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Differentiation of biologically important thiols, such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) is still a challenging task. Herein, we present a novel fluorescent chemodosimeter capable of selectively detecting Cys over other biothiols including Hcy and GSH and other amino acids by a facile thiol-Michael addition/transcyclization rearrangement cascade click process. The unique transcyclization step is critical for the selectivity as a result of the kinetically favorable formation of a six-membered ring with the Cys Michael adduct. Moreover, the probe adopts a distinctive dual quenching mechanism-photoinduced electron transfer (PET) and photoinduced intramolecular charge transfer (ICT) to deliver a drastic turn-on fluorescence response only at the Cys-selective transcylization step. The judicious selection of strong electron-withdrawing naphthalimide fluorophore with maleimide group enhances the electrophilicity and thus reactivity for the cascade process leading to fast detection and ultrasensitivity with a detection limit of 2.0 nm (S/N=3). The probe has demonstrated its practical utility potential in Cys imaging in live cells.

It is still challenging to sensitively detect protein biomarkers via surface-enhanced Raman scattering (SERS) technique owing to their low Raman activity. SERS tag-based immunoassay is usually applied; however, it is laborious and needs specific antibodies. Herein, an ultrasensitive and universal “Raman indicator” sensing strategy is proposed for protein biomarkers, with the aid of a glass capillary-based molecularly imprinted SERS sensor. The sensor consists of an inner SERS substrate layer for signal enhancement and an outer mussel-inspired polydopamine imprinted layer as a recognition element. Imprinted cavities have two missions: first, selectively capturing the target protein, and second, the only passageway of Raman indicator to access SERS substrate. Specific protein recognition means filling imprinted cavities and blocking Raman indicator flow. Thus, the quantity of captured protein can be reflected by the signal decrease of ultra-Raman active indicator molecule. The capillary sensor exhibited specific and reproducible detection at the level down to 4.1 × 10−3 μg L−1, for trypsin enzyme in as-received biological samples without sample preparation. The generality of the mechanism is confirmed by using three different protein models. This platform provides a facile, fast and general route for sensitive SERS detection of Raman inactive biomacromolecules, which offers great promising utility for in situ and fast point-of-care practical bioassay.

We report a versatile “turn-on” sensing strategy for the ultratrace level detection of Cd2+ ion based on structure-switching aptamer duplex which is immobilized on electrochemically reduced graphene (ERGO) electrode. This approach utilizes a methylene blue (MB) tagged aptamer probe (Apt) and its partially complimentary DNA (cDNA), designed to form aptamer duplex (cDNA-Apt) which is immobilized on the ERGO electrode. The double helix structure of cDNA-Apt on the ERGO electrode impedes the efficient electron transfer of MB to the electrode, due to the far proximity of MB and electrode, resulting in the decreased oxidation peak current of MB. The presence of analytes causes cDNA-Apt duplex to unwind and release the cDNA which can quantitatively facilitate the efficiency of electron transfer of MB, leading to the enhancement of electrochemical signal. It is noted that we can successfully achieve ultrasensitivity of the sensor owing to the large conformational change of Apt from the proposed aptamer architecture and “turn-on” mode sensing upon target binding. This sensing platform can be used to monitor Cd2+ from 1 fM to 1 nM and very low detection limit of 0.65 fM (S/N = 3), which is among the best in all the reported electrochemical aptasensors.

Graphical abstract, Highlights • Homo-oligomerisation may offer an unexpected variety of signal processing functions. • These include: dynamic signal encoding via oligomeric complex size. • Homeostatic regulation of monomer concentration. • Bistability via pseudo-multisite modification and multi-enzyme regulation. • The findings partly explain why homo-oligomerisation is so commonly found in evolution., Homo-oligomerisation of proteins is a ubiquitous phenomenon whose exact role remains unclear in many cases. To identify novel functions, this paper provides an exploration of general dynamical mathematical models of homo-oligomerisation. Simulation and analysis of these models show that homo-oligomerisation on its own allows for a remarkable variety of complex dynamic and steady state regulatory behaviour such as transient overshoots or homeostatic control of monomer concentration. If post-translational modifications are considered, however, conventional mass action kinetics lead to thermodynamic inconsistencies due to asymmetric combinatorial expansion of reaction routes. Introducing a conservation principle to balance rate equations re-establishes thermodynamic consistency. Using such balanced models it is shown that oligomerisation can lead to bistability by enabling pseudo-multisite modification and kinetic pseudo-cooperativity via multi-enzyme regulation, thereby constituting a novel motif for bistable modification reactions. Due to these potential signal processing capabilities, homo-oligomerisation could play far more versatile roles in signal transduction than previously appreciated.

Harnessing ultrasensitivity from optical structures to detect tiny changes in the targeted samples is the main goal of scientists in the field of sensor design. In this study, an uncommon rhombus-shape plasmonic structure is proposed for providing blue-shift ultrasensitivity. The physical origin of this optical response relies on multi-faces of gold rhombus and their electromagnetic coupling with their induced images in a high-refractive-index substrate (Si3N4). A characteristic of blue-shift emerges as the Fano resonance in the reflection spectrum. We have experimentally shown that this novel structure has the surface sensitivity to the refractive index difference in the order of 10−5. These characteristics have been applied for non- and conditioned- cell culture medium with refractive differences in this order.This level of sensitivity is interesting for enhanced fingerprinting of minute quantities of targeted molecules and interfacial ion redistribution.

Biosensors that incorporate nanomaterials and nanofabrication techniques enable molecular detection of chemical and biological macromolecules with a high degree of specificity and ultrasensitivity. Here, we present a novel fabrication process that yields a nanostructure capable of detecting biological macromolecules. The extended core nanocoax (ECC) structure builds on a previously reported nanocoaxial-based sensor. The fabrication of the device incorporates an extended inner pillar, with controllable extension above the annulus and into the surrounding solution. This new design eliminates structural constraints inherent in the original nanocoax architecture. We also provide results demonstrating improvement in biosensing capability. Specifically, we show the capability of the new architecture to detect the B subunit of the Vibrio cholerae toxin at improved sensitivity (100 pg/ml) in comparison to optical enzyme-linked immunosorbant assay (1 ng/ml) and previously reported coaxial nanostructures (2 ng/ml).

