Probe Access Research Articles

Pitfalls and challenges of peptide nucleic acid immobilisation on carbon surfaces for sequence-specific capturing of nucleic acid biomarkers

Nucleic acid sensors based on a peptide nucleic acid (PNA) probe have seen a surge in interest since their discovery in the 1990s, and after the patent protecting them expired in 2013. The appeal of PNA as capture and/or sensing probes as an alternative to standard DNA or RNA oligonucleotides originates from their superior chemical stability and affinity for complementary oligonucleotides, as well as their increased responsiveness to single base mismatches. The implementation of PNA probes onto optical and electrochemical sensors has showed great promise although progress has been hampered by issues mostly associated with surface chemistry, probe accessibility and non-specific binding. Herein, we report on a systematic comparison between various PNA immobilisation strategies on carbon substrates based on both covalent and non-covalent chemistries. Besides the use of standard electrochemical techniques to characterise the extent of surface modification, the ability of immobilised PNAs to engage in chemical interactions with freely diffusing molecules was also investigated. Using original chemical tags, this study provides a unique insight into the impact of immobilisation chemistries on PNA's (bio)availability. Rapid immobilisation of biotinylated PNA oligomers on screen-printed carbon electrode (SPCE) coated with adsorbed polystreptavidin (pSA) demonstrated highest efficiency and ease in the preparation process. An original nucleic acid sensor using this immobilisation chemistry is reported that is based on a sandwich assay between a surface bound PNA capture probe and a freely diffusing electrochemically active PNA sensing probe.

Revealing the Mechanism Underlying 3D-AFM Imaging of Suspended Structures by Experiments and Simulations.

The invention of 3D atomic force microscopy (3D-AFM) has enabled visualizing subnanoscale 3D hydration structures. Meanwhile, its applications to imaging flexible molecular chains have started to be experimentally explored. However, the validity and principle of such imaging have yet to be clarified by comparing experiments and simulations or cross-observations with an alternative technique. Such studies are impeded by the lack of an appropriate model. Here, this difficulty is overcome by fabricating 3D carbon nanotube (CNT) structures flexible enough for 3D-AFM, large enough for scanning electron microscopy (SEM), and simple enough for simulations. SEM and 3D-AFM observations of the same model provide unambiguous evidence to support the possibility of imaging overlapped nanostructures, such as suspended CNT and underlying platinum (Pt) nanodots. Langevin dynamics simulations of such 3D-AFM imaging clarify the imaging mechanism, where the flexible CNT is laterally displaced to allow the AFM probe access to the underlying structures. These results consistently show that 3D-AFM images are affected by the friction between the CNT and AFM nanoprobe, yet it can be significantly suppressed by oscillating the cantilever. This study reinforces the theoretical basis of 3D-AFM for imaging various 3D self-organizing systems in diverse fields, from life sciences to interface sciences.

Facile semisynthesis of histone H3 enables nucleosome probes for investigation of histone H3K79 modifications

Histone H3K79 modifications are essential to regulate chromatin structure and gene transcription, but understanding of the molecular mechanisms is limited. Because H3K79 is at globular domain, short histone peptide cannot mimic H3K79 in chromatin. Instead, reconstituted nucleosome-based chemical tools are ideally used to investigate H3K79 modifications. In consequence, H3K79-modified histone H3 with additional chemical handles are required, but such synthesis is challenging and laborious. Here we report a facile semisynthesis method that enables multifunctional histone H3 readily available. H3K79-containing fragment is short for straight peptide synthesis that was later ligated to recombinant expressed H3 fragments for full-length product in large scale. As a result, nucleosomes with H3K79 modifications as well as photo-reactive group and affinity tag were obtained to investigate potential binding proteins. We believe this method that enhances synthetic accessibility of nucleosome probes will accelerate understanding of the underexplored H3K79 modifications.

Fibronectin Conformations after Electrodeposition onto 316L Stainless Steel Substrates Enhanced Early-Stage Osteoblasts' Adhesion but Affected Their Behavior.

