Functionalized tetrahedral DNA frameworks for the capture of circulating tumor cells.

Identification and characterization of circulating tumor cells (CTCs) from blood samples of patients with cancer can help monitor parameters such as disease stage, disease progression and therapeutic efficiency. However, the sensitivity and specificity of current multivalent approaches used for CTC capture is limited by the lack of control over the ligands' position. In this Protocol Update, we describe DNA-tetrahedral frameworks anchored with aptamers that can be configured with user-defined spatial arrangements and stoichiometries. The modified tetrahedral DNA frameworks, termed 'n-simplexes', can be used as probes to specifically target receptor-ligand interactions on the cell membrane. Here, we describe the synthesis and use of n-simplexes that target the epithelial cell adhesion molecule expressed on the surface of CTCs. The characterization of the n-simplexes includes measuring the binding affinity to the membrane receptors as a result of the spatial arrangement and stoichiometry of the aptamers. We further detail the capture of CTCs from patient blood samples. The procedure for the preparation and characterization of n-simplexes requires 11.5 h, CTC capture from clinical samples and data processing requires ~5 h per six samples and the downstream analysis of captured cells typically requires 5.5 h. The protocol is suitable for users with basic expertise in molecular biology and handling of clinical samples.

Solid state ionics enabled ultra-sensitive detection of thermal trace with 0.001K resolution in deep sea.

The deep sea remains the largest uncharted territory on Earth because it's eternally dark under high pressure and the saltwater is corrosive and conductive. The harsh environment poses great difficulties for the durability of the sensing method and the device. Sea creatures like sharks adopt an elegant way to detect objects by the tiny temperature differences in the seawater medium using their extremely thermo-sensitive thermoelectric sensory organ on the nose. Inspired by shark noses, we designed and developed an elastic, self-healable and extremely sensitive thermal sensor which can identify a temperature difference as low as 0.01 K with a resolution of 0.001 K. The sensor can work reliably in seawater or under a pressure of 110 MPa without any encapsulation. Using the integrated temperature sensor arrays, we have constructed a model of an effective deep water mapping and detection device.

Triggering ZT to 0.40 by Engineering Orientation in One Polymeric Semiconductor.

Breaking the thermoelectric (TE) trade-off relationship is an important task for maximizing the TE performance of polymeric semiconductors. Existing efforts have focused on designing high-mobility semiconductors and achieving ordered molecular doping, ignoring the critical role of the molecular orientation during TE conversion. Herein, the achievement of ZT to 0.40 is reported by fine-tuning the molecular orientation of one diketopyrrolopyrrole (DPP)-based polymer (DPP-BTz). Films with bimodal molecular orientation yield superior doping efficiency by increasing the lamellar spacing and achieve increased splitting between the Fermi energy and the transport energy to enhance the thermopower. These factors contribute to the simultaneous improvement in the Seebeck coefficient and electrical conductivity in an unexpected manner. Importantly, the bimodal film exhibits a maximum power factor of up to 346 µW m-1 K-2 , >400% higher than that of unimodal films. These results demonstrate the great potential of molecular orientation engineering in polymeric semiconductors for developing state-of-the-art organic TE (OTE) materials.

Advances in materials and devices for mimicking sensory adaptation.

Adaptive devices, which aim to adjust electrical behaviors autonomically to external stimuli, are considered to be attractive candidates for next-generation artificial perception systems. Compared with typical electronic devices with stable signal output, adaptive devices possess unique features in exhibiting dynamic fitness to varying environments. To meet this requirement, increasing efforts have been made focusing on developing new materials, functional interfaces and novel device geometry for sensory perception applications. In this review, we summarize the recent advances in materials and devices for mimicking sensory adaptation. Keeping this in mind, we first introduce the fundamentals of biological sensory adaptation. Thereafter, the recent progress in mimicking sensory adaptation, such as tactile and visual adaptive systems, is overviewed. Moreover, we suggest five strategies to construct adaptive devices. Finally, challenges and perspectives are proposed to highlight the directions that deserve focused attention in this flourishing field.

An Oligonucleotide-Distortion-Responsive Organic Transistor for Platinum-Drug-Induced DNA-Damage Detection.

Organic transistor with DNA-damage evaluation ability can open up novel opportunities for bioelectronic devices. Even though trace amounts of drugs can cause cumulative gene damage in vivo, the extremely low occurrence proportion makes them hardly transduced into detectable electric signals. Here, an ultrasensitive DNA-damage sensor based on an oligonucleotide-distortion-responsive organic transistor (DROT) is reported by creating controllable conformation change of double-stranded DNA on the surface of organic semiconductors. In combination with interfacial charge redistribution and efficient signal amplification, the DROT provides an ultrasensitive single-site DNA-damage response with 20.5 s even upon 1 × 10-12 m cisplatin. The high generalizability of this DROT to three generations of classical platinum drugs and gene-relevant DNA damage is demonstrated. A biochip is further designed for intelligent damage analysis in complex environments, which holds the potential for high-throughput biotoxicity evaluation and drug screening in the future.

