Machine learning-assisted liquid crystal-based aptasensor for the specific detection of whole-cell Escherichia coli in water and food.

We have developed a rapid, facile liquid crystal (LC)-based aptasensor for E. coli detection in water and juice samples. A textile grid-anchored LC platform was used with specific aptamers adsorbed via a cationic surfactant, cetyltrimethylammonium bromide (CTAB), on the LC surface. The presence of E. coli dissociates the aptamers from CTAB and restores the dark signal induced by the surfactant. Using polarized microscopy, the images of the LCs in the presence of various concentrations of E. coli were captured and analyzed using image analysis and machine learning (ML). The artificial neural networks (ANN) and extreme gradient boosting (XGBoost) rendered the best results for water samples (R2 = 0.986 and RMSE = 0.209) and juice samples (R2 = 0.976 and RMSE = 0.262), respectively. The platform was able to detect E. coli with a detection limit (LOD) of 6 CFU mL-1.

Colorimetric and fluorescent dual-biosensor based on zirconium and preasodium metal-organic framework (zr/pr MOF) for miRNA-191 detection.

MicroRNAs (miRNAs) are associated with certain types of cancer, tumor stages, and responses to treatment, thus efficient methods are required to identify them quickly and accurately. Abnormal expression of microRNA-191 (miR-191) has been linked to particular cancers and several other health conditions, such as diabetes and Alzheimer's disease. In this study, a new dual-biosensor based on the zirconium and preasodium-based metal-organic framework (Zr/Pr MOF) was developed for the rapid, ultrasensitive, and selective detection of miRNA-191. The synthesized Zr/Pr MOF exhibited peroxidase-like activity and fluorescence properties. Our dual method involves monitoring the fluorescence and peroxidase activity of metal-organic frameworks (MOFs) in the presence of miRNAs. The Zr/Pr MOF can catalyze hydrogen peroxide (H2O2) to oxidize the chromogenic substrate 3, 3', 5, 5'-tetramethylbenzidine (TMB) to produce blue oxidized TMB (oxTMB), which exhibits ultraviolet absorption at 660 nm. However, the addition of a label-free miRNA-191 probe caused a significant change in fluorescence intensity and absorbance, indicating the binding of single-stranded miRNAs to the MOF through van der Waals interactions and π-π stacking. The presence of the target miRNA-191 caused the probe to be released from the surface of the MOF owing to hybridization, which increased the peroxidase-like activity of Zr/Pr-MOF. Both response signals showed acceptable linear relationship and low detection limits. Fluorescence and colorimetry have an LOD of 0.69 and 8.62 pM, respectively. This study demonstrates the reliability and sensitivity of miRNA identification in human serum samples.

COVID-19 electrochemical immunosensor with Ag-MOF: Rapid and high-selectivity nasal swab testing for effective detection.

Early detection of the coronavirus is acknowledged as a crucial measure to mitigate the spread of the pandemic, facilitating timely isolation of infected individuals, and disrupting the transmission chain. In this study, we leveraged the properties of synthesized Ag-MOF, including high porosity and increased flow intensity. Electrochemical techniques such as cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were employed to develop an economical and portable sensor with exceptional selectivity for COVID-19 detection. The methodology involves the deposition of Ag-MOF onto the surface of a Glassy Carbon Electrode (GCE), which resulted in a progressive augmentation of electric current. Subsequently, the targeted antibodies were applied, and relevant tests were conducted. The sensor demonstrated the capacity to detect the virus within a linear range of 100 fM to 10 nM, boasting a noteworthy Limit of Detection (LOD) of 60 fM. The entire detection process could be completed in a brief duration of 20 min, exhibiting high levels of accuracy and precision, outperforming comparable techniques in terms of speed and efficacy.

Ti3C2/Ni/Sm-based electrochemical glucose sensor for sweat analysis using bipolar electrochemistry.