A highly sensitive fluorescence biosensing system was designed for the detection of trace amounts of arsenic(III) (As3+) based on target-triggered successive signal amplification strategy. The specific recognition between the target As3+ and the aptamer sequence results in the release of the blocking DNA to trigger the subsequent signal amplification steps. Exonuclease III (Exo III)-mediated DNA recycling digest process is introduced into the sensing system to generate numerous Mg2+-dependent DNAzymes. After magnetic separation, the active DNAzyme with multiple turnovers could catalyze the continuous cleavage of the fluorophore-quencher-functionalized substrate strands, thus yielding a significantly amplified fluorescence signal for target detection. Due to the synergetic signal amplification of Exo III and DNAzyme, the fluorescent biosensor exhibits ultrasensitivity for As3+ monitoring, with a detection limit of 2 pM. Our established biosensor also displays excellent selectivity toward the target As3+ an...

High-fidelity mapping of amyloid-β (Aβ) plaques is critical for the early detection of Alzheimer’s disease. However, in vivo probing of Aβ plaques by commercially available thioflavin derivatives (ThT or ThS) has proven to be extremely limited, as evident by the restriction of enrichment quenching effect, low signal-to-noise (S/N) ratio, and poor blood–brain barrier (BBB) penetrability. Herein, we demonstrate a rational design strategy of near-infrared (NIR) aggregation-induced emission (AIE)-active probes for Aβ plaques, through introducing a lipophilic π-conjugated thiophene-bridge for extension to NIR wavelength range with enhancement of BBB penetrability, and tuning the substituted position of the sulfonate group for guaranteeing specific hydrophilicity to maintain the fluorescence-off state before binding to Aβ deposition. Probe QM-FN-SO3 has settled well the AIE dilemma between the lipophilic requirement for longer emission and aggregation behavior from water to protein fibrillogenesis, thus making ...

Biological switches are wide spread in many biological systems. Among them, the switch of the bacterial flagellar motor has been, for years, a focus of special interest. This is because it affects a mechanical process rather than a chemical reaction, it controls the direction of rotation of a rotary motor rather than being an on/off switch, and it is exceptionally ultrasensitive. Yet, the molecular mechanism underlying the function of this switch has remained unknown. Here we resolved unique features of this mechanism: On the one hand, it is tightly regulated by multiple means, involving three binding sites and two different covalent modifications, with the binding specificity being dictated by the type of covalent modification and by a strict binding sequence. On the other hand, it endows the motor with flexibility as it involves an intermediate stage of brief switches that provides a “go/no go” situation, in which the motor can either proceed to a stable rotation in the new direction or shift back to the original direction. This intermediate stage appears to be a means of a swimming cell to produce angular deflection while maintaining directional persistence. Furthermore, we show by mathematical modeling that such a mechanism provides ultrasensitivity. This unique combination of tight regulation, flexibility, and ultrasensitivity make this mechanism of special interest.

MAPK (mitogen-activated protein kinase) signaling pathways regulate a variety of biological processes through multiple cellular mechanisms. In most of these processes, such as apoptosis, MAPKs have a dual role since they can act as activators or inhibitors, depending on the cell type and the stimulus. In this review, we present the main pro- and anti-apoptotic mechanisms regulated by MAPKs, as well as the crosstalk observed between some MAPKs. We also describe the basic signaling properties of MAPKs (ultrasensitivity, hysteresis, digital response), and the presence of different positive feedback loops in apoptosis. We provide a simple guide to predict MAPKs’ behavior, based on the intensity and duration of the stimulus. Finally, we consider the role of MAPKs in osmostress-induced apoptosis by using Xenopus oocytes as a cell model. As we will see, apoptosis is plagued with multiple positive feedback loops. We hope this review will help to understand how MAPK signaling pathways engage irreversible cellular decisions.

Upon depletion of nutrients, Myxococcus xanthus forms mounds on a solid surface. The differentiation of rod-shaped cells into stress-resistant spores within mounds creates mature fruiting bodies. The developmental process can be perturbed by the addition of nutrient medium before the critical period of commitment to spore formation. The response was investigated by adding a 2-fold dilution series of nutrient medium to starving cells. An ultrasensitive response was observed, as indicated by a steep increase in the spore number after the addition of 12.5% versus 25% nutrient medium. The level of MrpC, which is a key transcription factor in the gene regulatory network, correlated with the spore number after nutrient medium addition. The MrpC level decreased markedly by 3 h after adding nutrient medium but recovered more after the addition of 12.5% than after 25% nutrient medium addition. The difference in MrpC levels was greatest midway during the period of commitment to sporulation, and mound formation was restored after 12.5% nutrient medium addition but not after adding 25% nutrient medium. Although the number of spores formed after 12.5% nutrient medium addition was almost normal, the transcript levels of “late” genes in the regulatory network failed to rise normally during the commitment period. However, at later times, expression from a reporter gene fused to a late promoter was higher after adding 12.5% than after adding 25% nutrient medium, consistent with the spore numbers. The results suggest that a threshold level of MrpC must be achieved in order for mounds to persist and for cells within to differentiate into spores. IMPORTANCE Many signaling and gene regulatory networks convert graded stimuli into all-or-none switch-like responses. Such ultrasensitivity can produce bistability in cell populations, leading to different cell fates and enhancing survival. We discovered an ultrasensitive response of M. xanthus to nutrient medium addition during development. A small change in nutrient medium concentration caused a profound change in the developmental process. The level of the transcription factor MrpC correlated with multicellular mound formation and differentiation into spores. A threshold level of MrpC is proposed to be necessary to initiate mound formation and create a positive feedback loop that may explain the ultrasensitive response. Understanding how this biological switch operates will provide a paradigm for the broadly important topic of cellular behavior in microbial communities.

