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Protein SUMOylation is a kind of post-translational modification that closely involves numerous physiological and pathological processes. Because of the important role of SUMOylation in living cells, sensitivity characterization of SUMOylation is urgent for the understanding of SUMOylatin-mediated cellular function. Due to the low- expression of SUMOylated substrate in living cells, a sensitive and selective sensing platform for the assay SUMOylation modification events is highly desired. In this work, we implemented SUMOylation using purified p53 by using the proximity ligation assay (PLA) method. GST-tagged SUMO 1 was employed for the SUMOylation assay. Using the sensing platform, we obtain P53 SUMOylation with ultrasensitivity and selectivity, which equally 83 copy P53 in 1 μL solution. Other forms of SUMOylation are also amenable to profiling by this well-designed strategy. Our PLA sensing platform will also provide a more coherent referential experience on the cellular protein-protein interaction responses that mainly measure expression levels of individual proteins or protein-protein interactions.

Graphene has been widely used in photodetectors; however its photoresponsivity is limited due to the intrinsic low absorption of graphene. To enhance the graphene absorption, a waveguide structure with an extended interaction length and plasmonic resonance with light field enhancement are often employed. However, the operation bandwidth is narrowed when this happens. Here, a novel graphene-based all-fiber photodetector (AFPD) was demonstrated with ultrahigh responsivity over a full near-infrared band. The AFPD benefits from the gold-enhanced absorption when an interdigitated Au electrode is fabricated onto a Graphene-PMMA film covered over a side-polished fiber (SFP). Interestingly, the AFPD shows a photoresponsivity of >1 × 104 A/W and an external quantum efficiency of >4.6 × 106% over a broadband region of 980–1620 nm. The proposed device provides a simple, low-cost, efficient, and robust way to detect optical fiber signals with intriguing capabilities in terms of distributed photodetection and on-line power monitoring, which is highly desirable for a fiber-optic communication system.

Both the nonclose-packed structure and the large refractive index contrast of guanine nanocrystals and cytosols in iridophores play a vital role in the dynamic camouflage of chameleons, including the bright skin color and color tuning sensitivity to external stimulus. Here, the nonclose-packed photonic crystals consisting of ZnS nanospheres and polymers, which have similar refractive indices with guanine nanocrystals and cytosols, respectively, are constructed by a two-step filling strategy. ZnS@SiO2 nanospheres are self-assembled to build intermediate close-packed photonic crystals followed by filling polymers in their interstices. The nonclose-packed photonic crystal is successfully achieved when the silica portion is etched by HF solution and refilled by polymers. Excitingly, the stimulus response of the designed photonic crystal is as sensitive as the skin of chameleons due to the similar contrast of refractive indices and nonclose-packed structure. The reflection peak of the structure can blue-shift more than 200 nm as the temperature increases from 30 to 55 °C or under 20% compressional strain. This work not only builds the nonclose-packed photonic crystals by introducing a two-step filling strategy but also proves that high refractive contrast in photonic crystals is an effective strategy to achieve ultrasensitivity, which is highly desirable for various applications.

Background The ultrasensitive monitoring strategy of zearalenone (ZEN) is essential and desirable for food safety and human health. In the present study, a coupling of gold nanoparticles-DNA barcode and direct competitive immunoassay-based real-time polymerase chain reaction signal amplification (RT-IPCR) for ZEN close to the sensitivity of PCR-like levels is described and evaluated. Results The RT-IPCR benefited from the use of a DNA barcode and RT-PCR detection strategy, thus resulting in ultrasensitive and simple detection for ZEN. Under the optimal RT-IPCR, the linear range of detection was from 0.5 to 1000 pg mL-1 and the limit of detection was 0.5 pg mL-1 , which was 400-fold lower than the enzyme-linked immunosorbent assay. The detection procedure was simplified and the detection time was shortened. The specificity, accuracy and precision of the RT-IPCR confirmed a high performance. ZEN-positive contamination levels were from 0.056 to 152.12 ng g-1 by the RT-IPCR, which was demonstrated to be highly reliable by liquid chromatography-tandem mass spectrometry. Conclusion The proposed RT-IPCR could be used as an alternative for detecting ZEN with satisfactory ultrasensitivity, simplicity, low cost and high-throughput. The present study could provide a strategy for the ultrasensitive detection of the small molecule with a simple and practical approach, which has significant appeal and application prospects.

A flexible SERS chip fully covered with silver (Ag) nanoparticles is obtained using a one-step metal thermal deposition on Kapton tape, which exhibits ultrasensitivity, high-uniformity, and importantly long-term SERS stability for up to 4 months under polyethylene terephthalate (PET) film packaging. The practicality is further demonstrated using pesticide residue detection on fruit surfaces.

Inspired by the self-adaptive nature of living organisms, the development of stimuli-responsive polymers that can sense and respond to surrounding microenvironments has received increasing interest. To date, stimuli-responsive polymers have found promising applications in terms of smart coatings, actuators, photolithography, imaging and diagnosis, and so on. Notwithstanding significant progress, it remains a grand challenge to improve the sensitivity of stimuli-responsive polymers toward external stimuli, which is, however, highly desirable in practical applications when the input signals are very weak. To circumvent this problem, self-propagating polymers have been developed, which enable the amplification of input signals in a positive feedback manner, thereby exponentially elevating the sensitivity. In this minireview, we summarize the recent design strategies and potential applications of self-propagating polymers by integrating feedback signal amplification modules, which have shown promising applications ranging from fundamental research to industry.

Bacterial chemotaxis allows bacteria to sense the chemical environment and modulate their swimming behavior accordingly. Although the intracellular chemotaxis signaling pathway has been studied extensively, experimental studies are still lacking that could provide direct link from the pathway output (the intracellular concentration of the phosphorylated form of the response regulator phosphorylated CheY (CheY-P)) to single-cell swimming behavior. Here, we measured the swimming behavior of individual Escherichia coli cells while simultaneously detecting the intracellular CheY-P concentration, thereby providing a direct relationship between the intracellular CheY-P concentration and the single-cell run-and-tumble behavior. The measured relationship is consistent with the ultrasensitivity of the motor switch and a “veto model” that describes the interaction among individual flagella, although contribution from the voting mechanism could not be ruled out.

Organic semiconductor (OSC)-based gas detection has attracted considerable attention due to the facile manufacturing process and effective contact with target chemicals at room temperature. However, OSCs intrinsically suffer from inferior sensing and recovery capability due to lack of functional sites and deep gas penetration into the film. Here, we describe an interpenetrating polymer semiconductor nanonetwork (IPSN) channel possessing unreacted silanol (Si-OH) groups on its surface to overcome bottlenecks that come from OSC-based chemodetection. On the top of the IPSN, moreover, we introduced electron-donating amine (NH2) groups as a chemical receptor because they strongly interact with the electron-withdrawing nature of NO2 gas. The NH2-IPSN-based field-effect transistor exhibited high-performance chemodetection such as ultrasensitivity (990% ppm-1 at 5 ppm) and excellent NO2 selectivity against other toxic gases. Impressively, the gas recovery was significantly improved because the NH2 chemical receptors anchored on the surface of the IPSN suppress deep gas penetration into the film. This work demonstrates that our NO2 chemodetection is expected to provide inspiration and guideline for realization of practical gas sensors in various industries and daily life.

