Environment‐Recognizing DNA‐Computation Circuits for the Intracellular Transport of Molecular Payloads for mRNA Imaging

Programming intelligent DNA nanocarriers for the targeted transport of molecular payloads in living cells has attracted extensive attention. In vivo activation of these nanocarriers usually relies on external light irradiation. An interest is emerging in the automatic recognition of intracellular surroundings by nanocarriers and their in situ activation under the control of programmed DNA‐computation circuits. Herein, we report the integration of DNA circuits with framework nucleic acid (FNA) nanocarriers that consist of a truncated square pyramid (TSP) cage and a built‐in duplex cargo containing an antisense strand of the target mRNA. An i‐motif and ATP aptamer embedded in the TSP are employed as logic‐controlling units to respond to H⁺ and ATP inside cellular compartments, triggering the release of the sensing element for fluorescent mRNA imaging. Logic‐controlled FNA devices could be used to target drug delivery, enabling precise disease treatment.     

Catalytic Molecular Imaging of MicroRNA in Living Cells by DNA‐Programmed Nanoparticle Disassembly

Molecular imaging is an essential tool for disease diagnostics and treatment. Direct imaging of low‐abundance nucleic acids in living cells remains challenging because of the relatively low sensitivity and insufficient signal‐to‐background ratio of conventional molecular imaging probes. Herein, we report a class of DNA‐templated gold nanoparticle (GNP)–quantum dot (QD) assembly‐based probes for catalytic imaging of cancer‐related microRNAs (miRNA) in living cells with signal amplification capacity. We show that a single miRNA molecule could catalyze the disassembly of multiple QDs with the GNP through a DNA‐programmed thermodynamically driven entropy gain process, yielding significantly amplified QD photoluminescence (PL) for miRNA imaging. By combining the robust PL of QDs with the catalytic amplification strategy, three orders of magnitude improvement in detection sensitivity is achieved in comparison with non‐catalytic imaging probe, which enables facile and accurate differentiation between cancer cells and normal cells by miRNA imaging in living cells.     

Focusing and stabilization of bis‐intercalating dye–DNA complexes for high‐sensitive CE‐LIF DNA analysis

Multiple labeling of nucleic acids by intercalative dyes is a promising method for ultrasensitive nucleic acid assays. The properties of the fast dissociation and instability of dye–DNA complexes may prevent from their wide applications in CE‐LIF nucleic acid analysis. Here, we describe an optimum CE focusing method by using appropriately paired sample and separation buffers, Tris‐glycine buffer and Tris‐glycine‐acetic acid buffer. The developed method was applied in both uncoated and polyacrylamide coated fused‐silica capillary‐based CE‐LIF analysis while the sample and separation buffers were conversely used. The complexes of intercalative dye benzoxazolium‐4‐pyridinium dimer and dsDNA were greatly focused (separation efficiency: 1.8 million theoretical plates per meter) by transient isotachophoresis mechanism in uncoated capillary, and moderately focused by transient isotachophoresis in combination of field amplified sample stacking and further stabilized by the paired buffer in polyacrylamide coated capillary. Based on the developed focusing strategy, an ultrasensitive DNA assay was developed for quantitation of calf thymus dsDNA (from 0.02 to 2.14 pM). By the use of an excitation laser power as low as 1 mW, the detection limits of calf thymus dsDNA (3.5 kb) are 7.9 fM in concentration and 2.4×10^?22 mol (150 molecules) in mass. We further demonstrate that the non‐gel sieving CE‐LIF analysis of DNA fragments can be enhanced by the same strategy. Since the presented strategy can be applied to uncoated and coated capillaries and does not require special device, it is also reasonable to extend to the applications in chip‐based CE DNA analysis.     