The aryl hydrocarbon receptor (AhR) is a nuclear receptor that facilitates a wide transcriptional response and causes a variety of adaptive and maladaptive physiological functions. Such functions are entirely dependent on the type of ligand activating it, and therefore, the nuances in the activation of this receptor at the single-cell level have become a research interest for different pharmacological and toxicological applications. Here, we investigate the activation of the AhR by diverse classes of compounds in a Hepa1c1c7-based murine hepatoma cell line. The exogenous compounds analyzed produced different levels of ultrasensitivity in AhR activation as measured by XRE-coupled EGFP production and analyzed by both flow cytometric and computational simulation techniques. Interestingly, simulation experiments reported herein were able to reproduce and quantitate the natural single-cell stochasticity inherent to mammalian cell lines as well as the ligand-specific differences in ultrasensitivity. Classical AhR modulators 2,3,7,8-tetrachlorodibenzodioxin (10− 1–105 pM), PCB-126 (10− 1–107 pM), and benzo[a]pyrene (10− 1–107 pM) produced the greatest levels of single-cell ultrasensitivity and most maximal responses, while consumption-based ligands indole-3-carbinol (103–109 pM), 3,3′-diindolylmethane (103–108 pM), and cannabidiol (103–108 pM) caused low-level AhR activation in more purely graded single-cell fashions. All compounds were tested and analyzed over a 24 h period for consistency. The comparative quantitative results for each compound are presented within. This study aids in defining the disparity between different types of AhR modulators that produce distinctly different physiological outcomes. In addition, the simulation tool developed for this study can be used in future studies to predict the quantitative effects of diverse types of AhR ligands in the context of pharmacological therapies or toxicological concerns.

Stress exists ubiquitously and is critically important for the manufacturing industry. Due to the ultrasensitive mechanoresponse of the emission of 1,1,2,2,-tetrakis(4-nitrophenyl)ethane (TPE-4N), a luminogen with aggregation-induced emission characteristics, the visualization of stress/strain distributions on metal specimens with a pure organic fluorescent material is achieved. Such a fluorescence mapping method enjoys the merits of simple setup, real-time, full-field, on-site, and direct visualization. Surface analysis shows that TPE-4N can form a nonfluorescent, crystalline uniform film on the metal surface, which cracks into fluorescent amorphous fragments upon mechanical force. Therefore, the invisible information of the stress/strain distribution of the metal specimens are transformed to visible fluorescent signals, which generally matches well but provides more details than software simulation. Remarkably, fatigue crack propagation in stainless steel and aluminum alloy can be observed and predicted clearly, further demonstrating the ultrasensitivity and practicability of TPE-4N.

In this work, we report the fast and ultrasensitive detection of a nerve agent simulant in the gas phase, diethyl chlorophosphate (DCP), by using carbazole-based nanofibers from 1. When exposed to trace DCP, the formed pyridine-phosphorylated product in 1 nanofibers can cause amplified ratiometric fluorescence responses, i.e., amplified fluorescence quenching via quenching excitons within the diffusion length of 1 nanofibers and simultaneously amplified turn-on fluorescence responses via harvesting excitons within the diffusion length to give the intramolecular charge transfer (ICT) emission at a longer wavelength. On the basis of these amplified ratiometric fluorescence responses, detection of DCP with fast response (ca. 3 s), ultrasensitivity (4 ppb), and improved selectivity is achieved.

The dimerization and high-order oligomerization of transcription factors has endowed them with cooperative regulatory capabilities that play important roles in many cellular functions. However, such advanced regulatory capabilities have not been fully exploited in synthetic biology and genetic engineering. Here, we engineered a C-terminally fused oligomerization domain to improve the cooperativity of transcription factors. First, we found that two of three designed oligomerization domains significantly increased the cooperativity and ultrasensitivity of a transcription factor for the regulated promoter. Then, seven additional transcription factors were used to assess the modularity of the oligomerization domains, and their ultrasensitivity was generally improved, as assessed by their Hill coefficients. Moreover, we also demonstrated that the allosteric capability of the ligand-responsive domain remained intact when fusing with the designed oligomerization domain. As an example application, we showed that the engineered ultrasensitive transcription factor could be used to significantly improve the performance of a "stripe-forming" gene circuit. We envision that the oligomerization modules engineered in this study could act as a powerful tool to rapidly tune the underlying response profiles of synthetic gene circuits and metabolic pathway controllers.

New dual electrochemical and fluorescence sensitive curcumin-graphene quantum dots sensing platform coated on the transparent Indium-Tin-Oxide electrode was developed to sense APOe4 DNA, responsible of Alzheimer's disease. Curcumin molecule with its dual fluorescence and electrochemical properties was electropolymerized on GQDs-ITO surface. EDC/NHS chemistry was used to covalently immobilize an amino-substituted DNA probe via a malonic acid spacer. Quenching of curcumin signals following hybridized DNA complex was employed to quantify APOe4 DNA. Amperometric studies revealed an ultrasensitive behavior toward the formation of DNA complex with a sensitivity of 4.74 nA.mL.pg −1 and a limit of detection as low as 0.48 pg mL −1. The platform exhibits very good performances such as repeatability, reproducibility, selectivity and long storage stability. Fluorescence results were established for the support and the complementarity of electrochemical results. Founded results confirmed the ultrasensitivity of platform with comparable performances. Recorded results in human blood plasma demonstrated the high efficacy of curcumin system sensing even in the clinical matrix.

Understanding signaling and other complex biological processes requires elucidating the critical roles of intrinsically disordered proteins (IDPs) and regions (IDRs), which represent ∼30% of the proteome and enable unique regulatory mechanisms. In this review, we describe the structural heterogeneity of disordered proteins that underpins these mechanisms and the latest progress in obtaining structural descriptions of conformational ensembles of disordered proteins that are needed for linking structure and dynamics to function. We describe the diverse interactions of IDPs that can have unusual characteristics such as "ultrasensitivity" and "regulated folding and unfolding". We also summarize the mounting data showing that large-scale assembly and protein phase separation occurs within a variety of signaling complexes and cellular structures. In addition, we discuss efforts to therapeutically target disordered proteins with small molecules. Overall, we interpret the remodeling of disordered state ensembles due to binding and post-translational modifications within an expanded framework for allostery that provides significant insights into how disordered proteins transmit biological information.