Surface-enhanced Raman resonant scattering (SERRS) tags encoded with near-infrared (NIR) Raman reporters showed great potential for in vivo detection owing to their ultrasensitivity. However, in vivo signal stability of such tags is a remaining problem due to the lack of suitable silica coating method because the weakly adsorbed NIR reporters tend to detach from traditional gold nanosubstrates in the ethanol-rich and high pH conditions, which are commonly used for silica coating. Herein, we propose a silica coating method for NIR SERRS tags by using waxberry-like gold nanoparticles (NPs) as substrates. The lipid bilayer of the NPs played a crucial role in the coating, which can encapsulate the NIR Raman reporter via hydrophobic interactions and prevent the interference from a harsh medium. Thus, the silica-coated tags well preserved ultrasensitivity of bare tags and simultaneously gained satisfactory signal stability in vivo. Moreover, the coating method is compatible for the encapsulation of a variety of thiol group-free NIR reporters (as exemplified by DTTC, Cy7, IR792, and DIR), relying on which a tag-pair with distinguishable peaks can be screened (labeling with DTTC and Cy7, respectively). In vivo duplexing detection revealed that the tag-pair-labeled liposome was cleared faster in the liver than polydopamine NPs within one mouse. The developed method paves an easy way for gaining high-quality SERRS tags and will promote their in vivo multiplex analysis and diagnostics applications.

Facilitates chromatin transcription (FACT) is a histone chaperone that functions as a nucleosome remodeler and a chaperone. The two subunits of FACT, Spt16 and SSRP1, mediate multiple interactions between the subunits and components of the nucleosome. Among the interactions, the role of the DNA-binding domain in SSRP1 has not been characterized. We reported previously that the DNA-binding domain in Drosophila SSRP1 (dSSRP1) has multiple casein kinase II phosphorylation sites, and the DNA binding affinity of the domain changes sigmoidally in response to the degree of phosphorylation ("ultrasensitive response"). In this report, we explored the molecular mechanisms for the ultrasensitive response of the DNA-binding domain in dSSRP1 using the shortest fragment (AB-HMG, residues 434-624) responsible for nucleosome binding. AB-HMG contains two intrinsically disordered (ID) regions: the N-terminal part rich in acidic residues (AID) and the C-terminal part rich in basic residues (BID) followed by the HMG box. NMR and coarse-grained molecular dynamics simulations revealed a phosphorylation-dependent change in intramolecular contacts between the AID and BID-HMG, which is mediated by a hinge bending motion of AB-HMG to enable the ultrasensitive response. Ultrasensitivity generates two distinct forms of dSSRP1, which are high- and low-affinity nucleosome-binding forms. Drosophila FACT (dFACT) switches function according to the degree of phosphorylation of the AID in dSSRP1. We propose that dFACT in various phosphorylation states functions cooperatively to facilitate gene regulation in the context of the chromatin.

The bimodal waveguide (BiMW) biosensor is an innovative common path interferometric sensor based on the evanescent field detection principle. This biosensor allows for the direct detection of virtually any biomolecular interaction in a label-free scheme by using specific biorecognition elements. Due to its inherent ultrasensitivity, it has been employed for the monitoring of relevant nucleic-acid sequences such as mRNA transcripts or microRNAs present at the attomolar-femtomolar concentration level in human samples. The application of the BiMW biosensor to detect these nucleic acids can be a powerful analytical tool for diagnosis and prognosis of complex illnesses, such as cancer, where these biomarkers play a major role. The BiMW sensor is fabricated using standard silicon-based microelectronics technology, which allows its miniaturization and cost-effective production, meeting the requirements of portability and disposability for the development of point-of-care (PoC) sensing platforms. In this chapter, we describe the working principle of the BiMW biosensor as well as its application for the analysis of nucleic acids. Concretely, we show a detailed description of DNA functionalization procedures and the complete analysis of two different RNA biomarkers for cancer diagnosis: (1) the analysis of mRNA transcripts generated by alternative splicing of Fas gene, and (2) the detection of miRNA 181a from urine liquid biopsies, for the early diagnosis of bladder cancer. The biosensing detection is performed by a direct assay in real time, by monitoring the changes in the intensity pattern of the light propagating through the BiMW biosensor, due to the hybridization of the target with the specific DNA probe previously functionalized on the BiMW sensor surface., ICN2 is supported by the “Severo Ochoa Centers of Excellence” Program from the Spanish Ministry of Science (MINECO) (Grant No. SEV-2017-0706). Dr. Cesar S. Huertas is a recipient of an RMIT Vice Chancellor’s Postdoctoral Fellowship.

Background RhoA is a master regulator of cytoskeletal contractility, while nitric oxide (NO) is a master regulator of relaxation, e.g., vasodilation. There are multiple forms of cross-talk between the RhoA/ROCK pathway and the eNOS/NO/cGMP pathway, but previous work has not studied their interplay at a systems level. Literature review suggests that the majority of their cross-talk interactions are antagonistic, which motivates us to ask whether the RhoA and NO pathways exhibit mutual antagonism in vitro, and if so, to seek the theoretical implications of their mutual antagonism. Results Experiments found mutual antagonism between RhoA and NO in epithelial cells. Since mutual antagonism is a common motif for bistability, we sought to explore through theoretical simulations whether the RhoA-NO network is capable of bistability. Qualitative modeling showed that there are parameters that can cause bistable switching in the RhoA-NO network, and that the robustness of the bistability would be increased by positive feedback between RhoA and mechanical tension. Conclusions We conclude that the RhoA-NO bistability is robust enough in silico to warrant the investment of further experimental testing. Tension-dependent bistability has the potential to create sharp concentration gradients, which could contribute to the localization and self-organization of signaling domains during cytoskeletal remodeling and cell migration.

Perovskite materials have shown ultrasensitivity to atmospheres especially to O2, which is due to the passivation effect of the surface defects. Identification of the chemical nature of the surface...