Control of Biocatalytic Transformations by Programmed DNA Assemblies

This study demonstrates the self‐assembly of inhibitor/enzyme‐tethered nucleic acid fragments or enzyme I‐, enzyme II‐modified nucleic acids into functional nanostructures that lead to the controlled inhibition of the enzyme or the activation of an enzyme cascade. In one system, the anti‐cocaine aptamer subunits are modified with monocarboxy methylene blue (MB⁺) as the inhibitor and with choline oxidase (ChOx). The cocaine‐induced self‐assembly of the aptamer subunits complex results in the inhibition of ChOx by MB⁺. In a further configuration, two nucleic acids of limited complementarity are functionalized at their 3′ and 5′ ends with glucose oxidase (GOx) and horseradish peroxidase (HRP), respectively, or with MB⁺ and ChOx. In the presence of a target DNA sequence, synergistic complementary base‐pairing occurs, thus leading to stable supramolecular Y‐shaped nanostructures of the nucleic acid units. A GOx/HRP bienzyme cascade or the programmed inhibition of ChOx by MB⁺ is demonstrated in the resulting nucleic acid nanostructures. A quantitative theoretical model that describes the nucleic acid assemblies and that results in the inhibition of ChOx by MB⁺ or in the activation of the GOx/HRP cascade, respectively, is provided.     

Bridging the Two Worlds: A Universal Interface between Enzymatic and DNA Computing Systems

Molecular computing based on enzymes or nucleic acids has attracted a great deal of attention due to the perspectives of controlling living systems in the way we control electronic computers. Enzyme‐based computational systems can respond to a great variety of small molecule inputs. They have the advantage of signal amplification and highly specific recognition. DNA computing systems are most often controlled by oligonucleotide inputs/outputs and are capable of sophisticated computing as well as controlling gene expressions. Here, we developed an interface that enables communication of otherwise incompatible nucleic‐acid and enzyme‐computational systems. The enzymatic system processes small molecules as inputs and produces NADH as an output. The NADH output triggers electrochemical release of an oligonucleotide, which is accepted by a DNA computational system as an input. This interface is universal because the enzymatic and DNA computing systems are independent of each other in composition and complexity.     

Dual‐signal amplification strategy for ultrasensitive chemiluminescence detection of PDGF–BB in capillary electrophoresis

Many efforts have been made toward the achievement of high sensitivity in capillary electrophoresis coupled with chemiluminescence detection (CE‐CL). This work describes a novel dual‐signal amplification strategy for highly specific and ultrasensitive CL detection of human platelet‐derived growth factor–BB (PDGF–BB) using both aptamer and horseradish peroxidase (HRP) modified gold nanoparticles (HRP–AuNPs–aptamer) as nanoprobes in CE. Both AuNPs and HRP in the nanoprobes could amplify the CL signals in the luminol–H₂O₂ CL system, owing to the excellent catalytic behavior of AuNPs and HRP in the CL system. Meanwhile, the high affinity of aptamer modified on the AuNPs allows detection with high specificity. As proof‐of‐concept, the proposed method was employed to quantify the concentration of PDGF–BB from 0.50 to 250 fm with a detection limit of 0.21 fm. The applicability of the assay was further demonstrated in the analysis of PDGF–BB in human serum samples with acceptable accuracy and reliability. The result of this study exhibits distinct advantages, such as high sensitivity, good specificity, simplicity, and very small sample consumption. The good performances of the proposed strategy provide a powerful avenue for ultrasensitive detection of rare proteins in biological sample, showing great promise in biochemical analysis. Copyright © 2015 John Wiley & Sons, Ltd.     

Environment-Recognizing DNA-Computation Circuits for the Intracellular Transport of Molecular Payloads for mRNA Imaging

Programming intelligent DNA nanocarriers for the targeted transport of molecular payloads in living cells has attracted extensive attention. In vivo activation of these nanocarriers usually relies on external light irradiation. An interest is emerging in the automatic recognition of intracellular surroundings by nanocarriers and their in situ activation under the control of programmed DNA-computation circuits. Herein, we report the integration of DNA circuits with framework nucleic acid (FNA) nanocarriers that consist of a truncated square pyramid (TSP) cage and a built-in duplex cargo containing an antisense strand of the target mRNA. An i-motif and ATP aptamer embedded in the TSP are employed as logic-controlling units to respond to H⁺ and ATP inside cellular compartments, triggering the release of the sensing element for fluorescent mRNA imaging. Logic-controlled FNA devices could be used to target drug delivery, enabling precise disease treatment.     