An oxidase-like mimic system based on facilely synthesized Ag@Ag3PO4 microcubes (Ag@Ag3PO4MCs) was designed and utilized to detect mercury ions with high selectivity and ultrasensitivity. Ag@Ag3PO4MCs with an average size of ca. 1.6 μm were synthesized by the reaction of [Ag(NH3)2](+) complex and Na2HPO4 and subsequent photoreduction under ultraviolet light. The as-prepared Ag@Ag3PO4MCs can effectively catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) and o-phenylenediamine (OPD) in the presence of dissolved oxygen in slightly acidic solution, exhibiting oxidase-like activities rather than peroxidase-like activity. Interestingly, the introduction of Ag nanoparticles (AgNPs) on the surfaces of Ag3PO4MCs can dramatically enhance the oxidase-like activities due to a synergistic effect between AgNPs and Ag3PO4MCs, as evidenced by the faster oxidation speed of TMB and OPD than that of native Ag3PO4MCs in the presence of dissolved oxygen. The enzyme kinetics can be well-explained by the Michaelis-Menten equation. As "poisoning" inhibitor, Hg(2+) ions can inhibit the enzyme reaction catalyzed by Ag3PO4MCs or Ag@Ag3PO4MCs. On the basis of this effect, a colorimetric Hg(2+) sensor was developed by the enzyme inhibition reaction of Ag3PO4MCs or Ag@Ag3PO4MCs. The excellent specific interaction of Hg-Ag or Hg(2+)-Ag(+) provides high selectivity for Hg(2+) over interfering metal ions. Meanwhile, the sensitivity of this sensor to Hg(2+) is extremely excellent with a limit of detection as low as 0.253 nM for Ag@Ag3PO4MCs. Considering the advantages of low detection limit, low cost, facile preparation, and visualization, the colorimetric Ag@Ag3PO4MCs sensor shows high promise for the testing of Hg(2+) in water samples.

A surface atomic-ligand exchange method is applied the first time in the construction of photodetectors (PDs) based on PbS quantum dots (QDs) for ultrasensitivity. The device thus produces a high photosensitivity to visible and near-infrared light with a photoresponsivity up to 7.5 × 103 A W−1 and a high stability in air. In particular, these PbS-QD-based PDs show the capability of following a pulse light with a frequency up to 100 kHz well at a relatively fast response time/recovery time of ≈4/40 μs, much faster than most previous QD-based PDs. The short response time is attributed to modification for the surface of the PbS-QDs by cetyltrimethylammonium bromide treatment, which effectively improves the contact between the QDs and the Au electrodes, leading to extracting a high carrier mobility (≈0.142 cm2 V−1 s−1). These findings show the great potential of PbS-QDs as high-speed nano-photodetectors, and, more importantly, demonstrate the importance of the surface atomic-ligand exchange method in the construction of QD-based devices.

In the simplest case, substrate competition arises if two ligands compete for access to a single binding site of a receptor protein (or enzyme). If the two ligands exhibit different binding affinities the competition becomes biased: as long as the receptor concentration remains lower than that of the high-affinity ligand the latter blocks all of the available binding sites so that the concentration of the complex comprising the low-affinity ligand remains low. The latter only rises if the receptor concentration is increased beyond that of the high-affinity ligand. Depending on the binding affinity of the low-affinity ligand this increase may then occur in an ultrasensitive manner. Similar behavior has been observed in a phosphorylation/dephosphorylation cycle involved in cell-cycle regulation. However, a steady state analysis shows that in this case the threshold concentration is modulated by the catalytic rate constants for phosphorylation and dephosphorylation of the high-affinity substrate. As a consequence, there exists a trade-off between the dynamic range of the system as measured by the maximal phosphorylation level of the substrate and the sensitivity of the system as measured by the position of the threshold. Using the ratio of the binding affinities as a small parameter we derive explicit expressions for the stimulus-response curves as a function of the receptor (or enzyme) concentration as well as conditions for the occurrence of ultrasensitivity. Interestingly, the network motifs investigated in this study are described by structurally similar steady state equations indicating that the analysis presented here may be extendable for analyzing substrate competition in more complex regulatory networks.

Colloidosomes are robust microcapsules attractive for molecular sensing because of their characteristic micron size, large specific surface area, and dual-phase stability. However, current colloidosome sensors are limited to qualitative fluorogenic receptor-based detection, which restrict their applicability to a narrow range of molecules. Here, we introduce plasmonic colloidosome constructed from Ag nanocubes as an emulsion-based 3D SERS platform. The colloidosomes exhibit excellent mechanical robustness, flexible size tunability, versatility to merge, and ultrasensitivity in SERS quantitation of food/industrial toxins down to sub-femtomole levels. Using just 0.5 μL of sample volumes, our plasmonic colloidosomes exhibit >3000-fold higher SERS sensitivity over conventional suspension platform. Notably, we demonstrate the first high-throughput multiplex molecular sensing across multiple liquid phases.

A pair of reversible photochemical reactions correlates their reactant and product specifically, and such a correlation uniquely distinguishes their correlated signal from others that are not linked by this reversible reaction. Here a nanoparticle-shielded fluorophore is photodriven to undergo structural dynamics, alternating between a green-fluorescence state and a red-fluorescence state. As time elapses, the fluorophore can be in either state but not both at the same time. Thus, the red fluorescence is maximized while the green fluorescence is minimized and vice versa. Such an antiphase dual-color (AD) corelationship between the red and green fluorescence maxima as well as between their minima can be exploited to greatly improve the signal-to-noise ratio, thus enhancing the ultimate detection limit. Potential benefits of this correlation include elimination of all interferences originating from single-color dyes and signal amplification of AD photoswitching molecules by orders of magnitude.