Under oxidative and electrophilic stresses, cells launch an NRF2-mediated transcriptional antioxidant program. The activation of NRF2 depends on a redox sensor, KEAP1, which promotes the ubiquitination and degradation of NRF2. While a great deal has been learned about this duo, its quantitative signaling properties are largely unexplored. Here we examined these properties, including half-life, maximal activation, and response steepness (ultrasensitivity) of NRF2, through mathematical modeling. The models describe the binding of KEAP1 and NRF2 via ETGE and DLG motifs, NRF2 production, KEAP1-dependent and independent NRF2 degradation, and perturbations by different classes of NRF2 activators. Simulations revealed at the basal condition, NRF2 is sequestered by KEAP1 and the KEAP1-NRF2 complex is distributed comparably in an ETGE-bound (open) state and an ETGE and DLG dual-bound (closed) state. When two-step ETGE binding is considered, class I–V, electrophilic NRF2 activators shift the balance to a closed state incompetent to degrade NRF2, while the open and closed KEAP1-NRF2 complexes transition from operating in cycle mode to equilibrium mode. Ultrasensitive NRF2 activation (a steep rise of free NRF2) can occur when NRF2 nearly saturates KEAP1. The ultrasensitivity results from zero-order degradation through DLG binding and protein sequestration through ETGE binding. Optimal abundances of cytosolic and nuclear KEAP1 exist to maximize ultrasensitivity. These response characteristics do not require disruption of DLG binding as suggested by the hinge-latch hypothesis. In comparison, class VI NRF2 activators cause a shift to the open KEAP1-NRF2 complex and ultimately its complete dissociation, resulting in a fast release of NRF2 followed by stabilization. However, ultrasensitivity is lost due to decreasing free KEAP1 abundance. In summary, by simulating the dual role of KEAP1, i.e., sequestering and promoting degradation of NRF2, our modeling provides novel quantitative insights into NRF2 activation, which may help design novel NRF2 modulators and understand the oxidative actions of environmental stressors., Graphical abstract Class I–V electrophilic compounds induce increased formation of closed KEAP1-NRF2 complex and an abrupt increase in nuclear NRF2. By titrating KEAP1, class VI protein-protein interaction inhibitors induce dissociation of NRF2 from KEAP1 and a gradual increase in nuclear NRF2. Mathematical modeling captures the two different NRF2-activating mechanisms and predicts quantitatively different dose-response profiles of NRF2 activation.Image 1, Highlights • Steep (ultrasensitive) NRF2 activation can occur when it rises to saturate KEAP1. • Ultrasensitivity results from zero-order degradation and protein sequestration. • Optimal cytosolic and nuclear KEAP1 abundances exist for maximal ultrasensitivity. • Open and closed KEAP1-NRF2 complexes transition in cycle and equilibrium mode. • NRF2 activation by KEAP1-NRF2 interaction inhibitors is more gradual.

The evaluation of true penicillin allergy is significant to reduce its occurrence and the overdiagnosis before anti-infective treatment. However, the currently available methods with high specificity still face the problem of low sensitivity, thereby easily leading to false negatives. Herein, an alkyne responsive surface-enhanced Raman scattering (SERS) immunosensor is reported for ultrasensitive detection of penicillin allergen penicilloyl protein (P-protein) by using Au-Ag alloy nanoparticles@(antibody + alkyne probe) (as SERS immunoprobe) together with Ag nanofilm modified by antibody (as SERS capture substrate). The SERS immunoassay integrates the interference-free Raman response of high wavenumber region (2212 cm−1) and specific capture antibody with high affinity to selectively recognize P-protein from complicated sample. Meanwhile, the target-induced near-field coupling effect between localized surface plasmon resonances of individual SERS immunoprobe and capture substrate enables the detection of P-protein as low as pg/mL level, and the limit of detection can reach 0.329 pg/mL that is about 6 orders of magnitude lower than the limit defined protein residue (causing penicillin allergy). With the ultrasensitivity and specific selectivity, the proposed SERS immunoassay platform can precisely evaluate the content of P-protein in blood sample or penicillin drugs. It will be a potential tool to monitor allergic reaction to penicillin and better understand the mechanism of penicillin hypersensitivity.

Growth feedback, the inherent coupling between the synthetic gene circuit and the host cell growth, could significantly change the circuit behaviors. Previously, a diverse array of emerged behaviors, such as growth bistability, enhanced ultrasensitivity, and topology-dependent memory loss, were reported to be induced by growth feedback. However, the influence of the growth feedback on the circuit functions remains underexplored. Here, we reported an unexpected oscillatory behavior of a self-activation gene circuit induced by nutrient-modulating growth feedback. Specifically, after dilution of the activated self-activation switch into the fresh medium with moderate nutrient, its gene expression first decreases as the cell grows and then shows a significant overshoot before it reaches the steady states, leading to oscillation dynamics. Fitting the data with a coarse-grained model suggests a nonmonotonic growth-rate regulation on gene production rate. The underlying mechanism of the oscillation was demonstrated by a molecular mathematical model, which includes the ribosome allocation towards gene production, cell growth, and cell maintenance. Interestingly, the model predicted a counterintuitive dependence of oscillation amplitude on the nutrition level, where the highest peak was found in the medium with a moderate nutrient but was not observed in rich nutrient. We experimentally verified this prediction by tuning the nutrient level in the culture medium. We did not observe significant oscillatory behavior for toggle switch, suggesting that the emergence of oscillatory behavior depends on circuit network topology. Our results demonstrated a new nonlinear emergent behavior mediated by growth feedback, which depends on the ribosome allocation between gene circuit and cell growth.

Hedgehog signaling is fundamental in animal embryogenesis, and its dysregulation causes cancer and birth defects. The pathway is triggered when the Hedgehog ligand inhibits the Patched1 membrane receptor, relieving repression that Patched1 exerts on the GPCR-like protein Smoothened. While it is clear how loss-of-function Patched1 mutations cause hyperactive Hedgehog signaling and cancer, how other Patched1 mutations inhibit signaling remains unknown. Here, we develop quantitative single-cell functional assays for Patched1, which, together with mathematical modeling, indicate that Patched1 inhibits Smoothened enzymatically, operating in an ultrasensitive regime. Based on this analysis, we propose that Patched1 functions in cilia, catalyzing Smoothened deactivation by removing cholesterol bound to its extracellular, cysteine-rich domain. Patched1 mutants associated with holoprosencephaly dampen signaling by three mechanisms: reduced affinity for Hedgehog ligand, elevated catalytic activity, or elevated affinity for the Smoothened substrate. Our results clarify the enigmatic mechanism of Patched1 and explain how Patched1 mutations lead to birth defects.

Switch-like behaviours in biochemical networks are of fundamental significance in biological signal processing, and exist as two distinct types: ultra-sensitivity and bistability. Here we propose two new models of a reversible covalent-modification cycle with positive autoregulation (PAR), a motif structure that is thought to be capable of both ultrasensitivity and bistability in different parameter regimes. These new models appeal to a modelling framework that we call complex-complete , which accounts fully for the molecular complexities of the underlying signalling mechanisms. Each of the two new models encodes a specific molecular mechanism for PAR. We demonstrate that the modelling simplifications for PAR models that have been used in previous work, which rely on Michaelian approximations, are unable to accurately recapitulate the qualitative signalling responses supported by our detailed models. Strikingly, we show that complex-complete PAR models are capable of new qualitative responses such as one-way switches and a ‘prozone’ effect, depending on the specific PAR-encoding mechanism, which are not supported by Michaelian simplifications. Our results highlight the critical importance of accurately representing the molecular details of biochemical signalling mechanisms, and strongly suggest that the Michaelian approximation is inadequate for predictive models of enzyme-mediated chemical reactions with added regulations such as PAR.

