Experimental Detection Limit Research Articles

High-Performance Trichloroacetic Acid Sensor Based on the Intramolecular Hydrogen Bond Formation and Disruption of a Specially Designed Fluorescent o-Carborane Derivative in the Film State.

Discriminative and sensitive detection of environmentally important and health-related trichloroacetic acid (TCA) suffers from various problems such as bulky instruments and time-consuming operation as well as complex sample processing. Herein, we present a rapid, sensitive, and specific method for the detection of gaseous TCA using a fluorescent single-molecule array. An o-carborane-based benzothiazole derivative (CB-BT-OCH3) with specific fluorescence properties was specifically designed and utilized to fabricate a film-based single-molecule array. It was revealed that the fluorescent film is photochemically stable and extremely sensitive to TCA vapor, depicting an observable fluorescence color change from green to blue. The experimental detection limit is 0.2 ppm, which is lower than the safety limit (1 ppm) required by the threshold limit values and biological exposure indices. In addition, the film could show detectable intensity change within 0.2 s. On the basis of multiple signal responses, a conceptual two-channel-based fluorescent TCA sensor was developed. Importantly, the proposed conceptual sensor paves a new route to the development of specific fluorescent film-based sensor arrays with a single fluorophore as sensing units.

An apta-aggregation based machine learning assay for rapid quantification of lysozyme through texture parameters.

A novel assay technique that involves quantification of lysozyme (Lys) through machine learning is put forward here. This article reports the tendency of the well- documented Ellington group anti-Lys aptamer, to produce aggregates when exposed to Lys. This property of apta-aggregation has been exploited here to develop an assay that quantifies the Lys using texture and area parameters from a photograph of the elliptical aggregate mass through machine learning. Two assay sets were made for the experimental procedure: one with high Lys concentration between 25–100 mM and another with low concentration between 1–20 mM. The high concentration set had a sample volume of 10 μl while the low concentration set had a higher sample volume of 100 μl, in order to obtain the statistical texture values reliably from the aggregate mass. The platform exhibited an experimental limit of detection of 1 mM and a response time of less than 10 seconds. Further, two potential operating modes for the aptamer were hypothesized for this aggregation property and the more accurate mode among the two was ascertained through bioinformatics studies.

High-Performance Ketone Sensing in Vapor Phase Enabled by o-Carborane-Modified Cyclometalated Alkynyl-Gold(III) Complex-Based Fluorescent Films.

Development of high-performance, low-power-consumption, small-sized detectors is a key issue for fabricating specific miniaturized chromatographs (GCs). Herein, we report, for the first-time, utilization of a film-based fluorescent sensor as a GC detector. In the studies, we designed a new o-carborane derivative of a known cyclometalated alkynyl-gold(III) complex, Au-CB. Unlike the parent gold(III) complex, the newly synthesized Au-CB depicted a remarkable aggregation-induced emission (AIE) property, enabling fabrication of a fluorescent film. The film emission is highly sensitive to the presence of ketones such as acetone, 2-pentanone, 3-pentanone, cyclopentanone, etc., in the air. It was demonstrated that the sensing performance of the film could be further improved by changing the film from a planar structure to a tubular one. Via combination with an earlier reported homemade sensory device, a conceptual film-based fluorescent sensor was developed, which demonstrated instant and fully reversible response to the ketones. The experimental detection limits for cyclohexanone and acetone could be lower than 0.08 and 13.0 ppm, respectively. Moreover, the sensor is super stable, as 24 h continuous illumination resulted in less than 1.0% reduction of the fluorescence emission, 50 successive sensings showed no observable decay in the performance, and more than 1 year of storage had no effect upon the property. Further studies demonstrated that the film sensor could be used as a GC detector with performance comparable to the commercial flame ionization detector (FID), which lays the foundation for future applications in specific miniaturized GCs because of its merits in size, power consumption, carrier gas, etc.

A photoelectrochemical enzyme biosensor based on functionalized hematite microcubes for rutin determination by square-wave voltammetry.