PNA-based probe for quantitative chemiluminescent in situ hybridisation imaging of cellular parvovirus B19 replication kinetics

To allow the ultrasensitive localization and the quantitative detection of parvovirus B19 nucleic acids in single infected cells at various times post-infection, a peptide nucleic acid (PNA)-based in situ hybridisation (ISH) assay with chemiluminescent detection has been developed. The assay is based on the use of a biotin-labelled PNA probe detected by a streptavidin-linked alkaline phosphatase and a chemiluminescent dioxetane phosphate derivative substrate. The luminescent signal was quantified and imaged with an ultrasensitive nitrogen-cooled CCD camera connected to an epifluorescence microscope. The assay was used to analyze the parvovirus B19 infection process in cell cultures and to quantify the amount of viral nucleic acids at different times after infection.The chemiluminescent ISH-PNA assay is characterized by high resolution providing a sharp localization of B19 nucleic acids within single cells, with higher sensitivity with respect to conventional colorimetric ISH detection. Thanks to the high detectability and wide linear range of chemiluminescence detection, an objective evaluation of the percentage of infected cells, which reached its maximum at 24 h after infection, following a B19 virus infectious cycle could be accurately evaluated. Chemiluminescence detection also allowed the quantitative analysis of viral nucleic acids at the single-cell level, showing a continuous increase of the content of viral nucleic acids in infected cells with time after infection.The developed chemiluminescent ISH-PNA assay could thus represent a potent tool for the assessment of viral infections and for the quantitative evaluation of the virus nucleic acid load of infected cells in virus studies and diagnostics.     

Silver Coated Platinum Core–Shell Nanostructures on Etched Si Nanowires: Atomic Layer Deposition (ALD) Processing and Application in SERS

A new method to prepare plasmonically active noble metal nanostructures on large surface area silicon nanowires (SiNWs) mediated by atomic layer deposition (ALD) technology has successfully been demonstrated for applications of surface‐enhanced Raman spectroscopy (SERS)‐based sensing. As host material for the plasmonically active nanostructures we use dense single‐crystalline SiNWs with diameters of less than 100 nm as obtained by a wet chemical etching method based on silver nitrate and hydrofluoric acid solutions. The SERS active metal nanoparticles/islands are made from silver (Ag) shells as deposited by autometallography on the core nanoislands made from platinum (Pt) that can easily be deposited by ALD in the form of nanoislands covering the SiNW surfaces in a controlled way. The density of the plasmonically inactive Pt islands as well as the thickness of noble metal Ag shell are two key factors determining the magnitude of the SERS signal enhancement and sensitivity of detection. The optimized Ag coated Pt islands on SiNWs exhibit great potential for ultrasensitive molecular sensing in terms of high SERS signal enhancement ability, good stability and reproducibility. The plasmonic activity of the core‐shell Pt//Ag system that will be experimentally realized in this paper as an example was demonstrated in numerical finite element simulations as well as experimentally in Raman measurements of SERS activity of a highly diluted model dye molecule. The morphology and structure of the core‐shell Pt//Ag nanoparticles on SiNW surfaces were investigated by scanning‐ and transmission electron microscopy. Optimized core–shell nanoparticle geometries for maximum Raman signal enhancement is discussed essentially based on the finite element modeling.     