The implantation of metallic orthopedic prostheses is increasingly common due to an aging population and accidents. There is a real societal need to implement new metal implants that combine durability, good mechanical properties, excellent biocompatibility, as well as affordable costs. Since the functionalization of low-cost 316L stainless steel substrates through the successive electrodeposition of a polypyrrole film (PPy) and a calcium phosphate deposit doped with silicon was previously carried out by our labs, we have also developed a bio-functional coating by electrodepositing or oxidating of fibronectin (Fn) coating. Fn is an extracellular matrix glycoprotein involved in cell adhesion and differentiation. Impacts of either electrodeposition or oxidation on the structure and functionality of Fn were first studied. Thus, electrodeposition is the technique that permits the highest deposition of fibronectin, compared to adsorption or oxidation. Furthermore, electrodeposition seems to strongly modify Fn conformation by the formation of intermingled long fibers, resulting in changes to the accessibility of the molecular probes tested (antibodies directed against Fn whole molecule and Fn cell-binding domain). Then, the effects of either electrodeposited Fn or oxidized Fn were validated by the resulting pre-osteoblast behavior. Electrodeposition reduced pre-osteoblasts' ability to remodel Fn coating on supports because of a partial modification of Fn conformation, which reduced accessibility to the cell-binding domain. Electrodeposited Fn also diminished α5 integrin secretion and clustering along the plasma membrane. However, the N-terminal extremity of Fn was not modified by electrodeposition as demonstrated by Staphylococcus aureus attachment after 3 h of culture on a specific domain localized in this region. Moreover, the number of pre-osteoblasts remains stable after 3 h culture on either adsorbed, oxidized, or electrodeposited Fn deposits. In contrast, mitochondrial activity and cell proliferation were significantly higher on adsorbed Fn compared with electrodeposited Fn after 48 h culture. Hence, electro-deposited Fn seems more favorable to pre-osteoblast early-stage behavior than during a longer culture of 24 h and 48 h. The electrodeposition of matrix proteins could be improved to maintain their bio-activity and to develop this promising, fast technique to bio-functionalize metallic implants.

Constant probe orientation for fast contact-based inspection of 3D free-form surfaces using (3+2)-axis inspection machines

A new probe optimization method for contact based (3+2)-axis inspection machines is proposed. Given an inspection path of a stylus on a free-form surface, an optimal orientation of the stylus is computed such that (i) the inclination angle of the stylus is within a given angular range with respect to the surface normal, (ii) the motion of the stylus is globally collision free, and (iii) the stylus remains constant in the coordinate system of the measuring machine. The last condition guarantees that the inspection motion requires only the involvement of the three translational axes of the measuring machine. The numerical simulations were validated through physical experiments on a testcase of a tooth of a bevel gear due to the surface complexity and probe accessibility. This optimized method was compared to 3-axis and 5-axis inspection strategies, showing that the fixed (3+2)-axis stylus returns more accurate inspection results compared to the traditional 3-axis approach and similar to 5-axis approach.

Development and validation of a radiofrequency ablation treatment planning system for vertebral metastases.

Bone-targeted radiofrequency ablation (RFA) is widely used in the treatment of vertebral metastases. While radiation therapy utilizes established treatment planning systems (TPS) based on multimodal imaging to optimize treatment volumes, current RFA of vertebral metastases has been limited to qualitative image-based assessment of tumour location to direct probe selection and access. This study aimed to design, develop and evaluate a computational patient-specific RFA TPS for vertebral metastases. A TPS was developed on the open-source 3D slicer platform, including procedural setup, dose calculation (based on finite element modelling), and analysis/visualization modules. Usability testing was carried out by 7 clinicians involved in the treatment of vertebral metastases on retrospective clinical imaging data using a simplified dose calculation engine. In vivo evaluation was performed in a preclinical porcine model (n = 6 vertebrae). Dose analysis was successfully performed, with generation and display of thermal dose volumes, thermal damage, dose volume histograms and isodose contours. Usability testing showed an overall positive response to the TPS as beneficial to safe and effective RFA. The in vivo porcine study showed good agreement between the manually segmented thermally damaged volumes vs. the damage volumes identified from the TPS (Dice Similarity Coefficient = 0.71 ± 0.03, Hausdorff distance = 1.2 ± 0.1mm). A TPS specifically dedicated to RFA in the bony spine could help account for tissue heterogeneities in both thermal and electrical properties. A TPS would enable visualization of damage volumes in 2D and 3D, assisting clinicians in decisions about potential safety and effectiveness prior to performing RFA in the metastatic spine.