A photoelectrochemical biosensing strategy for the highly sensitive detection of the flavonoid rutin was developed by synergizing the photoelectrocatalytic properties of hematite (α-Fe2O3) decorated with palladium nanoparticles (PdNPs) and the biocatalysis towards laccase-based reactions. The integration of α-Fe2O3.PdNPs with a polyphenol oxidase as a biorecognition element yields a novel biosensing platform. Under visible light irradiation, the photoactive biocomposite can generate a stable photocurrent, which was found to be directly dependent upon the concentration of rutin. Under the optimal experimental conditions, the cathodic photocurrent, measured at 0.33V vs. Ag/AgCl, from the square-wave voltammograms presented a linear dependence on the rutin concentration within the range of 0.008-30.0 × 10-8molL-1 (sensitivity: 1.7μA·(× 10-8M-1)·cm-2), with an experimental detection limit (S/N = 3) of 8.4 × 10-11molL-1. The proposed biosensor device presented good selectivity towards rutin in the presence of various organic compounds and inorganic ions, demonstrating the potential application of this biosensing platform in complex matrices. This bioanalytical device also exhibited excellent operational and analytical properties, such as intra-day (standard deviation, SD = 0.21%) and inter-day (SD = 1.30%) repeatability, and long storage stability (SD = 2.80% over 30days).Graphical abstract.

Attachment and Detection of Biofouling Yeast Cells Using Biofunctionalized Resonant Sensor Modality

This research addresses fuel biocontaminant sensing using resonant photonic gratings. The fundamental photonic resonance physics enables compact, economic sensors that provide a rapid, quantitative, and effective modality for detecting and monitoring biochemical bindings. Thus, we present resonant sensors and attendant surface chemistry targeting yeast cells as representative biofouling agents. We apply biorecognition elements (BREs) to functionalize the dielectric grating surface (TiO2) to produce a sensitive, quantitative wavelength shift-based assay for detecting Yarrowia yeast cells. Concanavalin A (Con A) is used as a BRE consisting of carbohydrate-binding proteins binding to ligands on the yeast cell surface. Corresponding resonance wavelength shifts are measured relative to concentration expressed in log10 [C] where C represents the concentration of cells in solution. The assay is shown to be highly specific for detecting Yarrowia cells in various concentrations. The experimental limit of detection (LOD) of the assay is 1.92 for yeast cells in culture media and 1.22 for cells in phosphate buffer saline (PBS). The comparisons of the various functionalization methods show that (3-aminopropyl)triethoxysilane (APTES) with glutardialdehyde (GA) and Con A protocol provides optimal detection of cells in PBS.

An Integral Recognition and Signaling for Electrochemical Assay of Protein Kinase Activity and Inhibitor by Reduced Graphene Oxide-Polydopamine-Silver Nanoparticle-Ti4+ Nanocomposite.

A novel electrochemical biosensing method for protein kinase (PKA) activity was demonstrated by using a reduced graphene oxide-polydopamine-silver nanoparticle-Ti4+ (rGO-PDA-AgNPs-Ti4+) nanocomposite. The obtained nanocomposite possessed an integral capability for phosphopeptide recognition and signal readout. The polydopamine modified reduced graphene oxide (rGO-PDA) was firstly prepared based on a self-polymerization method of dopamine. The silver ions were adsorbed onto polydopamine (PDA) layer and directly reduced into silver nanoparticles (AgNPs), which was used for electrochemical signal reporting. Then, the Ti4+ cations were attached onto the PDA layer for phosphopeptide recognition according to the strong coordination ability of PDA with Ti4+ and phosphate group. The prepared rGO-PDA-AgNPs-Ti4+ nanocomposites were characterized with different methods. The developed rGO-PDA-AgNPs-Ti4+ nanocomposites were then employed for electrochemical analysis of PKA-catalyzed kemptide phosphorylation. The sensitive detection toward PKA activity was realized with an experimental detection limit of about 0.01 U/mL. It may be also extended for the inhibitor evaluation. Thus, it provided a facile and sensitive means for electrochemical analysis of PKA activity and inhibitor screening.

Methane Detection with a Tungsten‐Calix[4]arene‐Based Conducting Polymer Embedded Sensor Array

AbstractThe detection of methane is important for industry, environment, and our daily life, but is made challenging by its small size, high volatility, and nonpolar nature. Herein, a tungsten‐capped calix[4]arene‐based p‐doped conducting polymer with hexafluorophosphate or perchlorate counter‐anions as a transducer is used to detect methane in dry air. The host–guest interaction between calixarene moieties within the polymer chain and methane molecules leads to the resistance variation of the polymer. The experimental limit of detection (LoD) of methane for the polymer‐based sensor is demonstrated to be less than 50 ppm at room temperature, and the extrapolated theoretical LoD of 2 ppm represents exceptional sensitivity to methane. Furthermore, the discrimination of methane from interfering volatile organic compounds is achieved by exploiting a sensor array using complementary chemiresistors and principal component analysis.