Ultrasensitivity is a ubiquitous emergent property of biochemical reaction networks. The design and construction of synthetic reaction networks exhibiting ultrasensitivity has been challenging, but would greatly expand the potential properties of life-like materials. Herein, we exploit a general and modular strategy to reversibly regulate the activity of enzymes using light and show how ultrasensitivity arises in simple out-of-equilibrium enzymatic systems upon incorporation of reversible photoswitchable inhibitors (PIs). Utilizing a chromophore/warhead strategy, PIs of the protease α-chymotrypsin were synthesized, which led to the discovery of inhibitors with large differences in inhibition constants (Ki) for the different photoisomers. A microfluidic flow setup was used to study enzymatic reactions under out-of-equilibrium conditions by continuous addition and removal of reagents. Upon irradiation of the continuously stirred tank reactor with different light pulse sequences, i.e., varying the pulse duration or frequency of UV and blue light irradiation, reversible switching between photoisomers resulted in ultrasensitive responses in enzymatic activity as well as frequency filtering of input signals. This general and modular strategy enables reversible and tunable control over the kinetic rates of individual enzyme-catalyzed reactions and makes a programmable linkage of enzymes to a wide range of network topologies feasible.

This article describes the enhanced sensitivity to a viscous medium by a molecular rotor based fluorophore (RBF), TPSI I. The TPSI I molecule is designed in such a way that it consists of a rotor and a fluorophore with a p-rich bridge between them. TPSI I is a light-responsive material in solution as well as in the solid state. The structural design of the molecule allows flexible rotation and photo-induced cis-trans isomerization both in the solid state as well as in solution. These combined attributes of TPSI I are responsible for the ultrasensitive viscosity response of the new material, which was verified through the Fo ̈rster-Hoffmann equation. According to this equation, the derived 'x' value is 1.02 (x is related to the sensitivity) which is the highest among the contemporary reports for RBFs. The facts were evidenced both by experimental as well as theoretical data. The ultrasensitivity towards viscosity was further analyzed in in vitro studies by detecting the subtle changes in the alteration of intracellular viscosity in normal and cancerous cells. An alteration of intracellular viscosity in cells treated with viscosity modula- tors was also confirmed using a previously well-established viscosity measurement technique, dynamic measurement through the piezoelectric patch. Our research offers a detailed mechanism to improve viscosity sensors and an efficient probe for detecting minute changes in intracellular viscosity.

Control processes are responsible for maintenance of the organism’s internal environment (homeostasis) and, therefore, inextricable from the very definition of living matter. Optimal homeostasis involves adaptation and relies on processing of signals from the environment. Signaling and control processes are considered that exhibit nonlinear effects such as an all-of-nothing response, switches, modulation of oscillations, and hysteresis. Examples are drawn from intracellular molecular systems, as well as multicellular and inter-organ control networks: protein kinase cascades with feedback, dynamics of the transmembrane electrochemical potential, regulation of gene expression by metabolism, and control of breathing.

Protein turnover is vital to cellular homeostasis. Many proteins are degraded efficiently only after they have been post-translationally “tagged” with a polyubiquitin chain. Ubiquitylation is a form of Post-Translational Modification (PTM): addition of a ubiquitin to the chain is catalyzed by E3 ligases, and removal of ubiquitin is catalyzed by a De-UBiquitylating enzyme (DUB). Nearly four decades ago, Goldbeter and Koshland discovered that reversible PTM cycles function like on-off switches when the substrates are at saturating concentrations. Although this finding has had profound implications for the understanding of switch-like behavior in biochemical networks, the general behavior of PTM cycles subject to synthesis and degradation has not been studied. Using a mathematical modeling approach, we found that simply introducing protein turnover to a standard modification cycle has profound effects, including significantly reducing the switch-like nature of the response. Our findings suggest that many classic results on PTM cycles may not hold in vivo where protein turnover is ubiquitous. We also found that proteins sharing an E3 ligase can have closely related changes in their expression levels. These results imply that it may be difficult to interpret experimental results obtained from either overexpressing or knocking down protein levels, since changes in protein expression can be coupled via E3 ligase crosstalk. Understanding crosstalk and competition for E3 ligases will be key in ultimately developing a global picture of protein homeostasis., Author summary Previous work has shown that substrates of Post-Translational Modification (PTM) cycles can have coupled responses if those substrates share enzymes. This implies that modifications leading to substrate degradation (e.g. ubiquitylation by an E3 ligase) could introduce coupling in concentrations of substrates sharing a ligase. Using mathematical models, we found adding protein turnover to a PTM cycle diminishes both sensitivity and ultrasensitivity, particularly in models admitting long ubiquitin chains. We also found that proteins sharing an E3 ligase can indeed have coupled changes in both expression and sensitivity to signals. These results imply that accounting for crosstalk in protein degradation networks is crucial for the interpretation of results from a wide variety of common experimental perturbations to living systems.

Nucleic acid detection technology based on polymerase chain reaction (PCR) and antibody detection based on immunochromatography still have many problems such as false negatives for the diagnosis of coronavirus disease 2019 (COVID-19). Therefore, it is of great importance to develop new techniques to improve the diagnostic accuracy of COVID-19. We herein developed an ultrasensitive, rapid, and duplex digital enzyme-linked immunosorbent assay (dELISA) for simultaneous detection of spike (S-RBD) and nucleocapsid (N) proteins of SARS-CoV-2 based on a single molecule array. This assay effectively combines magnetic bead encoding technology and the ultrasensitive detection capability of a single molecule array. The detection strategies of S-RBD protein and N-protein exhibited wide response ranges of 0.34–1065 pg/mL and 0.183–338 pg/mL with detection limits of 20.6 fg/mL and 69.8 fg/mL, respectively. It is a highly specific method for the simultaneous detection of S-RBD protein and N-protein and has minimal interference from other blood proteins. Moreover, the spike assay showed a satisfactory and reproducible recovery rate for the detection of S-RBD protein and N-protein in serum samples. Overall, this work provides a highly sensitive method for the simultaneous detection of S-RBD protein and N-protein, which shows ultrasensitivity and high signal-to-noise ratio and contributes to improve the diagnosis accuracy of COVID-19. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03435-z.