Engineering of Interfaces with Tetrahedra DNA Nanostructures for Biosensing Applications

AbstractThe probe‐target interactions in the interfaces are significantly critical to biosensing. However, the disordered arrangement of probes and nonspecific adsorption of proteins in the biosensing interfaces for conventional biosensors often restricted the accessibility and recognition efficiency of probes towards targets, leading to poor detection performances (e. g., sensitivity and selectivity). Engineering of biosensing interfaces with functional molecules or nanomaterials has provided a promising molecular toolkit for enhanced accessibility and efficient recognition of biosensing probes. Among them, DNA has been an appealing material for interface engineering, because of its unique merits of biocompatible, predictable hybridization, and unparallel self‐assembly ability. In particular, employing tetrahedra DNA nanostructures (TDNs) to engineer interfaces has been a powerful means to improve biosensor performance. Here, this review introduces the recent progress in TDN‐based interface engineering. Then, we summarize the roles of TDNs in tailoring the properties of different interfaces, including electrode surface, channel surface, cell surface, etc., and highlight their biosensing applications. Finally, scientific challenges and future perspectives of TDN‐engineered biosensing interface are also discussed.

Annular Dump Diffuser and Deswirl System for Back-Pressure Control in Engine-Scale Transonic Annular Cascade

Abstract A common requirement for turbomachinery testing facilities is the ability to independently control Mach number and Reynolds number. In practice, this means independent control of the inlet total pressure and exit static pressure in a test facility. In this paper, we present a solution to this problem with particular applicability to large-scale annular test facilities. We describe the design and commissioning of a combined annular dump diffuser and deswirl system for back-pressure control in environments with high-whirl transonic flow. The particular application was an annular cascade of nozzle guide vanes from a current civil engine. The purpose was to provide independent control of Mach number and Reynolds number, by controlling the back-pressure in the intermediate annular plenum which forms the dump diffuser. The dump diffuser is necessary to facilitate optical and probe access (without interaction effects) and to reduce the risk of exit static pressure disturbances (due to particular duct design). The system has been installed and validated in the engine component aerothermal (ECAT) facility at the University of Oxford. In this implementation of the system, the high-whirl transonic flow from the nozzle guide vanes passes through a short, parallel annular duct, and is dumped into an annular plenum, before being re-accelerated into a deswirl vane ring. The deswirl ring turns the flow to the axial direction. The flow is then discharged through a variable choke plate into a silencer. Conditioning the flow to have zero whirl at the choke plate reduces the sensitivity of the choke plate effective blockage to the whirl angle. The design, deswirl vane aerodynamic performance, and overall system performance are assessed with detailed experiments and 3D unsteady computational fluid dynamics predictions. The control of high-whirl transonic flow is notoriously challenging, and the deswirl system has application to exhaust conditioning in a number of applications including annular cascade experiments and rocket turbo-pump exhaust systems.