Valence‐Engineering of Quantum Dots Using Programmable DNA Scaffolds

Precise control over the valency of quantum dots (QDs) is critical and fundamental for quantitative imaging in living cells. However, prior approaches on valence control of QDs remain restricted to single types of valences. A DNA‐programmed general strategy is presented for valence engineering of QDs with high modularity and high yield. By employing a series of programmable DNA scaffolds, QDs were generated with tunable valences in a single step with near‐quantitative yield (>95 %). The use of these valence‐engineered QDs was further demonstrated to develop 12 types of topologically organized QDs‐QDs and QDs‐AuNPs and 4 types of fluorescent resonance energy transfer (FRET) nanostructures. Quantitative analysis of the FRET nanostructures and live‐cell imaging reveal the high potential of these nanoprobes in bioimaging and nanophotonic applications.     

Ultrasensitive Biosensing Platform Based on the Luminescence Quenching Ability of Plasmonic Palladium Nanoparticles

An ultrasensitive biosensing platform for DNA and protein detection is constructed based on the luminescence quenching ability of plasmonic palladium nanoparticles (PdNPs). By growing the particles into large sizes (ca. 30 nm), the plasmonic light absorption of PdNPs is broadened and extended to the visible range with extinction coefficients as high as 10⁹ L mol^?1 cm^?1, enabling complete quenching of fluorescent dyes that emit at diverse ranges and that are tagged to bioprobes. Meanwhile the nonspecific quenching of the dyes (not bound to probes) is negligible, leading to extremely low background signal. Utilizing the affinity of PdNPs towards bioprobes, such as single‐stranded (ss) DNA and polypeptide molecules, which is mainly assigned to the coordination interaction, nucleic acid assays with a quantification limit of 3 pM target DNA and protein assay are achieved with a simple mix‐and‐detect strategy based on the luminescence quenching‐and‐recovery protocol. This is the first demonstration of biosensing employing plasmonic absorption of nanopalladium, which offers pronounced sensing performances and can be reasonably expected for wide applications.     

Coupling Sensitive Nucleic Acid Amplification with Commercial Pregnancy Test Strips

The detection of nucleic acid biomarkers for point‐of‐care (POC) diagnostics is currently limited by technical complexity, cost, and time constraints. To overcome these shortcomings, we have combined loop‐mediated isothermal amplification (LAMP), programmable toehold‐mediated strand‐exchange signal transduction, and standard pregnancy test strips. The incorporation of an engineered hCG–SNAP fusion reporter protein (human chorionic gonadotropin‐O⁶‐alkylguanine‐DNA alkyltransferase) led to LAMP‐to‐hCG signal transduction on low‐cost, commercially available pregnancy test strips. Our assay reliably detected as few as 20 copies of Ebola virus templates in both human serum and saliva and could be adapted to distinguish a common melanoma‐associated SNP allele (BRAF V600E) from the wild‐type sequence. The methods described are completely generalizable to many nucleic acid biomarkers, and could be adapted to provide POC diagnostics for a range of pathogens.     

Monolayer Two‐dimensional Molecular Crystals for an Ultrasensitive OFET‐based Chemical Sensor

The sensitivity of conventional thin‐film OFET‐based sensors is limited by the diffusion of analytes through bulk films and remains the central challenge in sensing technology. Now, for the first time, an ultrasensitive (sub‐ppb level) sensor is reported that exploits n‐type monolayer molecular crystals (MMCs) with porous two‐dimensional structures. Thanks to monolayer crystal structure of NDI3HU‐DTYM2 (NDI) and controlled formation of porous structure, a world‐record detection limit of NH₃ (0.1 ppb) was achieved. Moreover, the MMC‐OFETs also enabled direct detection of solid analytes of biological amine derivatives, such as dopamine at an extremely low concentration of 500 ppb. The remarkably improved sensing performances of MMC‐OFETs opens up the possibility of engineering OFETs for ultrasensitive (bio)chemical sensing.     