An Electrochemical ATP Biosensor with Enzymes Entrapped within a PEDOT Film

AbstractThis paper reports on the electrochemical behavior of an ATP biosensor that utilizes glucose oxidase (GOx) and hexokinase (Hex) immobilized within the electroactive polymer, polyethylenedioxythiophene (PEDOT). This biosensor design detects ATP indirectly at 0.85 V vs. Ag/AgCl based on the oxidation current for enzymatically generated H2O2, and at −0.20 V; a potential at which improved analyte selectivity is achieved. The detection figures of merit at both detection potentials are a response time of 15±1 s, an experimental detection limit of 10.0±0.2 μmol L−1 (S/N=3), and a sensitivity in the range of 100–500 mA M−1cm−2.

High sensitive pyridine chemiresistive sensors based on azacyclobutane modified perylene bisimide derivatives

A novel bay brominated perylene bisimide derivative with azacyclobutane modified (PBI–1N) was designed and synthesized. The derivative had good solubility in organic solvents and excellent self-assembly properties.The networked structure of PBI-1N was transferred to the surface of the device to prepare the chemiresistive sensor, and its sensing performance was studied. There is a higher sensitivity for PBI-1N-based sensor with experimental detection limit of pyridine reaching 375 ppb. Moreover, the pyridine and its derivatives can be distinguished by introducing halogen bond interaction. This provides a useful reference for the molecular structure design of high performance organic semiconductors in the future.

Bloch surface waves biosensing in the ultraviolet wavelength range – Bragg structure design for investigating protein adsorption by in situ Kretschmann-Raether ellipsometry

We designed a Bragg mirror structure with an SiO2 top layer to create a resonance in the ultraviolet wavelength range, near the absorption peak position of various proteins. We demonstrate that the wavelength of enhanced sensitivity can be adjusted by proper design of the 1D photonic structure. The possibility to design the wavelength of enhanced sensitivity supports measurements of better selectivity, optimized for the absorption of the target material. Since the width of the resonant peak in the reflectance spectra can be sharper than those of plasmonics, and they can be positioned at more favourable regions of the instrument and material (e.g., in terms of intensity or selectivity), the sensitivity can exceed those of plasmon-enhanced measurements. In this study we demonstrate the main features of the concept at the example of in situ spectroscopic ellipsometry of fibrinogen adsorption in the Kretschmann-Raether configuration. We realized a resonant peak with a full width at half maximum of 3 nm near the wavelength of 280 nm, which coincides with the absorption maximum of fibrinogen. The influence of depolarization and surface roughness on the measurements, and the potential for improving the current experimental detection limit of 45 pg/mm2 is also discussed.

Direct measurements of electronic ground state ro-vibrationally excited D2 molecules produced on ECR plasma-facing materials by means of VUV-FT absorption spectroscopy

Ro-vibrationally excited molecules of deuterium are involved in non-equilibrium chemical reactions in divertor region of tokamak (see molecular assisted recombination) or in neutral beam injector to produce negative ion (see dissociative electron attachment) for fusion plasmas. These molecules produced both in a plasma volume and on surfaces are no longer in their electronic ground state X1∑g+(v”⩾0,J”) and populate non-uniformly different vibrational (v″) and rotational levels (J″). The high resolution VUV Fourier Transform spectrometer of the DESIRS beam line (SOLEIL synchrotron) is applied to directly scrutinize the ro-vibrationally excited levels of the D2 ground state from v″ = 0 to 10 and J″ up to 8 in an electron cyclotron resonance (ECR) cold plasma. This is performed for different plasma-facing materials, i.e. quartz, tantalum, tungsten, and stainless steel, in order to compare their relative impact in molecular excitation through recombinative desorption from surfaces. A significant effect of these materials on the absolute distribution of the vibrationally states has been found above v″ > 3. The experimental detection limit for Quartz is v″ = 7 whereas tungsten and stainlees steel excite the molecules up to v″ = 9 and tantalum up to v″ = 10. The use of a bare, temperature-controlled surface of Quartz as a reference, compared to metallic surfaces, allows us to determine the relative production of these excited levels. The recombinative desorption rate on tungsten and stainless steel compared to quartz is ~ 2.6 higher and reaches ~ 5 for tantalum.