Detection of miRNAs presents a particular challenge because of their limited size, high sequence homology and greatly various expression level. In this work, an ultrasensitive, label-free and isothermal miRNA detection was developed based on asymmertric hairpin probe, exonuclease I(Exo I) and SYBR Green I. The method employed asymmetric hairpin probe to perform cycled polymerization and Exo I to reduce background signal. In the presence of the target miRNA, the target triggers probe-mediated cycled polymerization reactions to generate lots of dsDNA products. The dsDNA product effectively prevents itself from being degraded by Exo I and permitted the insertion of more fluorescence dye into it to enlarge the fluorescence signal. In the absence of the target miRNA, there was no probe-mediated polymerization reaction, and the probe was digested by Exo I added, which minimized the intercalation of fluorescence dye to reduce the background signal. The combination of the probe-mediated cycled polymerization with the Exo I-assisted background reduction allows us to achieve a detection limit of 5×10−18 mol/L. Owing to its ultrasensitivity, excellent specificity, convenience and low-cost, this method might hold out great promise in miRNA detection.

In the era of systems biology and bigdata, reductionism is often viewed aspasse´. But it can come in handy, espe-cially when you have a theory to test.Despite decades of study, the field ofbiochemical signal transduction is stillrife with mechanistic controversies,and one reason is that even systemsof a few interacting components canexhibit complex and nonintuitive be-havior.Manymechanismsareproposed,but most tests are confounded by thecomplexityofthe underlyingbiologicalsystems in which they are tested.Agood,andperhapsevennecessaryfirststeptotestinganyproposedmechanism,is to recapitulate itin a minimalbiolog-ical model that removes as many of theconfounding variables as possible, suchas a transfected cell. Mukhopadhyayet al. (1) followed exactly such anapproach to testing their hypotheticalmechanism for ultrasensitivity in T cellreceptor signaling.A longstanding question in immunereceptor signaling is why the T cellantigen receptor (TCR; Fig. 1)issocomplex, and in particular, why itscytoplasmic domains contain such alarge number of tyrosine phosphoryla-tion sites (2). In fact, multisite phos-phorylation is a common feature ofmany receptor signaling systems, butthe T cell receptor is notable for thelarge total number of sites—20—andthat these sites occur as pairs in highlyconserved regions called tyrosine-based activation motifs (ITAMs) (3).The TCR has a total of 10 ITAMs,six of which occur in the subunitcomprising the z-chain homodimer.This high degree of apparent redun-dancy has motivated numerous investi-gations, but the functional role of thehigh ITAM multiplicity of the TCR re-mains an open question (4). Severalexperimental approaches have beenemployed to examine the role of TCRITAMs in vivo during T cell develop-ment, but these studies have oftenyielded contradictory results (5,6).Takingamoremechanisticapproach,Dusheketal.(7)proposedthatmultisitephosphorylation could enable the TCRsystem to generate sharp switchlike(or ultrasensitive) responses to inputs.Recent work has shown that TCRsignaling can exhibit sharp responsethresholds to input signals, but knownmechanisms for generating such beha-vior involve components considerablydownstream of the receptor itself (8).Dushek et al. (7) proposed a novelmechanism that incorporated diffusion-limited interactions among membrane-associated kinases and phosphatasesand their multisite substrate. The TCRz-dimer subunit with its 12 tyrosinesseemed like a natural system in whichto test their model, and they proposedexperiments based on a previouslydeveloped transfectant system ( 9).Not long after, James and Vale (10)used a different transfectant system toinvestigate the mechanism of T cellactivation, demonstrating that key fea-tures such as synapse formation andrecruitment of the cytosolic kinaseZAP-70 could be recapitulated in anonimmune cell with a minimal num-ber of transfected components. Sub-sequently, Hui and Vale (11) wentfurther along a reductionist path toinvestigate T cell signaling mecha-nisms, using a fully in vitro systemcomposed of purified proteins on lipo-somes to characterize the dynamics ofearly phosphorylation events involvingthe TCR z-chain and CD45 and the ki-nases Lck, which is primarily respon-sible for z-chain phosphorylation, andCsk, which regulates Lck. Althoughboth of these studies addressed mecha-nistic questions, they did not present amathematical framework with whichto analyze and further interpret thefindings.In the meantime, Mukhopadhyayet al. (12) further developed theircomputational model of multisiteTCR phosphorylation building on twokey hypotheses, supported by earlierexperimental work: (1) that z-ITAMsare phosphorylated in a specificsequence and (2) that ZAP-70 exhibitsincreasing affinity for each ITAM inthe sequence. Together, these wereshown to be necessary and sufficient

Metal‐topological insulator–metal subwavelength structure is proposed to achieve ultrasensitivity, room temperature terahertz photodetectors. Due to peculiar properties of topological surface state (TSS), direct detection of low energy photons can be enabled by a localized THz field, exhibiting both high sensitivity and fast response at the self‐powered and the bias mode, opening up a route for large area, fast imaging.

Many proteins exhibit dynamic activation patterns in the form of irregular pulses. Such behavior is typically attributed to a combination of positive and negative feedback loops in the underlying regulatory network. However, the presence of positive feedbacks is difficult to demonstrate unequivocally, raising the question of whether stochastic pulses can arise from negative feedback only. Here, we use the protein kinase A (PKA) system, a key regulator of the yeast pulsatile transcription factor Msn2, as a case example to show that irregular pulses of protein activity can arise from a negative feedback loop alone. Simplification to two variables reveals that a combination of zero-order ultrasensitivity, timescale separation between the activator and the repressor, and an effective delay in the feedback are sufficient to amplify a perturbation into a pulse. The same circuit topology can account for both activation and inactivation pulses, pointing toward a general mechanism of stochastic pulse generation. This work was supported by the Spanish Ministry of Economy and Competitiveness and FEDER (project FIS2015-66503-C3-1-P), and by the Generalitat de Catalunya (project 2017SGR1054). R.M.C. acknowledges financial support from La Caixa foundation. J.G.O. acknowledges support from the ICREA Academia programme and from the “Mar ́ıa de Maeztu” Programme for Units of Excellence in R&D (Spanish Ministry of Economy and Competitiveness, MDM-2014-0370). Y.L. acknowledges the support from National Natural Science Foundation of China (Grant No. 31771425) and the Thousand Young Talents Program of China. M.B.E. is a Howard Hughes Medical Institute Investigator. This work was supported by National Science Foundation grant #1547056.

