The synchronization of multiple signal amplifications for label-free and sensitive aptamer-based sensing of a protein biomarker.

The abnormal variation of the mucin 1 (MUC1) protein level is associated with the development of multiple cancers, and the monitoring of trace MUC1 can be useful for early disease diagnosis. Here, on the basis of the synchronization of DNA-fueled sequence recycling and dual rolling circle amplification (RCA), the establishment of a non-label and highly sensitive fluorescent aptamer-based detection strategy for the MUC1 protein biomarker is described. The target MUC1 binds the aptamer hairpin probe and causes its structure switching to release an ssDNA tail to trigger the recycling of the complex via two toehold-mediated strand displacement reactions under assistance of a fuel DNA. Such a recycling amplification leads to the formation of a partial dsDNA duplex with two primers at both ends, which cooperatively bind the circular DNA ring template to start the dual RCA to produce many G-quadruplex sequences. The protoporphyrin IX dye further associates with the G-quadruplex structures to show a dramatically elevated fluorescent signal for sensitively detecting MUC1 with a low detection limit of 0.5 pM. The established aptamer-based detecting strategy is also highly selective and can realize assay of MUC1 in diluted human serums, highlighting its potential for the detection of different protein biomarkers at low contents.

DNA-Origami-Based Fluorescence Brightness Standards for Convenient and Fast Protein Counting in Live Cells.

Fluorescence microscopy has been one of the most discovery-rich methods in biology. In the digital age, the discipline is becoming increasingly quantitative. Virtually all biological laboratories have access to fluorescence microscopes, but abilities to quantify biomolecule copy numbers are limited by the complexity and sophistication associated with current quantification methods. Here, we present DNA-origami-based fluorescence brightness standards for counting 5-300 copies of proteins in bacterial and mammalian cells, tagged with fluorescent proteins or membrane-permeable organic dyes. Compared to conventional quantification techniques, our brightness standards are robust, straightforward to use, and compatible with nearly all fluorescence imaging applications, thereby providing a practical and versatile tool to quantify biomolecules via fluorescence microscopy.

Coupling strand extension/excision amplification with target recycling enables highly sensitive and aptamer-based label-free sensing of ATP in human serum.

Detection of aberrant ATP concentrations with high sensitivity and selectivity is of critical importance for monitoring many biological processes and disease stages. By coupling extension/excision amplification with target recycling, we have established an aptamer-based method for label-free fluorescence ATP detection in human serum with high sensitivity. The ATP target molecules associate with the aptamer-containing double hairpin probes and cause conformational changes of the probes to initiate the cyclic strand extension/excision processes in the presence of polymerase, endonuclease and assistance sequences for the recycling of ATP and the production of a large number of G-quadruplex sequences. The organic dye thioflavin T subsequently binds these G-quadruplex sequences to yield substantially enhanced fluorescence emission for achieving highly sensitive detection of ATP down to 2.2 nM in the range of 5 to 200 nM without using any labels. The developed aptamer sensing method also exhibits high selectivity and allows the monitoring of ATP at low concentrations in diluted real samples, which offers promising opportunities to establish effective signal magnification means for the detection of various biomolecules at trace levels.

Programmable DNA Ring/Hairpin-Constrained Structure Enables Ligation-Free Rolling Circle Amplification for Imaging mRNAs in Single Cells.

Self-assembled functional DNA structures have proven to be excellent materials for designing and implementing a variety of nanoscale devices. We demonstrate here that a rationally designed and programmable DNA ring/hairpin-constrained structure can achieve in situ ligation-free rolling circle amplification (RCA), which further leads to highly specific, sensitive, and multicolor imaging of mRNA molecules in single cells. Such a structure aims at addressing current challenges in terms of simplicity, sensitivity, and multiplexing capability related to the detection and imaging of intracellular mRNA sequences. With this new DNA ring/hairpin-RCA approach, we are able to detect the target mRNAs with high sensitivity at the subpicomolar levels in vitro. Besides, the multiplexing capability of the DNA structures can be readily realized by barcoding the DNA rings and hairpins with distinct sequences. Due to the excellent sequence recognition ability of the hairpins, the DNA structures exhibit single-base variation discrimination capability for the target mRNA and can be used to image trace amounts of down-expressed mRNAs in single cells. Moreover, drug-dependent mRNA expression variations can also be clearly differentiated by these DNA structures, highlighting the great potential of such structures for early disease diagnosis and for screening possible therapeutic drugs.

A DNA-Fueled and Catalytic Molecule Machine Lights Up Trace Under-Expressed MicroRNAs in Living Cells.