Encapsulation and release of living tumor cells using hydrogels with the hybridization chain reaction.

Circulating tumor cells (CTCs) enable noninvasive liquid biopsy and identification of cancer. Various approaches exist for the capture and release of CTCs, including microfluidic methods and those involving magnetic beads or nanostructured solid interfaces. However, the concomitant cell damage and fragmentation that often occur during capture make it difficult to extensively characterize and analyze living CTCs. Here, we describe an aptamer-trigger-clamped hybridization chain reaction (atcHCR) method for the capture of CTCs by porous 3D DNA hydrogels. The 3D environment of the DNA networks minimizes cell damage, and the CTCs can subsequently be released for live-cell analysis. In this protocol, initiator DNAs with aptamer-toehold biblocks specifically bind to the epithelial cell adhesion molecule (EpCAM) on the surface of CTCs, which triggers the atcHCR and the formation of a DNA hydrogel. The DNA hydrogel cloaks the CTCs, facilitating quantification with minimal cell damage. This method can be used to quantitively identify as few as 10 MCF-7 cells in a 2-µL blood sample. Decloaking of tumor cells via gentle chemical stimulus (ATP) is used to release living tumor cells for subsequent cell culture and live-cell analysis. We also describe how to use the protocol to encapsulate and release cells of cancer cell lines, which can be used in preliminary experiments to model CTCs. The whole protocol takes ~2.5 d to complete, including downstream cell culture and analysis.

Programming bulk enzyme heterojunctions for biosensor development with tetrahedral DNA framework.

Protein-protein interactions are spatially regulated in living cells to realize high reaction efficiency, as seen in naturally existing electron-transfer chains. Nevertheless, arrangement of chemical/biochemical components at the artificial device interfaces does not possess the same level of control. Here we report a tetrahedral DNA framework-enabled bulk enzyme heterojunction (BEH) strategy to program the multi-enzyme catalytic cascade at the interface of electrochemical biosensors. The construction of interpenetrating network of BEH at the millimeter-scale electrode interface brings enzyme pairs within the critical coupling length (CCL) of ~10 nm, which in turn greatly improve the overall catalytic cascade efficiency by ~10-fold. We demonstrate the BEH generality with a range of enzyme pairs for electrochemically detecting clinically relevant molecular targets. As a proof of concept, a BEH-based sarcosine sensor enables single-step detection of the metabolic biomarker of sarcosine with ultrasensitivity, which hold the potential for precision diagnosis of early-stage prostate cancer.

Stepping gating of ion channels on nanoelectrode via DNA hybridization for label-free DNA detection.

Natural ion channels on cell membrane can gate the transport of ions and molecules by the conformational alteration of transmembrane proteins to regulate the normal physiological activities of cells. Inspired by the similarity of the conformation change under specific stimuli, here we introduce an ion channel gating model on a single nanoelectrode by anchoring DNA-gated switches on the very nanotip of gold nanoelectrode to mimic the response-to-stimulus behaviors of ion channels on bio-membranes. The surface-tethered DNA ion channels can be switched on by the Watson-Crick base pairing, which can alter the conformation of the tethered DNA from lying state to upright state. And these conformational alterations of the anchored DNA switches can effectively gate the transport of potassium ferricyanide onto the electrode interface. By continuously initiating the gates with DNA of different concentrations, we achieved the stepping gating of ion channels on a single nanoelectrode. Further, we demonstrated that the ion gating system on nanoelectrode showed excellent sensing performance. For example, the response kinetic was very fast with the signal saturation time of ~1 min, the reproducibility of the OFF/ON switch was robust enough to sustain for two cycles, and simultaneously, the specificity was high enough to distinguish complementary DNA and noncomplementary DNA. When used for label-free DNA detection, the limit of detection can be as low as 10 pM. This study provides a promising avenue to achieve label free and real-time detection of multiple biomolecules.

Molecular Threading-Dependent Mass Transport in Paper Origami for Single-Step Electrochemical DNA Sensors.

Molecular transport controls the efficiency of complex biological network systems such as cellular signaling system and cascade biomedical reaction. However, device fabrication for molecular sensing is often restricted by a low transport efficiency and complicated processing. Here, we report a molecular threading-dependent transport system using three-dimensional (3D) paper origami enabling the directional transport of biomolecules. We demonstrate that framework nucleic acid-based interface engineering allows orthogonal molecular recognition and enzymatic reaction with programmed order on site. We thus develop a single-step electrochemical DNA sensor for quantitative analysis with 1 picomolar sensitivity within 60 min. Our sensor can discriminate a mismatched target at the level of a single base mismatch. Our study shows a great potential toward the development of a biomimetic molecular transport system for point-of-care and precision diagnosis.

