The proposed strategy exhibited a high sensitivity to antibiotics with a detection limit of 33 and 48 fM (S/N = 3) towards CAP and OTC, respectively. Moreover, this aptasensor showed promising applications for the detection of other analytes.

A regenerative ratiometric electrochemical biosensor for selective detecting Hg²âº based on Y-shaped/hairpin DNA transformation.

Inspired by dual-signaling ratiometric mechanism which could reduce the influence of the environmental change, a novel, convenient, and reliable method for the detection of mercury ions (Hg(2+)) based on Y-shaped DNA (Y-DNA) was developed. Firstly, the Y-DNA was formed via the simple annealing way of using two different redox probes simultaneously, omitting the multiple operation steps on the electrode. The Y-DNA was immobilized on the gold electrode surface and then an obvious ferrocene (Fc) signal and a weak methylene blue (MB) signal were observed. Upon addition of Hg(2+), the Y-DNA structure was transformed to hairpin structure based on the formation of T-Hg(2+)-T complex. During the transformation, the redox MB gets close to and the redox Fc gets far away from the electrode surface, respectively. This special design allows a reliable Hg(2+) detection with a detection range from 1 nM to 5 µM and a low detection limit down to 0.094 nM. Furthermore, this biosensor exhibits good selectivity and repeatability, and can be easily regenerated by using L-cysteine. This study offers a simple and effective method for designing ratiometric biosensors for detecting other ions and biomolecules.