A Novel Hybrid Plasmonic Resonator With High Quality Factor and Large Free Spectral Range

Whispering gallery (WG) mode resonators are experiencing a surge in the application of biosensing, attributing to high speed, flexibility, low cost, multi-analyte detection and compatibility with extra optical elements. The decoration of localized surface plasmon nanostructures to a dielectric WG mode resonator offers unprecedented sensitivity compared with a pure dielectric resonator. However, at the moment, the experimental quality (Q) factors of dielectric micro rings always achieve the value of 104 to 106, whereas the only reported hybrid counterpart possesses the Q factor of 300 and most studies are focused on the theoretical design. The resulting experimental detection limits (DLs) are in the range of 10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">-6</sup> 10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">-5</sup> RIU, which is one order of magnitude lower than the best value of hybrid resonators. The hybrid resonator should possess the experimental Q factor of 5000 to 10000 so as to match their purely dielectric rivals. We demonstrated a novel hybrid plasmonic WG mode resonator working on the multimode resonance mechanism. Under the condition of the multimode resonance, predominant field was distributed on the waveguide surface resulting in an improved bulk sensitivity. The multimode resonance promoted satisfied resonance frequency reduced and thus a high Q factor and a low value of DL could be achieved in the experiment. Afterwards, the resonator was made via micro-and nanofabrication. It was shown that the experimental Q factor of 5685 and the free spectral range larger than 35 nm, which were superior to the previously reported value to our best knowledge. Hypoglycemia and Hyperglycemia will produce noticeable organ damage over time. Early detection of glucose concentration is crucial for clinical diagnosis. The proposed resonator was used for the detection of glucose solution. The experimental sensitivity was 428 nm/RIU and the DL was 1.31 × 10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">-5</sup> RIU. The device with a footprint of 1500 μm × 12.7 μm, is able to integrate with other optical elements and developed as a commodity in the future.

Ultra-fine nickel sulfide nanoclusters @ nickel sulfide microsphere as enzyme-free electrode materials for sensitive detection of lactic acid

Abstract A new facile fabrication of ultrafine nanoclusters of nickel-sulfides with an average diameter of ~3.4 nm on NiS microsphere (NiS-NC@NiS-MS) on bare nickel foam (NiF) substrate (designated as NiS-NC@NiS-MS/NiF) using one-step electrochemical strategy for the direct electrochemical oxidation and sensing of lactic acid (LA) is demonstrated. The dimension, surface morphology, crystalline nature, chemical composition, and electrochemical characteristics of the NiS Nanoclusters @ NiS Microsphere are systematically studied with field-emission scanning electron microscope (FE-SEM), high-resolution transmission electron microscope (HR-TEM), X-ray diffraction, X-ray photoelectron spectroscopy (XPS), and cyclic voltammetric measurements. The developed NiS-NC@NiS-MS/NiF electrode exhibits an excellent enzyme-mimic electrocatalytic activity towards the lactic acid oxidation at the less positive potential of ~0.45 V (vs Ag/AgCl)) and high catalytic anodic current of ~1.7 mA. The present enzyme-mimic NiS-NC@NiS-MS catalyst based sensor presents an experimental lowest detection limit of 0.5 μM, high sensitivity of 0.39 μA μM−1 with a rapid sensing response, and a linear range from 0.5 μM to 85.5 μM. Owing to the uniform and high dispersion of small NiS nanoclusters, abundant electrochemical active sites, and improved mass-transfer kinetics, the NiS-NC@NiS-MS/NiF electrode delivers a high performance lactic acid sensing. The developed non-enzymatic lactic acid biosensor based on NiS nanoclusters shows an outstanding anti-interference ability in the presence of various potential interferences (uric acid (UA), ascorbic acid (AA), paracetamol (PA), Mg2+, Na+ and Ca2+), good reproducibility, and long-term durability. Moreover, the present sensor is successfully tested the lactic acid sensing in practical human urine samples.

Photoelectrochemical Immunosensor for Sensitive Quantification of Prostate Specific Antigen in Human Serum Samples Exploiting BaTiO3−CdS

AbstractA novel label‐free photoelectrochemical immunosensor was developed for the determination of prostate‐specific antigen (PSA) based on fluorine‐doped tin oxide (FTO) electrode modified with barium titanate and cadmium sulfide (BaTiO3−CdS/FTO). The photoelectrochemical current density of the BaTiO3 platform sensitized with CdS for the ascorbic acid (AA) donor molecule was higher than that observed in the absence of CdS. The BaTiO3−CdS/FTO platform was modified with PSA antibodies to produce the anti‐PSA/BaTiO3−CdS/FTO immunosensor, which was incubated in solutions of PSA to inhibit the photocurrent density of the immunosensor for AA. The photocurrent density of the anti‐PSA/BaTiO3−CdS/FTO immunosensor presented a linear relationship with the logarithm of the PSA concentration ranging from 1 pg mL−1 to 1 μg mL−1 with good sensitivity. The proposed method presented an experimental detection limit of 1 pg mL−1 (S/N=3). The immunosensor was successfully employed for the determination of PSA in blood serum samples, with recoveries between 98.7 %–104.4 %.