Plasmon Coupling Effect-Enhanced Imaging of Metal Ions in Living Cells Using DNAzyme Assembled Core-Satellite Structures.

We demonstrate a core-satellite plasmonic nanoprobe assembled via metal-ion-dependent DNA-cleaving DNAzyme linker for imaging intercellular metal ion based on plasmon coupling effect at a single-particle level. As metal ions are present in the system, the DNAzyme linker will be cleaved, and thus, disassembly of the core-satellite nanoprobes occurs, which results in distinct blue shift of the scattering spectra of Au core-satellite probes and naked color change of the scattering light. This change in scattering spectra has been supported by theoretical simulations. As a proof of concept, sensitive detection of Cu2+ with a limit of detection down to 67.2 pM has been demonstrated. The nanoprobes have been further utilized for intracellular Cu2+ imaging in living cells. The results demonstrate that the present strategy provides a promising platform for detection and imaging of metal ions in living cells and could be potentially applied to imaging other interesting target molecules simply by substituting the oligonucleotide sequence.

DNA Nanotechnology-Enabled Interfacial Engineering for Biosensor Development.

Biosensors represent biomimetic analytical tools for addressing increasing needs in medical diagnosis, environmental monitoring, security, and biodefense. Nevertheless, widespread real-world applications of biosensors remain challenging due to limitations of performance, including sensitivity, specificity, speed, and reproducibility. In this review, we present a DNA nanotechnology-enabled interfacial engineering approach for improving the performance of biosensors. We first introduce the main challenges of the biosensing interfaces, especially under the context of controlling the DNA interfacial assembly. We then summarize recent progress in DNA nanotechnology and efforts to harness DNA nanostructures to engineer various biological interfaces, with a particular focus on the use of framework nucleic acids. We also discuss the implementation of biosensors to detect physiologically relevant nucleic acids, proteins, small molecules, ions, and other biomarkers. This review highlights promising applications of DNA nanotechnology in interfacial engineering for biosensors and related areas.

Highly Efficient Capture and Electrochemical Release of Circulating Tumor Cells by Using Aptamers Modified Gold Nanowire Arrays.

The effective capture and release of circulating tumor cells (CTCs) is of significant importance in cancer prognose and treatment. Here we report a highly efficient method to capture and release human leukemic lymphoblasts (CCRF-CEM) using aptamers modified gold nanowire arrays (AuNWs). The gold nanowires, showing tunable morphologies from relatively random pillar deposit to relatively uniform arrays, were fabricated by electrochemical deposition using anodic aluminum oxide (AAO) as template. Upon simply being modified with aptamers by Au-S chemistry, the AuNWs exhibit higher specificity to target cells. Also compared to flat gold substrate, the AuNWs with nanostructure can capture target cells with much higher capture yield. Moreover, the captured CCRF-CEM cells can be released from AuNWs efficiently with little damage through an electrochemical desorption process. We predict that our strategy has great potential in providing a simple and economical platform for CTCs isolation, cancer diagnosis, and therapy.

DNA Hydrogel with Aptamer-Toehold-Based Recognition, Cloaking, and Decloaking of Circulating Tumor Cells for Live Cell Analysis.

Circulating tumor cells (CTCs) contain molecular information on the primary tumor and can be used for predictive cancer diagnostics. Capturing rare live CTCs and their quantification in whole blood remain technically challenging. Here we report an aptamer-trigger clamped hybridization chain reaction (atcHCR) method for in situ identification and subsequent cloaking/decloaking of CTCs by porous DNA hydrogels. These decloaked CTCs were then used for live cell analysis. In our design, a DNA staple strand with aptamer-toehold biblocks specifically recognizes epithelial cell adhesion molecule (EpCAM) on the CTC surface that triggers subsequent atcHCR via toehold-initiated branch migration. Porous DNA hydrogel based-cloaking of single/cluster of CTCs allows capturing of living CTCs directly with minimal cell damage. The ability to identify a low number of CTCs in whole blood by DNA hydrogel cloaking would allow high sensitivity and specificity for diagnosis in clinically relevant settings. More significantly, decloaking of CTCs using controlled and defined chemical stimuli can release living CTCs without damages for subsequent culture and live cell analysis. We expect this liquid biopsy tool to open new powerful and effective routes for cancer diagnostics and therapeutics.

Sensitive assay of protease activity on a micro/nanofluidics preconcentrator fused with the fluorescence resonance energy transfer detection technique.