An innovative electrochemical sensor is introduced that utilizes bipolar electrochemistry on a paper substrate for detecting glucose in sweat. The sensor employs a three-dimensional porous nanocomposite (MXene/NiSm-LDH) formed by decorating nickel-samarium nanoparticles with double-layer MXene hydroxide. These specially designed electrodes exhibit exceptional electrocatalytic activity during glucose oxidation. The glucose sensing mechanism involves enzyme-free oxidation of the analyte within the sensor cell, achieved by applying an appropriate potential. This leads to the reduction of K3Fe(CN)6 in the reporter cell, and the resulting current serves as the response signal. By optimizing various parameters, the measurement platform enables the accurate determination of sweat glucose concentrations within a linear range of 10 to 200 µM. The limit of detection (LOD) for glucose is 3.6 µM (S/N = 3), indicating a sensitive and reliable detection capability. Real samples were analysed  to validate the sensor's efficiency, and the results obtained were both promising and encouraging.

New application of bimetallic Ag/Pt nanoplates in a colorimetric biosensor for specific detection of E. coli in water.

A fast and sensitive aptasensor was developed using nanoplates with peroxidase activity as a novel approach. E. coli detection is described using a silver/platinum nanoplate (Ag/Pt NPL) that interacts with an oligonucleotide aptamer as a bioreceptor. The size of the Ag/Pt NPLs was about 42 nm according to the FE-SEM images. The EDS result indicates that a thin layer of Pt ions was coated on the surface of the Ag NPLs. This nanobiosensor has the ability to specifically bind to E. coli, increasing the peroxidase activity of the apt-Ag/Pt NPL. Finally, the blue color of the solution in the contaminated water samples was increased in the presence of 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate and H2O2. The assay can be completed in 30 min and the presence of E. coli levels can be distinguished with the naked eye. The absorbance at 652 nm is proportional to pathogen concentration from 10 to 108 CFU·mL-1, with a detection limit of 10 CFU·mL-1. The percent recovery for the water samples spiked with E. coli is 95%. The developed assay should serve as a general platform for detecting other pathogenic bacteria which affect water and food quality. The proposed E. coli detection strategy has appealing characteristics such as high sensitivity, simple operation, short testing time, and low cost.

Unmasking the Electrochemiluminescence Properties of Ternary Mn/Fe/Co Metals Doped Porous g-C3N4 Fiber-like Nanostructure.

Graphitic-phase carbon nitride (g-C3N4) materials have exhibited increasingly remarkable performance as emerging electrochemiluminescence (ECL) emitters, owing to their unique optical and electronic properties; however, the ECL merits of porous g-C3N4 nanofibers doped with ternary metals are not yet explored. Deciphering the ECL properties of trimetal-doped g-C3N4 nanofibers could provide an exquisite pathway for ultrasensitive sensing and imaging with impressive advantages of minimal background signal, great sensitivity, and durability. Herein, we rationally synthesized g-C3N4 nanofibers doped atomically with Mn, Fe, and Co elements (Mn/Fe/Co/g-C3N4) in a one-pot via the protonation in ethanol and annealing process driven by the rolling up mechanism. The ECL performance of g-C3N4 with and without metal dopants was investigated and compared with standard Ru(bpy)32+ in the presence of potassium persulfate (K2S2O8) as the coreactant. Notably, g-C3N4 nanofibers doped with metal ions exhibited an ECL efficiency of 483% that was 4.83 times higher than that of Ru(bpy)32+. Mechanistic investigations unveiled that the g-C3N4 nanofibers possess a large surface area and, as a result, exhibit a reduced interfacial impedance within the porous microstructure. These factors contribute to the acceleration of charge transfer rates and the stabilization of charge carriers and excitons, ultimately facilitating the ECL process. This research endeavor may pave the way for a new hot research area and serves as a powerful tool for elucidating fundamental inquiries of ECL on one-dimensional g-C3N4 nanostructures.

Design of chitosan/boehmite biocomposite for the removal of anionic and nonionic dyes from aqueous solutions: Adsorption isotherms, kinetics, and thermodynamics studies.