The bacterial flagellar motor is capable of adapting to changes in the concentrations of extracellular chemical stimuli by changing the composition of the switch complex of the flagellar motor. Such remodeling-based adaptation complements the receptor-mediated adaptation in the chemotaxis network to help maintain high sensitivity in the response of the motor to phospho-CheY concentrations, despite cell-to-cell variability in the abundances of chemotaxis proteins. In this chapter, a modeling approach is described that explains the mechanisms of switch-remodeling and motor-mediated adaptation. The approach is based on observations of structural differences, associated with the direction of motor rotation, that modulate the strength of FliM/FliN binding within the switch. By modulating the number of CheY-P-binding sites within the motor, remodeling maximizes sensitivity over a range of signal levels.

We demonstrate a new semiconducting polymer dot: the poly(o-phenylenediamine)–Rhodamine B copolymer dot (Pp–RhB dot), which emits in the red wavelength range. The Pp–RhB dots can be used as an ultrasensitive fluorescence probe for NO2−in vivo and show high selectivity and ultrasensitivity (detection limit: 2.0 × 10−11 M) for NO2−. The fluorescence of Pp–RhB dots is decreased (φ = 0.014) as a result of fast photoinduced electron transfer (PET) between the modulator (poly(o-phenylenediamine)) and the transducer (RhB), but the N–NO2 bonding mode prevents PET, causing the fluorescence emission to be enhanced (φ = 0.92). This probe effectively avoids the influence of auto-fluorescence in biological systems and gave positive results when tested in both aqueous solution and living cells.

Recent advances have shown increasing designs of nucleic acid organizations via controlling the thermodynamics and kinetics of oligonucleotides. Nevertheless, deeper understanding and further applications of these DNA nanotechnologies are majorly hampered by the lack of effective analytical methodologies that are competent enough to investigate them. To deliver a potential solution, here we developed an innovative exploration that employed the emerging nanopore technique to characterize DNA organization at the single-molecule level and in completely homogeneous condition without covalent modification. With the help of counting and profiling the translocation-induced current drop of a DNA assembly structure passing through a conical glass nanopore (CGN), we have directly verified the formation of the individual double-helix concatemer generated from our model, hybridization chain reaction (HCR). Due to the ultrasensitivity of the nanopore technology, those concatemers that were difficult to observe on a conventional electrophoresis image were brought to light. The translocation duration time also provided the approximate length and folding information for the concatemers. These advantages were proven also applicable to structures with more sophisticated folding behaviors. Eventually, when coupling with an upstream reaction, CGN was further turned to a universal detector that was capable of even detecting other nucleic acid organization behaviors as well as targets that were unable to generate huge products. All of these results are expected to promote deeper study and applications of the nanopore technique in the field of nucleic acid nanotechnology.

Much of the complexity of regulatory networks derives from the necessity to integrate multiple signals and to avoid malfunction due to cross-talk or harmful perturbations. Hence, one may expect that the input-output behavior of larger networks is not necessarily more complex than that of smaller network motifs which suggests that both can, under certain conditions, be described by similar equations. In this review, we illustrate this approach by discussing the similarities that exist in the steady state descriptions of a simple bimolecular reaction, covalent modification cycles and bacterial two-component systems. Interestingly, in all three systems fundamental input-output characteristics such as thresholds, ultrasensitivity or concentration robustness are described by structurally similar equations. Depending on the system the meaning of the parameters can differ ranging from protein concentrations and affinity constants to complex parameter combinations which allows for a quantitative understanding of signal integration in these systems. We argue that this approach may also be extended to larger regulatory networks.

Metal ions play a vital role in many biological processes. An important factor in these processes is the dynamics of exchange between ion bound-water molecules and the bulk. Although structural and dynamical properties of labile waters bound to metal ions, such as Na+ and Ca2+, can be elucidated using molecular dynamics simulations, direct evaluation of rates of exchange of waters rigidly bound to high charge density Mg2+, has been elusive. Here, we report a universal relationship, allowing us to determine the water exchange time on metal ions as a function of valence and hydration radius. The proposed relationship, which covers times spanning 14 orders of magnitude, highlights the ultrasensitivity of water lifetime to the ion size, as exemplified by divalent ions, Ca2+ (∼100 ps) and Mg2+ (∼1.5 μs). We show that even when structures, characterized by radial distributions are similar, a small difference in hydration radius leads to a qualitatively different (associative or dissociative) mechanism of water ...

The CRISPR/Cas-based genome editing system has provided a powerful tool for control of gene activity within cells. Here, we model an experimentally plausible architecture harnessing the power of CRISPR/Cas to create a biomolecular bistable switch. The designed in vitro circuit is based on mutual repression of two genes together with two other activator genes. The repression is generated by the binding of catalytically dead endonuclease (dCas9) to the target gene mediated by a guide RNA. The activation is accomplished by use of an antiguide RNA partially complementary to the guide RNA. Using mathematical analysis of the model, we show that the proposed scheme is capable of exhibiting bistability. We further discuss ultrasensitivity of the regulatory modules, and their capacity to manage competition for dCas9 and downstream load.

The Goldbeter-Koshland model has been a paradigm for ultrasensitivity in biological networks for more than 30 years. Despite its simplicity the validity of this model is restricted to conditions when the substrate is in excess over the converter enzymes − a condition that is easy to satisfy in vitro, but which is rarely satisfied in vivo. Here, we analyze the Goldbeter-Koshland model by means of the total quasi-steady state approximation which yields a comprehensive classification of the steady state operating regimes under conditions when the enzyme concentrations are comparable to or larger than that of the substrate. Where possible we derive simple expressions characterizing the input-output behavior of the system. Our analysis suggests that enhanced sensitivity occurs if the concentration of at least one of the converter enzymes is smaller (but not necessarily much smaller) than that of the substrate and if that enzyme is saturated. Conversely, if both enzymes are saturated and at least one of the enzyme concentrations exceeds that of the substrate the system exhibits concentration robustness with respect to changes in that enzyme concentration. Also, depending on the enzyme’s saturation degrees and the ratio between their maximal reaction rates the total fraction of phosphorylated substrate may increase, decrease or change nonmonotonically as a function of the total substrate concentration. The latter finding may aid the interpretation of experiments involving genetic manipulations of enzyme and substrate abundances.