Enzyme assays are important for many applications including clinical diagnostics, functional proteomics, and drug discovery. Current methods for enzymatic activity measurement often suffer from low analytical sensitivity. We developed an ultrasensitive method for the detection of enzymatic activity using Single Molecule Arrays (eSimoa). The eSimoa assay is accomplished by conjugating substrates to paramagnetic beads and measuring the conversion of substrates to products using single molecule analysis. We demonstrated the eSimoa method for the detection of protein kinases, telomerase, histone H3 methyltransferase SET7/9, and polypeptide N-acetylgalactosaminyltransferase with unprecedented sensitivity. In addition, we tested enzyme inhibition and performed theoretical calculations for the binding of inhibitor to its target enzyme and show the need for an ultrasensitive enzymatic assay to evaluate the potency of tight binding inhibitors. The eSimoa assay was successfully used to determine inhibition constants of both bosutinib and dasatinib. Due to the ultrasensitivity of this method, we also were able to measure the kinase activities at the single cell level. We show that the eSimoa assay is a simple, fast, and highly sensitive approach, which can be easily extended to detect a variety of other enzymes, providing a promising platform for enzyme-related fundamental research and inhibitor screening.

Protein activity is often regulated by ligand binding or by post-translational modifications such as phosphorylation. Moreover, proteins that are regulated in this way often contain multiple ligand binding sites or modification sites, which can operate to create an ultrasensitive dose response. Here, we consider the contribution of the individual modification/binding sites to the activation process, and how their individual values affect the ultrasensitive behavior of the overall system. We use a generalized Monod-Wyman-Changeux (MWC) model that allows for variable conformational free energy contributions from distinct sites, and associate a so-called activation parameter to each site. Our analysis shows that the ultrasensitivity generally increases as the conformational free energy contribution from one or more sites is strengthened. Furthermore, ultrasensitivity depends on the mean of the activation parameters and not on their variability. In some cases, we find that the best way to maximize ultrasensitivity is to make the contribution from all sites as strong as possible. These results provide insights into the performance objectives of multiple modification/binding sites and thus help gain a greater understanding of signaling and its role in diseases., Author summary Multisite protein modification is ubiquitous in gene regulation and signal transduction, often in the form of multisite phosphorylation. Many models of multisite ultrasensitivity are available in the literature, but they usually assume that all sites contribute equally to the activation of the multisite target. In this work, we relax this assumption and carry out computational and mathematical analysis of a multisite system in which the conformational free energy contribution varies across sites. We find that the ultrasensitivity of the system tends to increase (with some exceptions) when the conformational free energy contributed by any given site is strengthened. Our analysis predicts that all active sites should have approximately the same conformational free energy contribution, a property observed in proteins with unstructured modification domains and bulk electrostatics. We were also able to predict from first principles an energy range of -2 to -4 kcal/mol per site that effectively maximizes ultrasensitive behavior. This prediction is consistent with experimental measurements in phosphorylation sites. Another strategy predicted by some of our models is to select a subset of the sites and activate them uniformly, while silencing other modification sites in the protein. This strategy is also observed experimentally in many multisite phosphorylation proteins.

The toehold switch consists of a cis-repressing switch RNA hairpin and a trans-acting trigger RNA. The binding of the trigger RNA to an unpaired toehold sequence of the switch hairpin allows for a branch migration process, exposing the start codon and ribosome binding site for translation initiation. In this work, we demonstrate that responses of toehold switches can be modulated by introducing an inhibitory hairpin that shortens the length of the unpaired toehold region. First, we investigated the effect of the toehold region length on output gene expression and showed that the second trigger RNA, which binds to the inhibitory hairpin, is necessary for output gene activation when the hairpin-to-hairpin spacing is short. Second, the apparent Hill coefficient was found generally to increase with decreasing hairpin-to-hairpin spacing or increasing hairpin number. This work expands the utility of toehold switches by providing a new way to modulate their response.

SummaryCellular decision making often builds on ultrasensitive MAPK pathways. The phosphorylation mechanism of MAP kinase has so far been described as either distributive or processive, with distributive mechanisms generating ultrasensitivity in theoretical analyses. However, the in vivo mechanism of MAP kinase phosphorylation and its regulation by feedback loops remain unclear. We thus characterized the regulation of the MAP kinase Hog1 in Saccharomyces cerevisiae, which is transiently activated in response to hyperosmolarity. Specifically, we combined Hog1 activation data from different modalities and multiple conditions. We constructed ODE models with different pathway topologies, which were then assessed via parameter estimation and model selection. Interestingly, our best fitting model switches between distributive and processive phosphorylation behavior via a positive feedback loop targeting the MAP kinase-kinase Pbs2. Simulations further suggest that this mixed mechanism is required not only for full sensitivity to stimuli, but also to ensure robustness to different perturbations.

Bioassays using a fluorophore and DNA aptamer have been extensively developed due to the ultrasensitivity of fluorophores and recognition ability of DNA aptamers. Conventional fluorescent aptamer-based sensors (aptasensors) require chemical labeling between the fluorophore and aptamer and is technologically impracical for various sensing and assay applications. A simple "mix and go" strategy has been introduced that uses label-free technology as a platform for sensor development. The biosensors comprise a fluorophore, a ssDNA aptamer, and eco-friendly graphene oxide (GO). In the absence of the sensor target, GO quenches the fluorescence of the fluorophore and single-strand DNA aptamer complex. When the target is added, the DNA aptamer conformationally turns into a duplex, G-quadruplexe, or other secondary structure. This structure change leads to release of GO by the fluorophore-aptamer-target complex, generating dramatic fluorescence recovery and amplification. With this sensing method, the DNA aptamer does not need to be chemically labeled. Therefore, flexible fluorophore indicators and ssDNA aptamers can be used in this label-free aptasensing strategy. In this review, we discuss various unlabeled fluorophores, including synthetic small molecular fluorophores and genetically encoded fluorescent proteins, as indicators for generating GO-based fluorescent DNA aptasensors for label-free bioassay.

An ultrasensitive and selective light-scattering (LS) aptasensor for prostate specific antigen (PSA) was developed based on bifunctional DNA decorated gold nanoparticles (Au NPs) by use of target stimuli-responsive assembly. Both binding sequence poly adenine (poly(dA), Domain I) and the recognition sequence aptamer (Domain II) of bifunctional ssDNA were in favor of the sensitivity for the fabricated light-scattering aptasensor due to modulated the lateral spacing of DNA on Au NPs surface and possessed strong antigen binding affinity, respectively. And they efficiently promoted the Au NPs aggregation and activated the signal of light-scattering of aptasensor. The formation of aptamer-PSA complex induced conformational transformation of the aptamer on the surface of the Au NPs-based aptasensor and then tuned the interparticle distance of gold nanoparticle assemblies. The larger scattering particles, the higher light-scattering intensity. Target stimuli-responsive aggregation behavior of Au NPs lit up the light-scattering signal of aptasensor. As the concentration of PSA increased, the light-scattering intensity gradually enhanced. The aptasensor response for PSA detection was in the linear range from 0.01 ng/mL to 20 ng/mL with the detection limit of 2 pg/mL. The thiol-free Au NPs-based light-scattering aptasensor has exceptional performances with ultrasensitivity and high selectivity besides the advantage of economic and facile fabrication. Moreover, the proposed target-activated aptasensor was applied for the detection of PSA in serum samples and demonstrated great potential in clinical applications.