This study introduces a chitosan/boehmite biocomposite as an efficient adsorbent for removing anionic Congo Red (CR) and non-ionic Bromothymol Blue (BTB) from water. Boehmite nanoparticles were synthesized using the Sol-gel method and then attached to chitosan particles using sodium tripolyphosphate through co-precipitation method. Characterized through FTIR, FE-SEM, BET, and XRD, the biosorbent displayed structural integrity with optimized pH conditions of 3 for CR and 4 for BTB, achieving over 90 % adsorption within 30 min. Pseudo second order kinetics model and Langmuir isotherm revealed monolayer sorption with capacities of 64.93 mg/g for CR and 90.90 mg/g for BTB. Thermodynamics indicated a spontaneous and exothermic process, with physisorption as the primary mechanism. The biosorbent demonstrated excellent performance and recyclability over five cycles, highlighting its potential for eco-friendly dye removal in contaminated waters.

Advancements in wearable technology for monitoring lactate levels using lactate oxidase enzyme and free enzyme as analytical approaches: A review.

Lactate is a metabolite that holds significant importance in human healthcare, biotechnology, and the food industry. The need for lactate monitoring has led to the development of various devices for measuring lactate concentration. Traditional laboratory methods, which involve extracting blood samples through invasive techniques such as needles, are costly, time-consuming, and require in-person sampling. To overcome these limitations, new technologies for lactate monitoring have emerged. Wearable biosensors are a promising approach that offers non-invasiveness, low cost, and short response times. They can be easily attached to the skin and provide continuous monitoring. In this review, we evaluate different types of wearable biosensors for lactate monitoring using lactate oxidase enzyme as biological recognition element and free enzyme systems.

A simple smartphone-assisted paper-based colorimetric biosensor for the detection of urea adulteration in milk based on an environment-friendly pH-sensitive nanocomposite.

Urea is a common milk adulterant that falsely increases its protein content. Excessive consumption of urea is harmful to the kidney, liver, and gastrointestinal system. The conventional methods for urea detection in milk are time-consuming, costly, and require highly skilled operators. So, there is an increasing demand for the development of rapid, convenient, and cost-efficient methods for the detection of urea adulteration in milk. Herein, we report a novel colorimetric paper-based urea biosensor, consisting of a novel environment-friendly nanocomposite of halloysite nanotubes (HNT), that urease enzyme and an anthocyanin-rich extract, as a natural pH indicator are simultaneously immobilized into its internal and external surfaces. The biosensing mechanism of this biosensor is based on anthocyanin color change, which occurs due to urease-mediated hydrolysis of urea and pH increment of the environment. The colorimetric signal of this biosensor is measured through smartphone-assisted analysis of the mean RGB (Red-Green-Blue) intensity of samples and is capable of detecting urea with a detection limit of 0.2 mM, and a linear range from 0.5 to 100 mM. This biosensor has demonstrated promising results for the detection of urea in milk samples, in the presence of other milk adulterants and interferents.

Fe3O4@SiO2@NiAl-LDH microspheres implication in separation, kinetic and structural properties of phenylalanine dehydrogenase.

Fe3O4@SiO2@NiAl-LDH three-components microsphere contains a Fe3O4@SiO2 magnetic core and a layered double hydroxide with nickel cation provide the binding ability to (His)-tagged-protein and exhibits high performance in protein separation and purification. The morphology and chemistry of the synthesized Fe3O4@SiO2@NiAl-LDH microspheres were characterized by energy-dispersive X-ray spectroscopy (EDX), scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared (FTIR), vibrating sample magnetometer (VSM), Dynamic light scattering (DLS). Purified enzyme was assesed with SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis and intrinsic fluorescence spectroscopy. In this study, the separation of phenylalanine dehydrogenase (PheDH) by Fe3O4@SiO2@NiAl -LDH was performed and the effect of microsphere was investigated on the kinetic and structural properties of PheDH. After purification, kinetic parameters such as Km, Vmax, Kcat, kcat/Km, optimum temperature, thermal stability, and and activation energy were evaluated and compared according to the mentioned methods. The interaction between the enzyme and the microsphere displayed a high performance in protein binding capacity. The results also revealed that the kinetic parameters of the enzyme changed in a dose-dependent manner in the presence of a microsphere. Moreover, the results of intrinsic fluorescence and Circular Dichroism (CD) confirmed the structural changes of the protein in the interaction with the microsphere.