Ultrasensitive response motifs, capable of converting graded stimuli into binary responses, are well-conserved in signal transduction networks. Although it has been shown that a cascade arrangement of multiple ultrasensitive modules can enhance the system's ultrasensitivity, how a given combination of layers affects a cascade's ultrasensitivity remains an open question for the general case. Here, we introduce a methodology that allows us to determine the presence of sequestration effects and to quantify the relative contribution of each module to the overall cascade's ultrasensitivity. The proposed analysis framework provides a natural link between global and local ultrasensitivity descriptors and it is particularly wellsuited to characterize and understand mathematical models used to study real biological systems. As a case study, we have considered three mathematical models introduced by O'Shaughnessy et al. to study a tunable synthetic MAPK cascade, and we show how our methodology can help modelers better understand alternative models. Fil: Altszyler Lemcovich, Edgar Jaim. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Ventura, Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Colman Lerner, Alejandro Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Chernomoretz, Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina

Stratified multisite ubiquitination of RIG-I provides a robust and optimal control of innate antiviral responses., The activation of retinoic acid–inducible gene I (RIG-I), an indispensable viral RNA sensor in mammals, is subtly regulated by ubiquitination. Although multiple ubiquitination sites at the amino terminus of RIG-I have been identified, their functional allocations in RIG-I activation remain elusive. We identified a stratified model for RIG-I amino-terminal ubiquitination, in which initiation at either Lys164 or Lys172 allows subsequent ubiquitination at other lysines, to trigger and amplify RIG-I activation. Experimental and mathematical modeling showed that multisite ubiquitination provides robustness in RIG-I–mediated type I interferon (IFN) signaling. Furthermore, the flexibly controlled ultrasensitivity and IFN activation intensity determine the specificity of the IFN-stimulated gene transcription and manipulate cell fate in antiviral immune response. Our work demonstrates that tunable type I IFN signaling can be regulated through multisite RIG-I ubiquitination and elucidates a new paradigm for dynamic regulation in RIG-I–mediated antiviral signaling.

A method is developed to synthesize surface-enhanced Raman scattering (SERS) materials capable of single-molecule detection, integrated with a microfluidic system. Using a focused laser, silver nanoparticle aggregates as SERS monitors are fabricated in a microfluidic channel through photochemical reduction. After washing out the monitor, the aggregates are irradiated again by the same laser. This key step leads to full reduction of the residual reactants, which generates numerous small silver nanoparticles on the former nanoaggregates. Consequently, the enhancement ability of the SERS monitor is greatly boosted due to the emergence of new “hot spots.” At the same time, the influence of the notorious “memory effect” in microfluidics is substantially suppressed due to the depletion of surface residues. Taking these advantages, two-step photoreduced SERS materials are able to detect different types of molecules with the concentration down to 10−13m. Based on a well-accepted bianalyte approach, it is proved that the detection limit reaches the single-molecule level. From a practical point of view, the detection reproducibility at different probing concentrations is also investigated. It is found that the effective single-molecule SERS measurements can be raised up to ≈50%. This microfluidic SERS with high reproducibility and ultrasensitivity will find promising applications in on-chip single-molecule spectroscopy.

A label-free photoelectrochemical (PEC) aptasensor for K+ was first constructed by direct self-assembly of the K+ aptamer onto the electrodeposited Mn-doped ZnO nanorods. In the presence of K+, the conformation of G-quadruplex changes to a K+-stabilized conformation, which can efficiently prevent the quercetin electron donor from reaching the functionalized photoanode, thus proportionately affecting the quercetin-enhanced photocurrent response. Under the optimal experimental conditions, the fabricated biosensor exhibited a linear response in the K+ concentration range of 0.012 to 12.32 nmol L-1, with a detection limit of 4.0 pmol L-1, which was 3-5 orders of magnitude lower than that of most of the recently reported methods. The presence of other ions did not interfere with the detection of K+, and the results in serum samples agreed well with those obtained by ICP-MS. Using K+ as the detection model, this novel PEC aptasensor exhibited a good performance in terms of ultrasensitivity, selectivity and simplicity, and it was economical. As regards the electrode modification, this work provided a convenient direct self-assembly strategy that did not require the use of ionic polymers such as PDDA as a binder. Thus, this study paves the way for the immobilization of molecular probes for label-free PEC aptasensing.

Here, we demonstrate a phosphorodiamidate morpholino oligos (PMO)-functionalized nanochannel biosensor for label-free detection of microRNAs (miRNAs) with ultrasensitivity and high sequence specificity. PMO, as a capture probe, was covalently anchored on the nanochannel surface. Because of the neutral character and high sequence-specific affinity of PMO, hybridization efficiency between PMO and miRNAs was enhanced, thus largely decreasing background signals and highly improving the detection specificity and sensitivity. The miRNAs detection was realized through observing the change of surface charge density when PMO/miRNAs hybridization occurred. Not only could the developed biosensor specifically discriminate complementary miRNAs (Let-7b) from noncomplementary miRNAs (miR-21) and one-base mismatched miRNAs (Let-7c), but also it could detect target miRNAs in serum samples. In addition, this nanochannel-based biosensor attained a reliable limit of detection down to 1 fM in PBS and 10 fM in serum sample, respectively. It is expected that such a new method will benefit miRNA detection in clinical diagnosis.

To meet the increasing demands for ultrasensitivity in monitoring trace amounts of low-abundance early biomarkers or environmental toxins, the development of a robust sensing system is urgently needed. Here, a novel signal cascade strategy is reported via an ultrasensitive polymeric sensing system (UPSS) composed of gold nanoparticle (gNP)-decorated polymer, which enables gNP aggregation in polymeric network and electrical conductance change upon specific aptamer-based biomolecular recognition. Ultralow concentrations of thrombin (10-18 m) as well as a low molecular weight anatoxin (165 Da, 10-14 m) are detected selectively and reproducibly. The biomolecular recognition induced polymeric network shrinkage responses as well as dose-dependent responses of the UPSS are validated using in situ real-time atomic-force microscopy, representing the first instance of real-time detection of biomolecular binding-induced polymer shrinkage in soft matter. Furthermore, in situ real-time confocal laser scanning microscopy imaging reveals the dynamic process of gNP aggregation responses upon biomolecular binding.