Routine detection of trace amounts of Pb2+ is of significant importance because it poses severe risks to human health and the environment. In this paper, a novel label-free fluorescence platform for the sensitive detection of Pb2+ has been successfully constructed by using Pb2+-specific 8–17 DNAzyme as the molecular recognition element, 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND) as the signal reporter, and exonuclease III (Exo III) as the signal amplifier. A good linearity between the fluorescence intensity and the logarithm of Pb2+ concentration was obtained in the range from 100 pM to 10 μM with a correlation coefficient (R2) of 0.99. The sensing system exhibited ultrasensitivity towards Pb2+ at low concentration (50 pM) without any labeling, modification, immobilization, or washing procedure. Besides, the proposed method was also highly selective for Pb2+ against other metal ions. This biosensor was robust and can be applied for the determination of Pb2+ in complex samples with desired recovery and good accuracy. With the successful demonstration of Pb2+ detection, the label-free biosensor can be readily expanded to monitor other targets in a simple, cost-effective, and ultrasensitive way by replacing the target-specific molecular recognition elements.

In this work, a label-free fluorescence biosensor for simple detection of the HIV-1 gene was proposed by using toehold-mediated strand displacement reactions (TMSDRs) combined with a non-enzymatic target recycling amplification strategy. In this system, two TMSDRs were used. In the presence of the HIV-1 gene, an autocatalytic DNA machine can be activated. This leads to the generation of numerous free G-rich sequences, which can associate with a fluorescent dye N-methylmesoporphyrin IX (NMM) to yield an amplified fluorescence signal for the target detection. This sensing platform showed a high sensitivity towards the HIV-1 gene with a detection limit as low as 1.9 pM without any labelling, immobilization, or washing steps. The designed sensing system also exhibits an excellent selectivity for the HIV-1 gene compared with other interference DNA sequences. Furthermore, the presented biosensor is robust and has been successfully applied for the detection of the HIV-1 gene in a real biological sample with satisfactory results, suggesting that this method is promising for simple and early clinical diagnosis of HIV infection. Thanks to its simplicity, cost-effectiveness and ultrasensitivity, our proposed sensing strategy provides a universal platform for the detection of other genes by substituting the target-recognition element.

Protein aggregation of alpha-synuclein (&alpha, Syn) is implicated in Parkinson&rsquo, s disease (PD), and, thus, &alpha, Syn aggregates are a potentially promising candidate biomarker for PD diagnosis. Here, we describe a simple and sensitive electrochemical sensor to monitor the aggregation of &alpha, Syn for early PD diagnosis. The sensor utilizes methylene blue (MB)-tagged aptamer (Apt) adsorbed on electrochemically reduced graphene oxide (ERGO) by &pi, &ndash, &pi, stacking. The binding of &alpha, Syn oligomer to the Apt induces desorption of the Apt from the ERGO surface, which leads to the electrochemical signal change. The resulting sensor allowed the highly sensitive and selective detection of &alpha, Syn oligomer according to the voltammetric change. Under optimized conditions, the linear range of detection was observed to be from 1 fM to 1 nM of the &alpha, Syn oligomer and the limit of detection (LOD) was estimated to be 0.64 fM based on S/N = 3. The sensor also showed good reproducibility and stability, enabling real sample analysis of the &alpha, Syn oligomer in human blood serum. With its ultrasensitivity and good performance for &alpha, Syn oligomer detection, the sensor provides one promising tool for the early diagnosis of PD.

In this work, a simple dichlororesorufin-based colorimetric and fluorescent probe R-Hg with the recognition receptor of carbonothioate was designed, synthesized, and exploited to determine mercury ions in aqueous solution and living cells as well as in zebrafish. After a series of analytical tests, probe R-Hg showed some excellent characteristics, such as ultrasensitivity, high selectivity, and good water solubility. The probe solution exhibited a very large fluorescence enhancement (about 150-fold) upon addition of Hg2+. Additionally, probe R-Hg exhibited excellent sensitivity, which could quantificationally detect Hg2+ at the nanomolar level, and the detection limit was 0.49 nM. Moreover, probe R-Hg could be used as a “naked-eye” probe for monitoring Hg2+, because the color of its solution transformed remarkably from yellow into pink with the participation of Hg2+. Importantly, probe R-Hg has potential application value and has been successfully applied to the determination of Hg2+ in practical water sa...

Thiophenols are a class of highly toxic pollutant compounds, which can cause severe damage to the central nervous system and other nervous systems after long-term exposure. Therefore, it is of significance to develop an effective method to detect trace thiophenols in the environment. Herein, we synthesized a dicyanoisophorone-based near-infrared fluorescent probe (probe 1) with large stokes shift (λem − λabs = 203 nm) for the selective detection of thiophenols. Probe 1 exhibited very high response speed (less than 200 S), sensitivity, and selectivity towards thiophenols, regardless of the presence of aliphatic thiols and other analytes. The detection limit was as low as 3.4 nM and the signal-to-background ratio of fluorescence intensity reached 30. Moreover, probe 1 displayed ultrasensitivity and high reliability in the quantitative detection of trace thiophenol in three river water samples.

Membrane-initiated steroid signaling (MISS) is a recently discovered aspect of steroidal control over cell function that has proved highly challenging to study due to its rapidity and ultrasensitivity to the steroid trigger [Chow RWY, Handelsman DJ, Ng MKC (2010) Endocrinology 151:2411-2422]. Fundamental aspects underlying MISS, such as receptor binding, kinetics of ion-channel opening, and production of downstream effector molecules remain obscure because a pristine molecular technology that could trigger the release of signaling steroids was not available. We have recently described a prototype DNA nanocapsule which can be programmed to release small molecules upon photoirradiation [Veetil AT, et al. (2017) Nat Nanotechnol 12:1183-1189]. Here we show that this DNA-based molecular technology can now be programmed to chemically trigger MISS, significantly expanding its applicability to systems that are refractory to photoirradiation.

Chromatin remodeling is an essential form of gene regulation that is involved in a variety of biological processes. We develop a theoretical model that takes advantage of percolation effects at the level of nucleosome interactions, which allows for ultrasensitive chromatin expansion. This model is non-cooperative and readily provides spatial bounds to the expansion region, preventing uncontrolled remodeling events. We explore different chromatin architectures and the ultrasensitivity of the chromatin density as a function of transcription factor concentration. We also compare our model with experimental data involving an inhibitor of nucleosome acetylation. These results suggest a novel mechanism for spatially-bounded chromatin remodeling and they provide means for quantitative comparisons between proposed models of chromatin architecture.