A fast and sensitive assay of protease activity on a micro/nanofluidics preconcentrator combining with fluorescence resonance energy transfer (FRET) detection technique has been developed in a homogeneous real-time format. First, the functionalized nanoprobes are formed by loading dye labeled protein onto gold nanoparticles (AuNPs), in which, the photoluminescence of donor dye was strongly quenched by AuNPs due to FRET mechanisms. For protease activity assay, the nanoprobes are enriched by a micro/nanofluidics preconcentrator. When the target protease is transported to the enriched nanoprobes, cleavage of protein occurs as a consequence of molecular recognition of enzyme to substrate. The release of cleavage fragments from AuNPs nanoprobes leads to the enhancement of fluorescence and enables the protease activity assay on the micro/nanofluidics chip. As a demonstration, digestion of fluorescein isothiocyanate labeled dog serum albumin (FITC-DSA) by trypsin was used as a model reaction. Because of the highly efficient preconcentration and space confinement effect, significantly increased protein cleavage rate and protease assay sensitivity can be achieved with enhanced enzyme activity. The present micro/nanofluidics platform fused with the FRET detection technique is promising for fast and sensitive bioanalysis such as immunoassay, DNA hybridization, drug discovery, and clinical diagnosis.

Insights into the "free state" enzyme reaction kinetics in nanoconfinement.

The investigation of enzyme reaction kinetics in nanoconfined spaces mimicking the conditions in living systems is of great significance. Here, a nanofluidics chip integrated with an electrochemical detector has been designed for studying "free state" enzyme reaction kinetics in nanoconfinement. The nanofluidics chip is fabricated using the UV-ablation technique developed in our group. The enzyme and substrate solutions are simultaneously supplied from two single streams into a nanochannel through a Y-shaped junction. The laminar flow forms in the front of the nanochannel, then the two liquids fully mix at their downstream where a homogeneous enzyme reaction occurs. The "free state" enzyme reaction kinetics in nanoconfinement can thus be investigated in this laminar flow based nanofluidics device. For demonstration, glucose oxidase (GOx) is chosen as the model enzyme, which catalyzes the oxidation of beta-d-glucose. The reaction product hydrogen peroxide (H2O2) can be electrochemically detected by a microelectrode aligning to the end of nanochannel. The steady-state electrochemical current responding to various glucose concentrations is used to evaluate the activity of the "free state" GOx under nanoconfinement conditions. The effect of liquid flow rate, enzyme concentration, and nanoconfinement on reaction kinetics has been studied in detail. Results show that the "free state" GOx activity increases significantly compared to the immobilized enzyme and bath system, and the GOx reaction rate in the nanochannel is two-fold faster than that in bulk solution, demonstrating the importance of "free state" and spatial confinement for the enzyme reaction kinetics. The present approach provides an effective method for exploiting the "free state" enzyme activity in nanospatial confinement.

Rapid protein concentration, efficient fluorescence labeling and purification on a micro/nanofluidics chip.

Fluorescence analysis has proved to be a powerful detection technique for achieving single molecule analysis. However, it usually requires the labeling of targets with bright fluorescent tags since most chemicals and biomolecules lack fluorescence. Conventional fluorescence labeling methods require a considerable quantity of biomolecule samples, long reaction times and extensive chromatographic purification procedures. Herein, a micro/nanofluidics device integrating a nanochannel in a microfluidics chip has been designed and fabricated, which achieves rapid protein concentration, fluorescence labeling, and efficient purification of product in a miniaturized and continuous manner. As a demonstration, labeling of the proteins bovine serum albumin (BSA) and IgG with fluorescein isothiocyanate (FITC) is presented. Compared to conventional methods, the present micro/nanofluidics device performs about 10(4)-10(6) times faster BSA labeling with 1.6 times higher yields due to the efficient nanoconfinement effect, improved mass, and heat transfer in the chip device. The results demonstrate that the present micro/nanofluidics device promises rapid and facile fluorescence labeling of small amount of reagents such as proteins, nucleic acids and other biomolecules with high efficiency.

Mussel-inspired anchoring for patterning cells using polydopamine.

This Article introduces a simple method of cell patterning, inspired by the mussel anchoring protein. Polydopamine (PDA), artificial polymers made from self-polymerization of dopamine (a molecule that resembles mussel-adhesive proteins), has recently been studied for its ability to make modifications on surfaces in aqueous solutions. We explored the interfacial interaction between PDA and poly(ethylene glycol) (PEG) using microcontact printing (µCP). We patterned PDA on several substrates such as glass, polystyrene, and poly(dimethylsiloxane) and realized spatially defined anchoring of mammalian cells as well as bacteria. We applied our system in investigating the relationship between areas of mammalian nuclei and that of the cells. The combination of PDA and PEG enables us to make cell patterns on common laboratorial materials in a mild and convenient fashion.