In this series of reviews, we are examining ultrasensitive responses, the switch-like input-output relationships that contribute to signal processing in a wide variety of signaling contexts. In the first part of this series, we explored one mechanism for generating ultrasensitivity, zero-order ultrasensitivity, where the saturation of two converting enzymes allows the output to switch from low to high over a tight range of input levels. In this second installment, we focus on three conceptually distinct mechanisms for ultrasensitivity: multisite phosphorylation, stoichiometric inhibitors, and positive feedback. We also examine several related mechanisms and concepts, including cooperativity, reciprocal regulation, coherent feed-forward regulation, and substrate competition, and provide several examples of signaling processes where these mechanisms are known or are suspected to be applicable.

Control over the sensitivity with which biomolecular receptors respond to small changes in the concentration of their target ligand is critical for the proper function of many cellular processes. Such control could likewise be of utility in artificial biotechnologies, such as biosensors, genetic logic gates, and "smart" materials, in which highly responsive behavior is of value. In nature, the control of molecular responsiveness is often achieved using "Hill-type" cooperativity, a mechanism in which sequential binding events on a multivalent receptor are coupled such that the first enhances the affinity of the next, producing a steep, higher-order dependence on target concentration. Here, we use an intrinsic-disorder-based mechanism that can be implemented without requiring detailed structural knowledge to rationally introduce this potentially useful property into several normally noncooperative biomolecules. To do so, we fabricate a tandem repeat of the receptor that is destabilized (unfolded) via the introduction of a long, unstructured loop. The first binding event requires the energetically unfavorable closing of this loop, reducing its affinity relative to that of the second binding event, which, in contrast occurs at a preformed site. Using this approach, we have rationally introduced cooperativity into three unrelated DNA aptamers, achieving in the best of these a Hill coefficient experimentally indistinguishable from the theoretically expected maximum. The extent of cooperativity and thus the steepness of the binding transition are, moreover, well modeled as simple functions of the energetic cost of binding-induced folding, speaking to the quantitative nature of this design strategy.

Quantitative studies of signal transduction systems have shown that ultrasensitive responses—switch-like, sigmoidal input/output relationships—are commonplace in cell signaling. Ultrasensitivity is important for various complex signaling systems, including signaling cascades, bistable switches, and oscillators. In this first installment of a series on ultrasensitivity, we survey the occurrence of ultrasensitive responses in signaling systems and examine how such responses can arise. We review why the simplest mass action systems exhibit Michaelian responses, and then move on to zero-order ultrasensitivity, a phenomenon that occurs when signaling proteins are operating near saturation. We also discuss the physiological relevance of zero-order ultrasensitivity to cellular regulation.

Reactive oxygen species (ROS) and cellular oxidant stress have long been associated with cancer. Unfortunately, the role of HClO in tumor biology is much less clear than for other ROS. Herein, we report a BODIPY-based HClO probe (BClO) with ultrasensitivity, fast response (within 1 s), and high selectivity, in which the pyrrole group at the meso position has an "enhanced PET" effect on the BODIPY fluorophore. The detection limit is as low as 0.56 nM, which is the highest sensitivity achieved to date. BClO can be facilely synthesized by a Michael addition reaction of acryloyl chloride with 2,4-dimethylpyrrole and applied to image the basal HClO in cancer cells for the first time and the time-dependent HClO generation in MCF-7 cells stimulated by elesclomol, an effective experimental ROS-generating anticancer agent.

In S. cerevisiae, following the Whole Genome Duplication (WGD), GAL1-encoded galactokinase retained its signal transduction function but lost basal expression. On the other hand, its paralogue GAL3, lost kinase activity but retained its signalling function and basal expression, thus making it indispensable for the rapid induction of the S. cerevisiae GAL switch. However, a gal3Δ strain exhibits delayed growth kinetics due to the redundant signalling function of GAL1. The subfunctionalization between the paralogues GAL1 and GAL3 is due to expression divergence and is proposed to be due to the alteration in the Upstream Activating Sequences (UASG ). We demonstrate that the GAL switch becomes independent of GAL3 by altering the interaction between Gal4p and Gal80p without altering the configuration of UASG . In addition to the above, the altered switch of S. cerevisiae loses ultrasensitivity and stringent glucose repression. These changes caused an increase in fitness in the disaccharide melibiose at the expense of a decrease in fitness in galactose. The above altered features of the ScGAL switch are similar to the features of the GAL switch of K. lactis that diverged from S. cerevisiae before the WGD.

The processive phosphorylation mechanism becomes important when there is macromolecular crowding in the cytoplasm. Integrating the processive phosphorylation mechanism with the traditional distributive one, we propose a mixed dual-site phosphorylation (MDP) mechanism in a single-layer phosphorylation cycle. Further, we build a degree model by applying the MDP mechanism to a three-layer mitogen-activated protein kinase (MAPK) cascade. By bifurcation analysis, our study suggests that the crowded-environment-induced pseudoprocessive mechanism can qualitatively change the response of this biological network. By adjusting the degree of processivity in our model, we find that the MAPK cascade is able to switch between the ultrasensitivity, bistability, and oscillatory dynamical states. Sensitivity analysis shows that the theoretical results remain unchanged within a reasonably chosen variation of parameter perturbation. By scaling the reaction rates and also introducing new connections into the kinetic scheme, we further construct a proportion model of the MAPK cascade to validate our findings. Finally, it is illustrated that the spatial propagation of the activated MAPK signal can be improved (or attenuated) by increasing the degree of processivity of kinase (or phosphatase). Our research implies that the MDP mechanism makes the MAPK cascade become a flexible signal module, and the coexistence of processive and distributive phosphorylation mechanisms enhances the tunability of the MAPK cascade.