Plasma drilling on Martian ice: Enabling efficient deep subsurface access to Mars' polar layered deposits

Mars' polar layered deposits (PLD) contain the best historical record of the Amazonian time period. Deep subsurface access at Mars' poles is challenging due to the restriction of Mars' atmospheric condition. Traditional terrestrial mechanical drilling methods using drilling fluids are not feasible in these conditions and the melting probe approach is very inefficient due to heat loss. For ice in Mars' polar condition, the increase in surface area due to cracks would greatly reduce its thermal conductivity. This will lead to a higher thermal efficiency and an easier access to polar layered deposits during melting or sublimation. The preliminary results with pulsed plasma ice drilling, using localized electric breakdown with electric energy input per pulse up to 80 ​J, were promising under both Mars and Earth conditions. To simulate Mars' polar condition, a vacuum vessel system was designed and implemented with a pulsed discharge circuit, ice plate, controlled liquid nitrogen cooling, and CO 2 gas feed-in. A simplified heat conduction model was established for estimating the heat conduction at various porosities for homogenous water ice and CO 2 gas mixture using COMSOL under two scenarios including constant thickness of mixture layer and constant thermal mass of the mixture layer under the heat source. The simulation results for both scenarios indicated more reduction of heat loss with an increasing porosity. Experimental studies of thermal conductivity measurement of granular ice under the Earth condition inside a freezer using a custom thermal needle probe with known porosity proved the concept that voids/cracks in ice can reduce the thermal conductivity. Based on simulation and experimental results, the classic melting probe model is extended with porous ice-CO 2 mixture to estimate the power consumption on Martian ice at varying descending speed. Plasma drilling with limited power is promising for accessing Mars' PLD and can also be expanded to planetary exploration in searching for life on other planets. • Plasma drilling method has no moving parts. • Pulsed plasma drilling improves the Rate of Penetration of the melting probe. • The cracks/voids in ice decreases its equivalent thermal conductivity. • Additional power requirement is acceptable within limited solar power access for Martian probe.

Spatial release from masking in anechoic and reverberant environments

Listening with both ears provides children with access to binaural and monaural cues that are helpful for understanding speech in competing babbles. Specifically, when the target and masker are spatially separated, children can gain an intelligibility benefit which is known as spatial release from masking (SRM). Recent work [Peng et al., 2021 JASA] suggested that school-age children demonstrated immature SRM using binaural cues that are distorted by reverberation. In this follow-up study, we further investigate the effect of reverberant distortion on individual auditory spatial cues, namely binaural and monaural head shadow cues. We compare SRM between adults and school-age children with typical hearing using the novel measure of minimum angle of separation (MAS) between target and masker, for which individual achieves a 20% intelligibility gain, in both virtually simulated anechoic and reverberant environments. MAS was measured in both binaural and monaural hearing conditions, as well asymmetric versus asymmetric masker displacement to probe access to various auditory cues of interest. Preliminary results show that adults demonstrate similar SRM in anechoic and in reverberation of T60 = ∼0.6 s, but better (smaller MAS) when additional spatial cues become available. Reverberation effect on children’s SRM will be discussed. [Work supported by NIH-NIDCD.]

Improved Methods for Single-Molecule Fluorescence In Situ Hybridization and Immunofluorescence in Caenorhabditis elegans Embryos.