A simple, precise, and highly sensitive detection method is urgently needed for diagnosis and monitoring of foodborne pathogens. To meet these requirements, we designed a micro-propulsion reactor based on the trans-cleavage activity of Cas12a and recombinase polymerase amplification (RPA) for simultaneous amplification and detection and named it “Cas12a-MPR”. This method can provide a contamination- and instrument-free nucleic acid rapid detection platform with precision and ultrasensitivity using CRISPR-Cas12a combined with RPA in the same vessel. For improved performance, the interaction between the CRISPR detection system and the RPA reagent was explored. Our data showed that the impact of CRISPR reagents on RPA was negligible, but magnesium ions played a vital role in the functioning of the CRISPR/Cas12a system. After exploring the interaction between the CRISPR detection system and the RPA reagent, the method was further optimized to specifically detect target DNA at the attomolar level and achieved a limit of detection of 10 CFU mL-1 at 37 °C. Compared to qPCR, our detection results exhibited 100 % accuracy for detection of Listeria monocytogenes contamination. All the results demonstrated that Cas12a-MPR has great promise for the detection of foodborne pathogens.

Nitroxyl (HNO) is a derivative of nitric oxide (NO) that plays an essential role in various biological and pharmacological events. Until now, the in situ trapping and specific detection of HNO in living samples is still challenging. In this project, we fabricated a novel BODIPY-based micellar nanoprobe for monitoring nitroxyl in vitro and in vivo in ratiometric mode in aqueous solution. The probe (P-BODIPY-N) contains an asymmetrical BODIPY dye for fluorescent signaling and a diphenylphosphinobenzoyl as the trigger moiety; then we encapsulated P-BODIPY-N into the hydrophobic interior of an amphiphilic copolymer (mPEG-DSPE) and prepared a novel BODIPY-based micellar nanoprobe: NP-BODIPY-N. As far as we know, this probe is the first reported ratiometric fluorescent nanoprobe for HNO, which exhibits ultrasensitivity, high selectivity, and good biocompatibility. Above all, this nanoprobe shows favorable cellular uptaken and was successfully used to detect intracellular HNO released by Angeli's salt in living cells and zebrafish larvae. These results indicate that our newly designed nanoprobe will provide a promising tool for the studies of HNO in living system.

In this work, a label-free fluorescence biosensor for ultrasensitive and simple detection of the mecA gene of Staphylococcus aureus was proposed by using an exonuclease III (Exo III)-assisted cascade signal amplification strategy. The 3' end-extruding hairpin probe (HP) acted as the target recognition element and the caged G-quadruplex was used as the signal reporter. Without the mecA gene, the HP probe cannot be digested by Exo III, as the G-rich sequences are blocked in the stem of the HP probe. In the presence of the mecA gene, the hybridization of the mecA gene with the 3' end-extruding HP probe triggers the digestion reaction of Exo III, liberating the mecA gene and the mecA gene analogue. Both the released mecA gene and the mecA gene analogue can hybridize with other HP probes and activate another round of the cleavage reaction. Consequently, the released free G-quadruplex is "lit up" by N-methylmesoporphyrin IX (NMM), displaying a dramatically enhanced fluorescence intensity. This sensing platform showed a high sensitivity towards the mecA gene with a detection limit as low as 2.4 fM without any labelling, immobilization, or washing steps. The designed sensing system also exhibits excellent selectivity for the mecA gene in the presence of other interfering DNA sequences. Furthermore, the presented biosensor is robust and has been successfully applied for the detection of the mecA gene in a real food sample with satisfactory results. Owing to its simplicity, cost-effectiveness and ultrasensitivity, our proposed sensing strategy provides a promising platform for the detection of other genes by substituting the target-recognition element.

In this work, a rapid, one-step and ultrasensitive signal-on fluorescence sensing for the detection of adenosine triphosphate (ATP) based on target-activated cascaded digestion amplification with Exo III aid was developed. Our strategy integrates the target-activated cascaded digestion amplification with Exo III assistance into a signal-on fluorescence sensing platform. This advance greatly enhances the specificity and significantly improves the sensitivity for ATP detection. Specifically, an arch probe containing an anti-ATP aptamer (Apt) and a trigger probe (T) is used for recognizing the target. In the presence of ATP, the cascaded digestion amplification with the help of Exo III was activated, generating a remarkably strong signal-on fluorescence response. Our strategy features several virtues. First, the background signal is effectively eliminated owing to the specially designed hairpin probes (one is named HAP, the other is named MB short for molecular beacon) successfully avoiding the effect of Exo III. Second, the combination of target-activation with Exo III-aided cascade digestion amplification is integrated with fluorescence sensing, affording high specificity and ultrasensitive detection of ATP. Third, our method achieved the reliable and accurate detection of ATP in human serum. Furthermore, under optimized conditions, the results suggest that the proposed fluorescence sensing exhibits excellent specificity and ultrasensitivity toward ATP with the limit of detection as low as 0.63 pM and the broad analytical range from 1 pM to 1.0 × 105 pM. This target-activated cascaded digestion amplification with Exo III assistance combined with the fluorescence sensing strategy paves a luciferous and practical way for detecting ATP and other analytes with trace amounts in bio-analysis and clinical biomedicine.

Long-term depression (LTD) and long-term potentiation (LTP) in the cerebellum are important for motor learning. However, the signaling mechanisms controlling whether LTD or LTP is induced in response to synaptic stimulation remain obscure. Using a unified model of LTD and LTP at the cerebellar parallel fiber-Purkinje cell (PF-PC) synapse, we delineate the coordinated pre- and postsynaptic signaling that determines the direction of plasticity. We show that LTP is the default response to PF stimulation above a well-defined frequency threshold. However, if the calcium signal surpasses the threshold for CaMKII activation, then an ultrasensitive “on switch” activates an extracellular signal-regulated kinase (ERK)-based positive feedback loop that triggers LTD instead. This postsynaptic feedback loop is sustained by another, trans-synaptic, feedback loop that maintains nitric oxide production throughout LTD induction. When full depression is achieved, an automatic “off switch” inactivates the feedback loops, returning the network to its basal state and demarcating the end of the early phase of LTD.

Signal amplification in biomolecular networks converts a linear input to a steeply sigmoid output and is central to a number of cellular functions including proliferation, differentiation, homeostasis, adaptation, and biological rhythms. One canonical signal amplifying motif is zero-order ultrasensitivity that is mediated through the posttranslational modification (PTM) cycle of signaling proteins. The functionality of this signaling motif has been examined conventionally by supposing that the total amount of the protein substrates remains constant, as by the classical Koshland–Goldbeter model. However, covalent modification of signaling proteins often results in changes in their stability, which affects the abundance of the protein substrates. Here, we use mathematical models to explore the signal amplification properties in such scenarios and report some novel aspects. Our analyses indicate that PTM-induced protein stabilization brings the enzymes closer to saturation. As a result, ultrasensitivity may emerge or is greatly enhanced, with a steeper sigmoidal response, higher magnitude, and generally longer response time. In cases where PTM destabilizes the protein, ultrasensitivity can be regained through changes in the activities of the involved enzymes or from increased protein synthesis. Importantly, ultrasensitivity is not limited to modified or unmodified protein substrates—when protein turnover is considered, the total free protein substrate can also exhibit ultrasensitivity under several conditions. When full enzymatic reactions are used instead of Michaelis–Menten kinetics for the modeling, the total free protein substrate can even exhibit nonmonotonic dose–response patterns. It is conceivable that cells use inducible protein stabilization as a strategy in the signaling network to boost signal amplification while saving energy by keeping the protein substrate levels low at basal conditions.