Programming Non-Nucleic Acid Molecules into Computational Nucleic Acid Systems

Nucleic acid (NA) computation has been widely developed in the past years to solve kinds of logic and mathematic issues in both information technologies and biomedical analysis. However, the difficulty to integrate non-NA molecules limits its power as a universal platform for molecular computation. Here, we report a versatile prototype of hybridized computation integrated with both nucleic acids and non-NA molecules. Employing the conformationally controlled ligand converters, we demonstrate that non-NA molecules, including both small molecules and proteins, can be computed as nucleic acid strands to construct the circuitry with increased complexity and scalability, and can be even programmed to solve arithmetical calculations within the computational nucleic acid system. This study opens a new door for molecular computation in which all-NA circuits can be expanded with integration of various ligands, and meanwhile, ligands can be precisely programmed by the nuclei acid computation.     

An Ultrasensitive Analytical Method Based on Chemiluminescence Sensing Electrochemical Parallel Catalytic Reaction Event

An ultrasensitive electrochemiluminescence (ECL) method on the combination of electrochemical parallel catalytic reaction and chemiluminesence signal sensing was proposed for improving ECL analytical characteristics using vanadate(V) as a representative. Vanadate(V) could be electrochemically reduced to generate vanadate(II) which could be chemically oxidized by potassium periodate to regenerate vanadate(V) and give parallel catalytic wave effect. Then, the reduced product of potassium periodate could react with butyl‐rhodamine B to emit a sensitive chemiluminescence signal. The chemiluminescence intensity was correlative with vanadate(V) concentration. The investigation on the electrochemical reaction rate constant (k⁰) confirmed that the speed of electrochemical reaction was faster than that of the subsequent chemiluminescence reaction. The possibility of the combination of electrochemical parallel catalytic reaction with chemiluminescence signal sensing was proved. The similar ECL behaviors could be observed at zirconia nanowires‐Nafion modified electrode. Because of the separation and enrichment effect of the modified electrode on vanadate(V), the selectivity and sensitivity was further improved greatly. Based on these findings, a new concept on the combination of electrochemical parallel catalytic reaction and chemiluminesence signal sensing was proposed and an ultrasensitive ECL method for the determination of vanadate(V) was developed at zirconia nanowires‐Nafion modified electrode. Under the optimum experimental conditions, the ECL intensity was linear with the concentration of vanadate(V) in the range of 2.0×10^?12 mol/L–2.0×10^?10 mol/L. The detection limit was 8.0×10^?13 mol/L, which was more than 6 orders of magnitude lower than that observed by electrochemical current transduction for electrochemical parallel catalytic reaction at zirconia nanowires‐Nafion modified electrode.     

Direct Detection of Circulating Tumor Cells in Whole Blood Using Time‐Resolved Luminescent Lanthanide Nanoprobes

The detection of circulating tumor cells (CTCs) is crucial to early cancer diagnosis and the evaluation of cancer metastasis. However, it remains challenging due to the scarcity of CTCs in the blood. Herein, we report an ultrasensitive platform for the direct detection of CTCs using luminescent lanthanide nanoprobes. These were designed to recognize the epithelial cell adhesion molecules on cancer cells, allowing signal amplification through dissolution‐enhanced time‐resolved photoluminescence (TRPL) and the elimination of short‐lived autofluorescence interference. This enabled the direct detection of blood breast‐cancer cells with a limit of detection down to 1 cell/well of a 96‐well plate. Moreover, blood CTCs (≥10 cells mL^?1) can be detected in cancer patients with a detection rate of 93.9 % (14/15 patients). We envision that this ultrasensitive detection platform with excellent practicality may provide an effective strategy for early cancer diagnosis and prognosis evaluation.     