Solid-Phase PCR-Amplified DNAzyme Activity for Real-Time FO-SPR Detection of the MCR-2 Gene.

The polymerase chain reaction (PCR) has been the gold standard molecular analysis technique for decades and has seen quite some evolution in terms of reaction components, methodology, and readout mechanisms. Nucleic acid enzymes (NAzymes) have been used to further exploit the applications of PCR, but so far the work was limited to the colorimetric G-quadruplex or fluorescent substrate cleaving NAzymes. In this study, a solid-phase, fiber optic surface plasmon resonance (FO-SPR) technique is presented as an alternative readout for PCR utilizing NAzymes. First, the surface cleavage activity of DNAzyme-extended amplicons (DNAzyme-amps) is established, followed by optimization of the PCR conditions, which are required for compatibility with the FO-SPR system. Next, by integrating the complement of a 10-23 DNAzyme into the primer pair, PCR-amplified DNAzyme-amps were generated, tested, and validated on qPCR for the detection of the antimicrobial resistance gene MCR-2. Once validated, this primer concept was developed as a one-step assay, driven by PCR-amplified DNAzymes, for FO-SPR-based sensitive and specific detection. Using gold nanoparticle labeled RNA-DNA hybrid strands as substrate for the DNAzyme, PCR-amplified DNAzyme-amps generated in the presence of MCR-2 gene were monitored in real-time, which resulted in an experimental limit of detection of 4 × 105 copy numbers or 6.6 fM. In addition, the DNAzyme-based FO-PCR assay was able to discriminate between the MCR-1 and MCR-2 genes, to further prove the specificity of this assay. Henceforth, this DNAzyme-based fiber optic PCR assay provides a universally applicable, real-time system for the detection of virtually any target NA, in a specific and sensitive manner.

Selective detection of cadmium ions using plasmonic optical fiber gratings functionalized with bacteria.

Environmental monitoring and potable water control are key applications where optical fiber sensing solutions can outperform other technologies. In this work, we report a highly sensitive plasmonic fiber-optic probe that has been developed to determine the concentration of cadmium ions (Cd2+) in solution. This original sensor was fabricated by immobilizing the Acinetobacter sp. around gold-coated tilted fiber Bragg gratings (TFBGs). To this aim, the immobilization conditions of bacteria on the gold-coated optical fiber surface were first experimentally determined. Then, the coated sensors were tested in vitro. The relative intensity of the sensor response experienced a change of 1.1 dB for a Cd2+ concentration increase from 0.1 to 1000 ppb. According to our test procedure, we estimate the experimental limit of detection to be close to 1 ppb. Cadmium ions strongly bind to the sensing surface, so the sensor exhibits a much higher sensitivity to Cd2+ than to other heavy metal ions such as Pb2+, Zn2+ and CrO42- found in contaminated water, which ensures a good selectivity.

SnS2 Quantum Dot-Based Optoelectronic Flexible Sensors for Ultrasensitive Detection of NO2 Down to 1 ppb.

Transition-metal dichalcogenides (TMDs) have gained intense interest for their outstanding optoelectronic and electrochemical characteristics, utilized in versatile applications such as gas sensors and photodetectors. However, TMD-based chemiresistors suffer from poor sensitivity at ppb-level detection, and the experimental detection limit fails to reach 1 ppb. Herein, SnS2 QD/graphene nanoheterostructures as functional flexible sensors are fabricated for NO2 gas and light detection at room temperature. The semiconductor type of the nanohybrids can be shifted between p-type and n-type by adjusting the proportion of the components, both of which exhibit excellent gas-sensing properties. The ppb-level NO2 detection is realized even under room temperature with superior sensitivity (860% to 125 ppb), fast response (114 s), and recovery (166 s). It also demonstrates ultrahigh sensitivity and broadband photodetection in the visible region. The photoresponsivity can reach upto 2.08 × 103 A/W under blue light illumination and under room temperature. Especially, the influence of light illumination of different wavelengths and intensities on gas-sensing performance is studied. Red light (1 mW/cm2) greatly enhances the sensitivity up to 5.1 folds, and the device performs obvious response to NO2 at concentrations as low as 1 ppb. Ab initio density functional theory calculation and band theories are applied to explain the interaction of the components and the effect of the light excitation inducing charge carriers on gas-sensing equilibrium.