Visualization of gene products in Caenorhabditis elegans has provided insights into the molecular and biological functions of many novel genes in their native contexts. Single-molecule fluorescence in situ hybridization (smFISH) and immunofluorescence (IF) enable the visualization of the abundance and localization of mRNAs and proteins, respectively, allowing researchers to ultimately elucidate the localization, dynamics, and functions of the corresponding genes. Whereas both smFISH and immunofluorescence have been foundational techniques in molecular biology, each protocol poses challenges for use in the C. elegans embryo. smFISH protocols suffer from high initial costs and can photobleach rapidly, and immunofluorescence requires technically challenging permeabilization steps and slide preparation. Most importantly, published smFISH and IF protocols have predominantly been mutually exclusive, preventing the exploration of relationships between an mRNA and a relevant protein in the same sample. Here, we describe protocols to perform immunofluorescence and smFISH in C. elegans embryos either in sequence or simultaneously. We also outline the steps to perform smFISH or immunofluorescence alone, including several improvements and optimizations to existing approaches. These protocols feature improved fixation and permeabilization steps to preserve cellular morphology while maintaining probe and antibody accessibility in the embryo, a streamlined, in-tube approach for antibody staining that negates freeze-cracking, a validated method to perform the cost-reducing single molecule inexpensive FISH (smiFISH) adaptation, slide preparation using empirically determined optimal antifade products, and straightforward quantification and data analysis methods. Finally, we discuss tricks and tips to help the reader optimize and troubleshoot individual steps in each protocol. Together, these protocols simplify existing workflows for single-molecule RNA and protein detection. Moreover, simultaneous, high-resolution imaging of proteins and RNAs of interest will permit analysis, quantification, and comparison of protein and RNA distributions, furthering our understanding of the relationship between RNAs and their protein products or cellular markers in early development. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Sequential immunofluorescence and single-molecule fluorescence in situ hybridization Alternate Protocol: Abbreviated protocol for simultaneous immunofluorescence and single-molecule fluorescence in situ hybridization Basic Protocol 2: Simplified immunofluorescence in C. elegans embryos Basic Protocol 3: Single-molecule fluorescence in situ hybridization or single-molecule inexpensive fluorescence in situ hybridization.

A simplified cascade‐based de‐embedding technique for on‐wafer microwave characterization of thin‐film resistors and transmission lines

AbstractA cascade‐based de‐embedding technique is presented for microwave characterization of thin‐film resistors and transmission lines. Parasitic effects of the probe pads, on‐chip interconnects and access via holes can be directly extracted using two devices under test without resorting to any additional Open/Thru dummy structures. The proposed matrix transformation provides a simplified de‐embedding methodology with a high precision similar to the conventional approaches using multiple dummy structures. The validity of the proposed technique has been demonstrated for GaN thin‐film resistors up to 65 GHz.

Touch Probe Measurement in Dimensional Metrology: A Review

Coordinate measuring machines (CMMs) are the standard displacement systems used for measurements in dimensional metrology. Since measurement with a touch probe mounted on a CMM provides high accuracy, repeatability, and reliability, it has been widely used for mechanical part inspection in manufacturing. The inspection process requires the use of several sensor orientations and optimal positioning of the part in order to measure all features. Recently, the field of probing path planning has become a huge and active research field. In this paper, various techniques aimed at generating the probe paths for part inspection are reviewed. Multiple issues related to the positioning of the part to maximise accessibility, analysis of probe accessibility to measure all inspection features, optimisation of the measurement sequence, distribution of measurement points, and collision avoidance are mentioned. The common research approaches and potential algorithms in this field are also discussed in this paper.

An optimized fixation method containing glyoxal and paraformaldehyde for imaging nuclear bodies.

The mammalian cell nucleus contains different types of membrane-less nuclear bodies (NBs) consisting of proteins and RNAs. Microscopic imaging has been widely applied to study the organization and structure of NBs. However, current fixation methods are not optimized for such imaging: When a fixation method is chosen to maximize the quality of the RNA fluorescence in situ hybridization (FISH), it often limits the labeling efficiency of proteins or affects the ultrastructure of NBs. Here, we report that addition of glyoxal (GO) into the classical paraformaldehyde (PFA) fixation step not only improves FISH signals for RNAs in NBs via augmented permeability of the fixed nucleus and enhanced accessibility of probes, but also largely preserves protein fluorescent signals during fixation and immunostaining. We also show that GO/PFA fixation enables the covisualization of different types of nuclear bodies with minimal impact on their ultrastructures under super-resolution microscopy.

Fabrication of a high-speed plasmonic reflection/transmission modulator

The fabrication of a high-speed plasmonic reflection/transmission modulator for operation at λ0 = 1550 nm is presented and described in detail. Front-side ground and signal contacts provide easy electrical probe access to the device, while allowing the transmission of light through the substrate. Modulation is based on enhanced perturbation of the effective refractive index of grating-coupled surface plasmon polaritons propagating along a metal–oxide–semiconductor structure on silicon. Fabrication steps include deposition of a plasmonic metal patch, deposition of Ohmic contacts, deposition of an Au nanograting coupler overlaid by e-beam lithography, and the application of an intermetal dielectric layer with metalized vias and metal electrical contacts. Current–voltage and capacitance–voltage characteristics verify the electrical integrity of the structure.