Ultrahighly Sensitive Homogeneous Detection of DNA and MicroRNA by Using Single‐Silver‐Nanoparticle Counting

DNA and RNA analysis is of high importance for clinical diagnoses, forensic analysis, and basic studies in the biological and biomedical fields. In this paper, we report the ultrahighly sensitive homogeneous detection of DNA and microRNA by using a novel single‐silver‐nanoparticle counting (SSNPC) technique. The principle of SSNPC is based on the photon‐burst counting of single silver nanoparticles (Ag NPs) in a highly focused laser beam (about 0.5 fL detection volume) due to Brownian motion and the strong resonance Rayleigh scattering of single Ag NPs. We first investigated the performance of the SSNPC system and then developed an ultrasensitive homogeneous detection method for DNA and microRNA based on this single‐nanoparticle technique. Sandwich nucleic acid hybridization models were utilized in the assays. In the hybridization process, when two Ag‐NP–oligonucleotide conjugates were mixed in a sample containing DNA (or microRNA) targets, the binding of the targets caused the Ag NPs to form dimers (or oligomers), which led to a reduction in the photon‐burst counts. The SSNPC method was used to measure the change in the photon‐burst counts. The relationship between the change of the photon‐burst counts and the target concentration showed a good linearity. This method was used for the assay of sequence‐specific DNA fragments and microRNAs. The detection limits were at about the 1 fM level, which is 2–5 orders of magnitude more sensitive than current homogeneous methods.     

An electrochemical signal 'off-on' sensing platform for microRNA detection

The abnormal expression of microRNAs (miRNAs) in many solid tumors makes miRNAs potential biomarkers for disease diagnosis and highlights the need for the sensitive and selective detection of miRNAs. In the present work, an 'off-on' signaling genosensor platform for miRNA-21 detection was well developed. This tactic was based on a locked nucleic acid-integrated nucleic acid hairpin probe, a biotin-labeled bridge DNA-AuNPs-bio-barcode signal amplification unit and enzymatic signal amplification. The test is simple, fast and ultrasensitive with a linear range of 0.01-700 pM. The detection limit was estimated to be 6 fM. The overexpression of miRNA-21 was confirmed in total RNA extracted from human hepatocarcinoma cells BEL-7402 and human HeLa cells compared with the control sample extracted from normal human hepatic L02 cells. This method does not need miRNA-21 labeling, isolation, enrichment or PCR amplification. The performance of the assay developed here could satisfy the need for rapid, easy, sensitive and specific early cancer diagnosis in clinical diagnostics.     

Layer‐by‐Layer Motif Architectures: Programmed Electrochemical Syntheses of Multilayer Mesoporous Metallic Films with Uniformly Sized Pores

Although multilayer films have been extensively reported, most compositions have been limited to non‐catalytically active materials (e.g. polymers, proteins, lipids, or nucleic acids). Herein, we report the preparation of binder‐free multilayer metallic mesoporous films with sufficient accessibility for high electrocatalytic activity by using a programmed electrochemical strategy. By precisely tuning the deposition potential and duration, multilayer mesoporous architectures consisting of alternating mesoporous Pd layers and mesoporous PdPt layers with controlled layer thicknesses can be synthesized within a single electrolyte, containing polymeric micelles as soft templates. This novel architecture, combining the advantages of bimetallic alloys, multilayer architectures, and mesoporous structures, exhibits high electrocatalytic activity for both the methanol oxidation reaction (MOR) and the ethanol oxidation reaction (EOR).     

Rapid,Quantitative, and Ultrasensitive Point‐of‐Care Testing: A Portable SERS Reader for Lateral Flow Assays in Clinical Chemistry

The design of a portable Raman/SERS‐LFA reader with line illumination using a custom‐made fiber optic probe for rapid, quantitative, and ultrasensitive point‐of‐care testing (POCT) is presented. The pregnancy hormone human chorionic gonadotropin (hCG) is detectable in clinical samples within only 2–5 s down to approximately 1.6 mIU mL^?1. This acquisition time is several orders of magnitude shorter than those of existing approaches requiring expensive Raman instrumentation, and the method is 15‐times more sensitive than a commercially available lateral flow assay (LFA) as the gold standard. The SERS‐LFA technology paves the way for affordable, quantitative, and ultrasensitive POCT with multiplexing potential in real‐world applications, ranging from clinical chemistry to food and environmental analysis as well as drug and biowarfare agent testing.