Bi-directional DNA Walking Machine and Its Application in an Enzyme-Free Electrochemiluminescence Biosensor for Sensitive Detection of MicroRNAs

Herein, a dual microRNA (miRNA) powered bi-directional DNA walking machine with precise control was developed to fabricate an enzyme-free biosensor on the basis of distance-based electrochemiluminescence (ECL) energy transfer for multiple detection of miRNAs. By using miRNA-21 as the driving force, the DNA walker could move forth along the track and generated quenching of ECL response due to the proximity between Au nanoparticles (AuNPs) and Mn²⁺ doped CdS nanocrystals (CdS:Mn NCs) film as the ECL emitters, realizing ultrasensitive determination of miRNA-21. Impressively, once miRNA-155 was introduced as the driving force, the walker could move back along the track automatically, and surface plasmon resonance (SPR) occurred owing to the appropriate large separation between AuNPs and CdS:Mn NCs, achieving an ECL enhancement and realizing ultrasensitive detection of miRNA-155. The bi-directional movement of the DNA walker on the track led to continuous distance-based energy transfer from CdS:Mn NCs film by AuNPs, which resulted in significant ECL signal variation of CdS:Mn NCs for multiple detection of miRNA-21 and miRNA-155 down to 1.51 fM and 1.67 fM, respectively. Amazingly, the elaborated biosensor provided a new chance for constructing controllable molecular nanomachines in biosensing, disease diagnosis, and clinical analysis

An “Off–On” Electrochemiluminescent Biosensor Based on DNAzyme-Assisted Target Recycling and Rolling Circle Amplifications for Ultrasensitive Detection of microRNA

In this study, an off–on switching of a dual amplified electrochemiluminescence (ECL) biosensor based on Pb²⁺-induced DNAzyme-assisted target recycling and rolling circle amplification (RCA) was constructed for microRNA (miRNA) detection. First, the primer probe with assistant probe and miRNA formed Y junction which was cleaved with the addition of Pb²⁺ to release miRNA. Subsequently, the released miRNA could initiate the next recycling process, leading to the generation of numerous intermediate DNA sequences (S2). Afterward, bare glassy carbon electrode (GCE) was immersed into HAuCl₄ solution to electrodeposit a Au nanoparticle layer (depAu), followed by the assembly of a hairpin probe (HP). Then, dopamine (DA)-modified DNA sequence (S1) was employed to hybridize with HP, which switching off the sensing system. This is the first work that employs DA to quench luminol ECL signal, possessing the biosensor ultralow background signal. Afterward, S2 produced by the target recycling process was loaded onto the prepared electrode to displace S1 and served as an initiator for RCA. With rational design, numerous repeated DNA sequences coupling with hemin to form hemin/G-quadruplex were generated, which could exhibit strongly catalytic toward H₂O₂, thus amplified the ECL signal and switched the ON state of the sensing system. The liner range for miRNA detection was from 1.0 fM to 100 pM with a low detection limit down to 0.3 fM. Moreover, with the high sensitivity and specificity induced by the dual signal amplification, the proposed miRNA biosensor holds great potential for analysis of other interesting tumor markers

An Electrochemical Biosensor for Sensitive Detection of MicroRNA-155: Combining Target Recycling with Cascade Catalysis for Signal Amplification

In this work, a new electrochemical biosensor based on catalyzed hairpin assembly target recycling and cascade electrocatalysis (cytochrome <i>c</i> (Cyt <i>c</i>) and alcohol oxidase (AOx)) for signal amplification was constructed for highly sensitive detection of microRNA (miRNA). It is worth pointing out that target recycling was achieved only based on strand displacement process without the help of nuclease. Moreover, porous TiO₂ nanosphere was synthesized, which could offer more surface area for Pt nanoparticles (PtNPs) enwrapping and enhance the amount of immobilized DNA strand 1 (S1) and Cyt <i>c</i> accordingly. With the mimicking sandwich-type reaction, the cascade catalysis amplification strategy was carried out by AOx catalyzing ethanol to acetaldehyde with the concomitant formation of high concentration of H₂O₂, which was further electrocatalyzed by PtNPs and Cyt <i>c</i>. This newly designed biosensor provided a sensitive detection of miRNA-155 from 0.8 fM to 1 nM with a relatively low detection limit of 0.35 fM

Numerical analysis of particle motion and collision of surround U-clevis in windy and sandy environment

This paper was reviewed and accepted by the APCWE-IX Programme Committee for Presentation at the 9th Asia-Pacific Conference on Wind Engineering, University of Auckland, Auckland, New Zealand, held from 3-7 December 2017

Dual microRNAs-Fueled DNA Nanogears: A Case of Regenerated Strategy for Multiple Electrochemiluminescence Detection of microRNAs with Single Luminophore

The determination of multiple biomarkers from cancer cells features a considerable step toward early diagnosis of cancers. However, realizing different biomarkers detection with single electrochemiluminescence (ECL) luminophore and regenerating the sensing platform remain a compelling goal. Herein, dual miRNAs-fueled DNA nanogears were designed for an enzyme-free ECL biosensor construction to perform the multiple sensitive detection of the microRNA (miRNA) biomarkers with single luminophore. The nanogears were assembled on CdS quantum dots (QDs) modified sensing surface. Using miRNA-21 as motive power, Au nanoparticles (AuNPs)-labeled nanogears B could be activated to roll against nanogear A, increasing the distance between AuNPs and CdS QDs. Thus, the significant ECL enhancement of CdS QDs was obtained owing to the ECL energy transfer between AuNPs and CdS QDs, simultaneously realizing the detection of miRNA-21. After the incubation of miRNA-155, nanogear B revolved against nanogear A continuously and realized the close-range of AuNPs and CdS QDs, resulting in the quenching of ECL intensity due to the Förster energy transfer and realizing the analysis of miRNA-155. The successive locomotion of the nanogears led to a significant ECL increasing for analysis of miRNA-21 down to 0.16 fM and a remarkable ECL suppression for determination of miRNA-155 down to 0.33 fM. Impressively, the proposed biosensor was able to be regenerated along with the gears roll against each other. In general, this enzyme-free strategy initiates a new thought to realize the multiple ECL detection with single luminophore, paving the way for applications of nanomachines in biosensing and clinical diagnosis