Boosting Electrochemiluminescence Immunoassay Sensitivity via Co-Pt Nanoparticles within a Ti3C2 MXene-Modified Single Electrode Electrochemical System on Raspberry Pi.

Point-of-care testing plays a crucial role in diagnostics within resource-poor areas, necessitating the utilization of portable and user-friendly devices. The adaptation of biosensors for point-of-care applications requires careful considerations, such as miniaturization, cost-effectiveness, and streamlined sample processing. In recent years, the electrochemiluminescence (ECL) immunoassay has gained significant attention due to its visual detection capabilities and ability to facilitate high-throughput analysis. However, the development of a practical and cost-effective ECL device remains a challenging task. This study presents the development of an integrated MXene-modified single-electrode electrochemical system (SEES) for visual and high-throughput ECL immunoassays incorporating a Raspberry Pi system. The SEES was designed by affixing a plastic sticker with multiple perforations onto a single carbon ink screen-printed electrode, which operates based on a resistance-induced potential difference. Leveraging the excellent adsorption and bioaffinity properties of the carbon ink screen-printed electrode, effective immobilization of antibodies was achieved. Furthermore, the incorporation of Co-Pt nanoparticles enhanced the ECL intensity and electron transfer kinetics, enabling the sensitive detection of SARS-CoV-2. The developed system comprised 18 individual reaction cells, allowing for simultaneous analysis while maintaining sample isolation. Impressively, the system achieved a remarkable minimum virus detection limit of 10-14 g mL-1, accompanied by a high R2 value of 0.9798. These findings highlight the promising potential of our developed system for efficient point-of-care testing in resource-limited settings.

Fluorescence self-assembled DNA hydrogel for the determination of prostate specific antigen by aggregation induced emission.

In this study, an aptamer-based, functionalized-DNA hydrogel system is developed for prostate-specific antigen (PSA) detection. A pure DNA hydrogel is constructed using specific DNA building blocks and an aptamer as a cross-linker. Firstly, silver nanoclusters (AgNCs) are constructed on the Y-shaped DNA (Y-DNA) building blocks. Then, the DNA hydrogel was formed via the addition of the cross-linker to the Y-DNA solution. In this case, the fluorescence emission of silver nanoclusters that have accumulated in the hydrogel increases due to aggregation-induced emission (AIE). The presence of PSA and its subsequent interaction with its specific aptamer dissolve the hydrogel structures, which leads to a low emission intensity. A great linear relationship was attained in this assay in the range of 0.05 to 8 ng mL-1 with a detection limit of 4.4 pg mL-1 for the detection of PSA. Additionally, the proposed aptasensor was successfully used to detect PSA in human serum samples. The recovery for different concentrations of PSA was in the range of 96.1% to 99.3%, and the RSD range was from 2.3% to 4.5%. Comparing our method to current ones in the field of PSA detection proves that our platform benefits from a simpler procedure, lower cost, and better efficiency, providing high potential for future clinical applications.

A dual-targeting nanobiosensor for Gender Determination applying Signal Amplification Methods and integrating Fluorometric Gold and Silver Nanoclusters.