Using infrared thermometry to improve irrigation scheduling on variable soils

An experiment was conducted to assess a modified Priestley and Taylor calculation (developed in a companion paper) that utilises surface temperature (Ts) to estimate evapotranspiration (ETMPT) and to assess the benefits of variable rate irrigation (VRI) based on ETMPT measurements. A ryegrass seed field with Low, Mid and High available water capacity soil zones was used. Four replicates of two irrigation treatments were established in each zone; uniform rate irrigation (URI) where the same amount of irrigation was applied to all three zones based on neutrone probe measurements; and VRI where ETMPT was measured and different amounts of irrigation applied to each zone to replace this. TS was measured continiously in each plot using stationary infrared radiometers, normalised difference vegetation index measured twice weekly with a hand-held instrument and meteorological variables were recorded in the field. Neutron probe access tubes were installed to 1.6m depth in all plots and monitored weekly to provide alternative evapotranspiration estimates (ETNP). The ETMPT decreased from 333mm in the high zone to 282 mm in the low zone. This in combination with faster canopy senescence and lower seed yields showed the low zone encountered water stress even though irrigated weekly. There was good agreement between ETMPT and ETNP in the VRI treatments and the High zone of the URI treatment. The higher values of ETNP in the Low and Med zones of the URI treatment were attributed to drainage. The URI treatment had 254.2 mm of irrigation applied to all zones during the season whereas VRI treatment had 183.4, 210.2 and 220.2 mm applied to the Low, Mid and High Zones, respectively. There, were no differences in ryegrass seed yield between the two irrigaiton treatments and less drainage from the VRI treatments. From this we conclude that VRI based on ETMPT is effective and field application of ETMPT is worth further development.

Analysis of Surgical Forces Required to Gain Access Using a Probe for Minimally Invasive Spine Surgery via Cadaveric-Based Experiments Towards Use in Training Simulators.

Virtual Reality haptic-based surgical simulators for training purposes have recently been receiving increased traction within the medical field. However, its future adoption is contingent on the accuracy and reliability of the haptic feedback. This study describes and analyzes the implementation of a set of haptic-tailored experiments to extract the force feedback of a medical probe used in minimally invasive spinal lumbar interbody fusion surgeries. Experiments to extract linear, lateral and rotational insertion, relaxation and extraction of the tool within the spinal muscles, intervertebral discs and lumbar nerve on two cadaveric torsos were conducted. Notably, mean force-displacement and torque-angular displacement curves describing the different tool-tissue responses were reported with a maximum force of 6.87 (±1.79) N at 40 mm in the muscle and an initial rupture force through the Annulus Fibrosis of 20.550 (±7.841) N at 6.441 mm in the L4/L5 disc. The analysis showed that increasing the velocity of the probe slightly reduced and delayed depth of the muscle punctures but significantly lowered the force reduction due to relaxation. Decreasing probe depth resulted with a reduction to the force relaxation drop. However, varying the puncturing angle of attack resulted with a significant effect on increasing force intensities. Finally, not resecting the thoracolumbar fascia prior to puncturing the muscle resulted with a significant increase in the force intensities. These results present a complete characterization of the input required for probe access for spinal surgeries to provide an accurate haptic response in training simulators.