Highly Ordered and Field-Free 3D DNA Nanostructure: The Next Generation of DNA Nanomachine for Rapid Single-Step Sensing

Herein, by directly using Watson–Crick base pairing, a highly ordered and field-free three-dimensional (3D) DNA nanostructure is self-assembled by azobenzene (azo)-functionalized DNA nippers in a few minutes, which was applied as a 3D DNA nanomachine with an improved movement efficiency compared to traditional Au-based 3D nanomachines due to the organized and high local concentration of nippers on homogeneous DNA nanostructure. Once microRNA (miRNA) interacts with the 3D nanomachine, the nippers “open” to hybridize with the miRNA. Impressively, photoisomerization of the azo group induces dehybridization/hybridization of the nippers and miRNA under irradiation at different wavelengths, which easily solves one main technical challenge of DNA nanotechnology and biosensing: reversible locomotion in one step within 10 min. As a proof of concept, the described 3D machine is successfully applied in the rapid single-step detection of a biomarker, which gives impetus to the design of new generations of mechanical devices beyond the traditional ones with ultimate applications in sensing analysis and diagnostic technologies

Electrochemiluminescent Graphene Quantum Dots as a Sensing Platform: A Dual Amplification for MicroRNA Assay

Graphene quantum dots (GQDs) with an average diameter as small as 2.3 nm were synthesized to fabricate an electrochemiluminescence (ECL) biosensor based on T7 exonuclease-assisted cyclic amplification and three-dimensional (3D) DNA-mediated silver enhancement for microRNA (miRNA) analysis. Herein, to overcome the barrier in immobilizing GQDs, aminated 3,4,9,10-perylenetetracarboxylic acid (PTCA–NH₂) was introduced to load GQDs through π–π stacking (GQDs/PTCA–NH₂), realizing the solid-state GQDs application. Furthermore, Fe₃O₄–Au core–shell nanocomposite (Au@Fe₃O₄) was adopted as a probe anchor to form a novel electrochemiluminescent signal tag of GQDs/PTCA–NH₂/Au@Fe₃O₄. The prepared ECL signal tag was decorated on the electrode surface, exhibiting excellent film-forming performance, good electronic conductivity, and favorable stability, all of which overcame the obstacle for applying GQDs in ECL biosensing and showed a satisfactory ECL response under the coreactant of S₂O₈^2– (peroxydisulfate). Afterward, hairpin probe modified on the electrode was opened by helper DNA, followed by assembling target to hybridize with the exposed stem of the helper DNA. Significantly, T7 exonuclease was employed to digest the DNA/RNA duplex and trigger the target recycling without asking for a specific recognition site in the target sequence, realizing a series of RNA/DNA detections by changing the sequence of the complementary DNA. At last, the ECL signal was further enhanced by silver nanoparticles (AgNPs)-based 3D DNA networks. After the two amplifications, the ECL signal of GQDs was extraordinarily increased and the prepared biosensor achieved a high sensitivity with the detection limit of 0.83 fM. The biosensor was also explored in real samples, and the result was in good accordance with the performance of quantitative real-time polymerase chain reaction (qRT-PCR). Considering the excellent sensitivity and applicability, we believe that the proposed biosensor is a potential candidate for nucleic acid biosensing

Detection of TUNEL-positive cells.

<p>A) Representative TUNEL staining of normal retina, injured retina 7 days after injury, and hUCBSC-transplanted retinas 7 days after surgery. TUNEL-positive cells were seen in inner nuclear layer and ganglion cell layer. Arrows indicated the TUNEL-positive cells (Brown). ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer. B) TUNEL-positive cells counting in retina in RGC layer at different time points in injury and hUCBSC transplanted rats. N = 5. ^*<i>p</i><0.05, ^**<i>p</i><0.01 <i>vs</i>. 3 hrs. ^#<i>p</i><0.05, ^##<i>p</i><0.01 <i>vs</i>. injury group.</p

Detection of CHOP mRNA expression.

<p>A) Representative RT-PCR of CHOP expression. CHOP mRNA expression was detected 3 hrs, 12 hrs, 24 hrs, 48 hrs and 7 days post surgery in the injury (Inj) and hUCBSC transplantation (Inj+SC) groups. B) Comparison of the band-pixel values of CHOP mRNA in injury and hUCBSC transplantation rats. ^*<i>p</i><0.001 <i>vs.</i> hUCBSC transplantation. N = 5.</p

Detection of GRP78 mRNA expression.

<p>A) Representative RT-PCR of GRP78 expression. GRP78 mRNA expression was detected 3 hrs, 12 hrs, 24 hrs, 48 hrs, 72 hrs and 7 days post surgery in the injury (Inj) and hUCBSC transplantation (Inj+SC) groups. B) Comparison of the band-pixel values of GRP78 mRNA in injury and hUCBSC transplantation rats. ^*<i>p</i><0.001 <i>vs</i>. injury group. N = 5.</p