A novel fluorescence biosensing strategy for ultrasensitive and specific detection of pathogenic bacteria based on target-triggered enzymatic repairing amplification (ERA) has been developed. This strategy relies on target–aptamer binding mediated ERA reaction, which is carried out cyclically with the help of polymerase and two DNA repairing enzymes, uracil-DNA glycosylase (UDG) and endonuclease IV (Endo IV) to produce amplified fluorescence signal. In our assay, the specially designed hairpin probe (HAP) is used as DNA template responsible for producing a great quantity of reporter oligonucleotides and secondary primers, which can initiate a new cycle of polymerization-repairing amplification. Moreover, by the combination of polymerase-catalyzed incorporation of lesion bases with UDG and Endo IV-assisted ERA, multiple cycle of amplification of the recognition event is achieved, enabling ultrasensitive detection of pathogenic bacteria. Under optimal conditions, this biosensor exhibits ultrasensitivity toward target pathogenic bacteria with detection limits of 9.86 cfu mL−1 and a detection range of 5 orders of magnitude. Additionally, the biosensor has the ability of combating nonspecific background. Furthermore, an archer probe containing the anti-target aptamer sequence and a primer sequence is designed, which translates the binding of target to aptamer into the presence of primer sequence, enabling the detection of various targets, such as protein, DNA, small molecular, and any substance possessing its aptamer. Hence, the proposed target-triggered ERA-based signal-on fluorescence sensing strategy indeed create a versatile and useful platform for detection of pathogenic bacteria, related food safety analysis and clinical diagnosis.

Developing some methods that can simply and effectively detect mercury ions (Hg2+) in the environment and biological systems are very important due to the problems of high toxicity and biological accumulation. Herein, we report a simple rhodol-derived colorimetric and fluorescent probe rhodol-Hg with a recognition receptor of carbonothioate for the specific determination of Hg2+. The color of probe rhodol-Hg solution changed remarkably from colorless to pink in the presence of Hg2+, thus rhodol-Hg could act as a "naked-eye" probe for Hg2+. Additionally, this probe exhibited high selectivity and ultrasensitivity in aqueous solution with the limit of detection (LOD) of 1.4 nM toward Hg2+, and the linear range was 0 - 0.8 μM determined by turn-on fluorescence spectrometry. Importantly, this probe has been successfully used for the detection of Hg2+ in environmental waters and living cells.

In this study, an ultrasensitive fluorescence turn-on assay for in situ sensing of intracellular microRNA (miRNA) was developed utilizing a carbon nitride nanosheet (CNNS) and a catalytic hairpin assembly (CHA). The CHA showed favourable signal amplification for low-level biomarkers, and CNNS was an excellent candidate as a fluorescence quencher and gene vector. Moreover, the hairpin DNA of CHA could be adsorbed onto the surface of CNNS. An enzyme-free fluorescence biosensor for ultrasensitive sensing of intracellular miRNA in cells based on CHA and CNNS was designed. When faced with target miRNA, the fluorescence was recovered due to the miRNA, which could trigger cycling of CHA circuits, leading to the production of a marked enhanced fluorescence signal. Compared with traditional methods, the proposed method is convenient, with low cytotoxicity, and high specificity and ultrasensitivity. It has promising potential for detection low-level biomarkers.

We propose a design strategy to fabricate a flexible bend sensor (BS) with ultrasensitivity toward airflow using all-poly(vinylidene fluoride) (PVDF) nanofiber web-based sensing elements and electrodes to monitor human respiration. The unique electrospinning (rotational speed of collector of 2000 rpm and tip-to-collector distance of 4 cm) with silver nanoparticle interfacing was introduced to prepare a Ag-doped oriented PVDF nanofiber web with high β-phase content as a sensing element (AgOriPVDF, β-phase crystallinity ∼44.5%). After that, a portion of the prepared AgOriPVDF was processed into a flexible and electrically conductive electrode through an electroless silver plating technique (SP-AgOriPVDF). Interestingly, the encapsulated AgOriPVDF BS with the SP-AgOriPVDF electrode exhibited superior piezoelectric bending response (open-circuit peak-to-peak output voltage, Vp–p ≈ 4.6 V) to injected airflow, which is more than 200 times higher than that of the unpackaged randomly aligned PVDF nanofiber web BS with a conductive tape electrode (Vp–p ≈ 0.02 V). In addition, the factors influencing the bend sensitivity of the BS such as the β-phase content, nanofiber orientation, flexibility of the electrode, and so forth were thoroughly analyzed and then discussed. We also demonstrated that the AgOriPVDF BS has sufficient capability to detect and identify various respiratory signals, presenting a great potential for wearable applications, for example, smart respiratory protective equipment.

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., Tetrahedral DNA framework-enabled bulk enzyme heterojunctions have been used to program biosensor interfaces. Here, the authors use DNA tetrahedrons to tether enzymes of an enzymatic cascade to gold electrodes, hence raising them over the bulk solution, which led to improved kinetics and sensitivity.

Emergence of fluorescence imaging with real-time and in situ manners has revolutionized the fields of tracing and defining enzymes in biological systems. β-galactosidase is a kind of enzyme that pl...

The unrepaired apurinic/apyrimidinic site (AP site) in mitochondrial DNA (mtDNA) promotes misincorporation of nucleotides and further causes serious damage for the living organism. Thus, accurate quantitative detection of AP sites in mtDNA in a rapid, highly sensitive, and highly selective fashion is important for the real-time evaluation of mtDNA oxidative damage. In this study, a targeting mtDNA ultrasensitive AP site-specific fluorescent rotor (BTBM-CN2) was designed by the strategy of molecular conformation torsion adjustment ratio fluorescent signal. The specific recognition reaction is activated when it encountered AP sites in mtDNA within 20 s, and BTBM-CN2 presented a "turn-on" red fluorescence signal at 598 nm. Then, about 100 s later, BTBM-CN2 emitted a new green fluorescence signal at 480 nm, which is mainly due to the activation of the rate-limiting reaction. With increasing numbers of AP sites (1-40 in 1 × 105 bp of mtDNA), the fluorescence emission at 598 nm decreased gradually, and the new emission at 480 nm increased. Intracellular experiments indicated that BTBM-CN2 could detect AP sites in mtDNA in a rapid and quantitative fashion with high selectivity and ultrasensitivity. On the basis of the emergence of the fluorescence signal at 480 nm and its signal strength, the cell whose mtDNA was damaged could be screened by flow cytometry and its degree of damage could be evaluated in real time by comet assay. Hence, the rotor may have potential applications varying from accurate and ultrasensitive detection of AP sites to the real-time evaluation of the oxidative damage in living cells.