The detection of specific intracellular microRNAs (miRNAs) in living cells can potentially provide insight into the causal mechanism of cancer metastasis and invasion. However, because of the characteristic nature of miRNAs in terms of small sizes, low abundance, and similarity among family members, it is a great challenge to monitor miRNAs in living cells, especially those with much lower expression levels. In this work, we describe the establishment of a DNA-fueled and catalytic molecule machinery in cell signal amplification approach for monitoring trace and under-expressed miRNAs in living cells. The presence of the target miRNA releases the hairpin sequences from the dsDNA (containing the fluorescence resonance energy transfer (FRET) pair-labeled and unfolded hairpin sequences)-conjugated gold nanoparticles (dsDNA-AuNPs), and the DNA fuel strands assist the recycling of the target miRNA sequences via two cascaded strand displacement reactions, leading to the operation of the molecular machine in a catalytic fashion and the release of many hairpin sequences. As a result, the liberated hairpin sequences restore the folded hairpin structures and bring the FRET pair into close proximity to generate significantly amplified signals for detecting trace miRNA targets. Besides, the dsDNA-AuNP nanoprobes have good nuclease stability and show low cytotoxicity to cells, and the application of such a molecular system for monitoring trace and under-expressed miRNAs in living cells has also been demonstrated. With the advantages of in cell signal amplification and reduced background noise, the developed method thus offers new opportunities for detecting various trace intracellular miRNA species.

Click chemistry-mediated cyclic cleavage of metal ion-dependent DNAzymes for amplified and colorimetric detection of human serum copper (II).

The determination of the level of Cu2+ plays important roles in disease diagnosis and environmental monitoring. By coupling Cu+-catalyzed click chemistry and metal ion-dependent DNAzyme cyclic amplification, we have developed a convenient and sensitive colorimetric sensing method for the detection of Cu2+ in human serums. The target Cu2+ can be reduced by ascorbate to form Cu+, which catalyzes the azide-alkyne cycloaddition between the azide- and alkyne-modified DNAs to form Mg2+-dependent DNAzymes. Subsequently, the Mg2+ ions catalyze the cleavage of the hairpin DNA substrate sequences of the DNAzymes and trigger cyclic generation of a large number of free G-quadruplex sequences, which bind hemin to form the G-quadruplex/hemin artificial peroxidase to cause significant color transition of the sensing solution for sensitive colorimetric detection of Cu2+. This method shows a dynamic range of 5 to 500 nM and a detection limit of 2 nM for Cu2+ detection. Besides, the level of Cu2+ in human serums can also be determined by using this sensing approach. With the advantages of simplicity and high sensitivity, such sensing method thus holds great potential for on-site determination of Cu2+ in different samples. Graphical abstract Sensitive colorimetric detection of copper (II) by coupling click chemistry with metal ion-dependentDNAzymes.

Target-triggered quadratic amplification for label-free and sensitive visual detection of cytokines based on hairpin aptamer DNAzyme probes.

The employment of DNAzyme probes for visual biodetections has received increasing interest recently due to the simple nature of this type of assay. However, achieving high sensitivity and detecting targets beyond nucleic acids remain two major challenges in DNAzyme-based visual detections. In this work, based on a new quadratic amplification strategy, we developed a sensitive and visual detection method for cytokines by using hairpin aptamer DNAzyme probes. The target cytokine, interferon γ (IFN-γ), associates with the aptamer sequences and unfolds the hairpin structure of the probes, leading to simultaneous recycling of the target IFN-γ (assisted by Bst-polymerase) and the DNA sequences (aided by λ exonuclease) to achieve quadratic amplification. This quadratic amplification results in the generation of numerous peroxidase-mimicking DNAzymes, which cause significantly intensified color change of the probe solution for highly sensitive detection of IFN-γ by the naked eye down to 50 pM. The proposed visual sensing method shows also high selectivity toward the target IFN-γ and can be performed in homogeneous solutions with using completely unmodified, synthetic aptamer DNAzyme probes. These distinct advantages of our developed assay protocol make it a potential platform for detecting various types of biomolecules with careful probe designs.

Metal ion-complexed DNA probe coupled with CRISPR/Cas12a amplification and AuNPs for sensitive colorimetric assay of metallothionein in fish.