Nanoscale heterojunctions of rGO-MoS2 composites for nitrogen dioxide sensing at room temperature

Chemiresistive sensors, employing binary and ternary hybrids of reduced graphene oxide (rGO), are developed to detect nitrogen dioxide (NO2) gas at parts per billion level (ppb) at room temperature. The sensors based on hierarchical structures of molybdenum disulphide (MoS2) sheets decorated rGO and further integration of it with silver nanoparticles (Ag NPs) exhibit improved sensing responses with lower detection limits than the unary counterpart (rGO). An increase of nearly 500% in sensing response is observed in the ternary hybrid device over rGO alone at a concentration of 1 ppm and a 1145% increase in response is observed at 104 ppm. The ternary hybrid device outperforms the binary and the unary counterparts in terms of sensitivity to NO2 over a wide concentration range from 1 ppm to 104 ppm. Additionally, the ternary hybrid device is highly selective to NO2 amongst other atmospheric pollutants like ammonia, sulphur dioxide and carbon monoxide. An experimental detection limit of 50 ppb is further achieved with this device which is lesser than the 53 ppb permissible limit declared by Environmental Protection Agency (EPA). A synergistic effect was achieved with the binary and the ternary hybrids with the electronic modulations at the nanoscale interfaces at the nanoheterojunctions playing a key role in selective and enhanced adsorption of NO2 at room temperature.

Magnetic dispersive solid phase extraction for simultaneous enrichment of cadmium and lead in environmental water samples

A new magnetic dispersive solid phase extraction (MDSPE) method and graphite furnace atomic absorption spectrometry (GFAAS) have been combined for the simultaneous enrichment of Cd and Pb from environmental water samples including seawater, and subsequent sequential determination of both analytes. For the preconcentration, a shell structured Fe3O4@graphene oxide nanospheres was synthetized and characterized. This nanosorbent presents a large contact area and has a high surface density of polar groups. With the optimized method, the experimental detection and determination limits, calculated as the concentration of analyte giving signals equivalent to three and ten times, respectively, the standard deviation of the blank plus the net blank intensity, were 0.005 µg L−1 (detection limit) and 0.017 µg L−1 (determination limit) for Cd; and 0.008 µg L−1 (detection limit) and 0.026 µg L−1 (determination limit) for Pb. The method offers good precisions (RSD < 3.5%) and a preconcentration factor of 200. The accuracy of the proposed method was verified using certified reference materials (SLRS-5, SPS-SW2) and by determining the analytes content in spiked seawater and tap water samples collected from Málaga (Spain). The determined values by external calibration were in good agreement with the certified values and the recoveries for the spiked samples were around 100% in all cases.

Dynamics Rationalize Proteolytic Susceptibility of the Major Birch Pollen Allergen Bet v 1.

Proteolytic susceptibility during endolysosomal degradation is decisive for allergic sensitization. In the major birch pollen allergen Bet v 1 most protease cleavage sites are located within its secondary structure elements, which are inherently inaccessible to proteases. The allergen thus must unfold locally, exposing the cleavage sites to become susceptible to proteolysis. Hence, allergen cleavage rates are presumed to be linked to their fold stability, i.e., unfolding probability. Yet, these locally unfolded structures have neither been captured in experiment nor simulation due to limitations in resolution and sampling time, respectively. Here, we perform classic and enhanced molecular dynamics (MD) simulations to quantify fold dynamics on extended timescales of Bet v 1a and two variants with higher and lower cleavage rates. Already at the nanosecond-timescale we observe a significantly higher flexibility for the destabilized variant compared to Bet v 1a and the proteolytically stabilized mutant. Estimating the thermodynamics and kinetics of local unfolding around an initial cleavage site, we find that the Bet v 1 variant with the highest cleavage rate also shows the highest probability for local unfolding. For the stabilized mutant on the other hand we only find minimal unfolding probability. These results strengthen the link between the conformational dynamics of allergen proteins and their stability during endolysosomal degradation. The presented approach further allows atomistic insights in the conformational ensemble of allergen proteins and provides probability estimates below experimental detection limits.