A dual-targeting nanobiosensor has been developed for the simultaneous detection of AMELX and AMELY genes based on the different fluorescence signals emitted from gold and silver nanoclusters, AuNCs and AgNCs respectively. In our design, both catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) have been used as isothermal, enzyme-free and simple methods for signal's amplification. The working principle is based on the initiation of a cascade of CHA-HCR reactions when AMELX is present, in which AuNCs, synthesized on the third hairpin, are aggregated on the surface of the dsDNA product, performing the phenomenon of aggregation induced emission (AIE) and enhancing their fluorescence signal. On the other hand, the presence of the second target, AMELY, is responsible for the enhancement of the fluorescence signal corresponding to AgNCs by the same phenomenon, via hybridizing to the free end of the dsDNA formed and at the same time to the probe of silver nanoclusters fixing it closer to the surface of the dsDNA product. Such a unique design has the merits of being simple, inexpensive, specific and stable and presents rapid results. The detection limits of this assay for AMELX and AMELY are as low as 3.16 fM and 23.6 fM respectively. Moreover, this platform showed great performance in real samples. The design has great promise for the application of dual-targeting nanobiosensors to other biomarkers.

Dual-emissive phenylalanine dehydrogenase-templated gold nanoclusters as a new highly sensitive label-free ratiometric fluorescent probe: heavy metal ions and thiols measurement with live-cell imaging.

Phenylalanine dehydrogenase (PheDH) has been proposed as an ideal protein scaffold for the one-step and green synthesis of highly efficient multifunctional gold nanoclusters. The PheDH-stabilized fluorescent gold nanoclusters (PheDH-AuNCs) with dual emission/single excitation exhibited excellent and long-term stability, high water solubility, large Stokes shift and intense photoluminescence. Selectivity studies demonstrated that the red fluorescence emission intensity of PheDH-AuNCs was obviously decreased in less than 10 min by the addition of mercury, copper, cysteine or glutathione under the single excitation at 360 nm, without significant change in the blue emission of the PheDH-AuNCs. Therefore, the as-prepared PheDH-AuNCs as a new excellent fluorescent probe were successfully employed to develop a simple, rapid, low cost, label- and surface modification-free nanoplatform for the ultrasensitive and selective detection of Hg2+, Cu2+, Cys and GSH through a ratiometric fluorescence system with wide linear ranges and detection limits of 1.6, 2.4, 160 and 350 nM, respectively which were lower than previous reports. In addition, the results showed that PheDH-AuNCs can be used for the detection of toxic heavy metal ions and small biomarker thiols in biological and aqueous samples with acceptable recoveries. Interestingly, PheDH-AuNCs also displayed a promising potential for live-cell imaging due to their low toxicity and great chemical- and photo-stability.

Paper-based colorimetric detection of COVID-19 using aptasenor based on biomimetic peroxidase like activity of ChF/ZnO/CNT nano-hybrid.

Corona Virus Disease 2019 (COVID-19) as the infectious disease caused the pandemic disease around the world through infection by SARS-CoV-2 virus. The common diagnosis approach is Quantitative RT-PCR (qRT-PCR) which is time consuming and labor intensive. In the present study a novel colorimetric aptasensor was developed based on intrinsic catalytic activity of chitosan film embedded with ZnO/CNT (ChF/ZnO/CNT) on 3,3',5,5'-tetramethylbenzidine (TMB) substrate. The main nanocomposite platform was constructed and functionalized with specific COVID-19 aptamer. The construction subjected with TMB substrate and H2O2 in the presence of different concentration of COVID-19 virus. Separation of aptamer after binding with virus particles declined the nanozyme activity. Upon addition of virus concentration, the peroxidase like activity of developed platform and colorimetric signals of oxidized TMB decreased gradually. Under optimal conditions the nanozyme could detect the virus in the linear range of 1-500 pg mL and LOD of 0.05 pg mL. Also, a paper-based platform was used for set up the strategy on applicable device. The paper-based strategy showed a linear range between 50 and 500 pg mL with LOD of 8 pg mL. The applied paper based colorimetric strategy showed reliable results for sensitive and selective detection of COVID-19 virus with the cost-effective approach.

The Chemical Space of Terpenes: Insights from Data Science and AI.