The accurate and sensitive detection of metallothionein (MT) is of great significance in the fields of biomedical, toxicological and environmental sciences. In this work, based on the high affinity interaction between MT and the heavy metal ions of Hg2+ and the significant signal amplification capability of Cas12a/crRNA enzyme as well, we report a simple and highly sensitive method for visual detection of MT, a biomarker in fish for heavy metal ion-induced water bio-pollution. The target MT molecules bind Hg2+ in the Hg2+- complexed hairpin DNA probes to unfold the hairpin structure into ssDNAs, which hybridize with the partial dsDNA duplexes via strand displacement to yield specific sequence-containing dsDNAs. Cas12a/crRNA recognizes these specific sequences to activate its enzyme activity to cyclically cleave the ssDNA linkers in the blue colored gold nanoparticle aggregates to transit their color into red to realize visual detection of MT. Owing to the signal amplification by Cas12a/crRNA, as low as 25 nM of MT can be visually detected with naked eye. In addition, our colorimetric detection method has high selectivity for MT against other interference proteins and can detect MT in the livers and kidneys of crucian carps bought from a local supermarket. Moreover, the developed assay overcomes the limitations of conventional MT detection methods in terms of complexity, high cost and low sensitivity and can therefore offer new methods for monitoring water bio-pollutions.

Electrochemical Performance and Microstructure Evolution of a Quasi-Solid-State Lithium Battery Prepared by Spark Plasma Sintering.

Solid-state lithium batteries are promising next-generation energy storage systems for electric vehicles due to their high energy density and high safety and require achieving and maintaining intimate solid-solid interfaces for lithium-ion and electron transport. However, the solid-solid interfaces may evolve over cycling, disrupting the ion and electron diffusion pathways and leading to rapid performance degradation. The development of solid-state lithium batteries has been hindered by the lack of fundamental understanding of the interfacial microstructure change over cycling and its relation to electrochemical properties. Herein, we prepared a quasi-solid-state lithium battery, 30%LiFePO4-55%Li1.5Al0.5Ge1.5(PO4)3-15%C| Li1.5Al0.5Ge1.5(PO4)3|Li, by spark plasma sintering, and employed it as a model system to reveal the microstructure evolution at the solid-solid interfaces with electrochemical performance of the batteries. The electrochemical assessment showed that the quasi-solid-state lithium battery exhibited a discharge specific capacity of about 150 mAh g-1 in the first 80 cycles and then experienced severe capacity attenuation afterward, accompanied by a gradual internal resistance increase. Scanning electron microscopy observation showed that more cracks were formed inside the solid-state electrolyte and at the solid-solid interfaces as the battery cycled from 10 to 67 and 157 cycles. Detailed microstructure and phase analysis by high-resolution transmission electron microscopy and selected area electron diffraction discovered that the crack formation and performance decay were mainly caused by (1) the volume change of the LiFePO4 composite cathode during cycling, (2) the grain expansion of the Li1.5Al0.5Ge1.5(PO4)3 solid-state electrolyte at its interface with lithium anode, and (3) the formation of a solid electrolyte interphase layer, comprising Li2CO3, LiF, and LiTFSI, at the cathode-solid-state electrolyte interface. These microstructure changes built up over repeated battery cycling, ultimately causing the structure collapse and battery failure. The microstructure evolution information is expected to guide the design of better structures and interfaces for solid-state lithium batteries.

Target-responsive triplex aptamer nanoswitch enables label-free and ultrasensitive detection of antibody in human serum via lighting-up RNA aptamer transcriptions.

Accurate and sensitive monitoring of the concentration change of anti-digoxigenin (Anti-Dig) antibody is of great importance for diagnosing infectious and immunological diseases. Combining a novel triplex aptamer nanoswitch and the high signal-to-noise ratio of lighting-up RNA aptamer signal amplification, a label-free and ultrasensitive fluorescent sensing approach for detecting Anti-Dig antibodies is described. The target Anti-Dig antibodies recognize and bind with the nanoswitch to open its triplex helix stem structure to release Taq DNA polymerase and short ssDNA primer simultaneously, which activates the Taq DNA polymerase to initiate downstream strand extension of ssDNA primer to yield specific dsDNA containing RNA promoter sequence. T7 RNA polymerase recognizes and binds to these promoter sequences to initiate RNA transcription reaction to produce many RNA aptamer sequences. These aptamers can recognize and bind with Malachite Green (MG) dye specifically and produce highly amplified fluorescent signal for monitoring Anti-Dig antibodies from 50 pM to 50 nM with a detection limit down to 33 pM. The method also exhibits high selectivity for Anti-Dig antibodies and can be used to discriminate trace Anti-Dig antibodies in diluted serum samples. Our method is superior to many immunization-based Anti-Dig antibody detection methods and thus holds great potential for monitoring disease progression and efficacy.