Interplay of electrostatic repulsion and surface grafting density on surface-mediated DNA hybridization

Single-molecule Förster Resonance Energy Transfer was used to observe the adsorption of fluorescently-labeled “target” DNA oligonucleotides and their association and hybridization with complementary DNA “probes” tethered to the surface as a function of surface grafting density. Ionic strength was varied systematically to disentangle the potentially competing effects of probe accessibility and electrostatic repulsion. At high ionic strength, when the Debye length was ~1 nm, the adsorption of target DNA was not significantly inhibited by the presence of tethered probe DNA, even at high grafting density, and the fraction of adsorbed target strands undergoing hybridization increased systematically with grafting density, leading to a dramatic increase in the net hybridization rate at high grafting density. However, at lower ionic strength, when the Debye length was ≥3 nm, the adsorption rate of target DNA decreased and the fraction of adsorbed target strands undergoing hybridization saturated at high probe grafting density (≥7,000 strands/µm2), presumably due to electrostatic repulsion. As a result, the net rate of hybridization exhibited a maximum as a function of grafting density. This has important consequences for the design of systems that optimize surface-mediated DNA hybridization under low-salt high-stringency conditions.

Miniaturisation of a peptide-based electrochemical protease activity sensor using platinum microelectrodes.

Proteases are ideal target biomarkers as they have been implicated in many disease states, including steps associated with cancer progression. Electrochemical peptide-based biosensors have attracted much interest in recent years. However, the significantly large size of the electrodes typically used in most of these platforms has led to performance limitations. These could be addressed by the enhancements offered by microelectrodes, such as rapid response times, improved mass transport, higher signal-to-noise and sensitivity, as well as more localised and less invasive measurements. We present the production and characterisation of a miniaturised electrochemical biosensor for the detection of trypsin, based on 25 μm diameter Pt microelectrodes (rather than the ubiquitous Au electrodes), benchmarked by establishing the equivalent Pt macroelectrode response in terms of quantitative response to the protease, the kinetics of cleavage and the effects of non-specific protein binding and temperature. Interestingly, although there was little difference between Au and Pt macroelectrode response, significant differences were observed between the responses of the Pt macroelectrode and microelectrode systems indicative of increased reproducibility in the microelectrode SAM structure and sensor performance between the electrodes, increased storage stability and a decrease in the cleavage rate at functionalised microelectrodes, which is mitigated by measurement at normal body temperature. Together, these results demonstrate the robustness and sensitivity of the miniaturised sensing platform and its ability to operate within the clinically-relevant concentration ranges of proteases in normal and disease states. These are critical features for its translation into implantable devices.

Encapsulation and Release of Recognition Probes Based on a Rigid Three-Dimensional DNA "Nanosafe-box" for Construction of a Electrochemical Biosensor.

Herein a rigid three-dimensional (3D) DNA "nanosafe-box" (DNB) for encapsulation and release of a recognition probe (N3) is designed to construct an electrochemical biosensor with the use of electroactive two-dimensional metal-organic framework (2D MOF) nanosheets as signal tags for ultrasensitive detection of mercury ion (Hg2+). Initially, N3 is locked in the 3D cavity of DNB by blocker DNA. After addition of target Hg2+, exonuclease III (Exo-III) digestion is initiated to the liberate DNA "key" (K); thereby, the free K triggers a strand displacement reaction for exposing the prelocked N3 to successfully ligate dibenzocyclooctyne (DBCO)-tagged anchor via metal-catalyst-free click chemistry, in which amounts of 2D MOF nanosheets containing Co(II) as electron mediator are introduced accompanied by significant electrochemical response. Compared with traditional linear or stem-loop DNA nanostructure, the well-designed 3D DNB possesses remarkably enhanced mechanical rigidity and structural stability, resulting in improved accessibility of probes and increased loading amounts of signal tags. More importantly, by this way of encapsulation and release of recognition probes, the background signal is decreased dramatically, leading to increased sensitivity of the proposed biosensor. Consequently, this electrochemical biosensor exhibits outstanding analytical performance for Hg2+ detection with a low detection limit of 33 fM and dynamic linear range of 0.1 pM to 10 nM. This strategy offers an ingenious method for detection of metal ions and biomarkers, possessing potential applications in environmental tests and clinical diagnosis.