Terpenes are a widespread class of natural products with significant chemical and biological diversity, and many of these molecules have already made their way into medicines. In this work, we employ a data science-based approach to identify, compile, and characterize the diversity of terpenes currently known in a systematic way, in a total of 59,833 molecules. We also employed several methods for the purpose of classifying terpene subclasses using their physicochemical descriptors. Light gradient boosting machine, k-nearest neighbours, random forests, Gaussian naïve Bayes and Multilayer perceptron were tested, with the best-performing algorithms yielding accuracy, F1 score, precision and other metrics all over 0.9, thus showing the capabilities of these approaches for the classification of terpene subclasses. These results can be important for the field of phytochemistry and pharmacognosy, as they allow the prediction of the subclass of novel terpene molecules, even when biosynthetic studies are not available.

Synthesis of cuttlebone/ carbon quantum dots/nickel oxide nanocomposite for visible light photodegradation of malachite green used for environmental remediation.

In recent years, the development of light-driven nanophotocatalysts has focused on efficiently eliminating organic pollutants. In this regard, the present work focuses on the photocatalytic removal of malachite green (MG) dye using cuttlebone powder (CB) modified with carbon quantum dots (CQDs)/nickel oxide (NiO) under visible light irradiation. Various techniques were used to characterize the proposed composite, including X-ray diffraction (XRD) and field emission scanning electron microscopy (FE-SEM) images. The optical properties of the synthesized CB/CQDs/NiO were analyzed by UV-VIS visible spectroscopy. Using central composite design (CCD), several effective parameters, including pH, dye concentration, amount of photocatalyst, and temperature degradation efficiency, were optimized to achieve the optimal condition for photocatalytic activity of CB/CQDs/NiO. The Langmuir-Hinshelwood model was employed to model the kinetics of the degradation of the dye, the resulting K being 0.378 min-1. The as synthesized nanocomposites could be efficiently removed from water by applying an external magnetic field. The test results indicate that the prepared CB/CQDs/NiO nanocomposite demonstrates excellent stability after four reaction cycles. Furthermore, the nanocomposite shows excellent photocatalytic activity, reducing 99.7% MGdye concentration within 12 min of visible light exposure.

An enzyme-free Ti3C2/Ni/Sm-LDH-based screen-printed-electrode for real-time sweat detection of glucose.

Here, we report the fabrication of an enzyme-free glucose sensor benefiting from nickel-samarium nanoparticles-decorated MXene layered double hydroxide (MXene/Ni/Sm-LDH). The electrochemical response of the MXene/Ni/Sm-LDH to glucose was studied via cyclic voltammetry (CV). The fabricated electrode has high electrocatalytic activity for glucose oxidation. The voltametric response of the MXene/Ni/Sm-LDH electrode to glucose was investigated by differential pulse voltammetry (DPV) that demonstrated an extended linear range of from 0.001 to 0.1 mM and 0.25-7.5 mM with a detection limit down to 0.24 µM (S/N = 3) and a sensitivity at 1673.54 µA mM-1 cm-2 1519.09 µA mM-1 cm-2 in concentrations of 0.01 mM and 1 mM respectively as well as good repeatability, high stability and applicability for the real sample analysis. Moreover, the as-fabricated sensor was applied to glucose detection in human sweat and showed promising results.

Smartphone image analysis-based fluorescence detection of tetracycline using machine learning.

Tetracycline (TC) is vastly used as a veterinary drug, making its detection highly important. We have aimed to develop a rapid detection method for TC. For this, BSA-protected Au/Ag bimetallic nanoclusters (BSA-BMNCs) were synthesized for the detection of TC in water and milk. The interaction of TC with BSA shifted the emission of the BMNCs from red to yellow as concentrations of TC increased. Images of the sensing platform were captured with various smartphones and the color and texture information was extracted to generate training datasets for water and milk samples. The datasets were used to train machine learning (ML) algorithms. A model using bagging and artificial neural networks (R2 = 0.994, NRMSE = 0.078) for water samples and one using bagging and decision trees (R2 = 0.999, NRMSE = 0.027) for milk samples were developed. This study shows the ability of ML algorithms for the development of rapid sensors for the detection of food analytes.

Fabrication and evaluation of optical nanobiosensor based on localized surface plasmon resonance (LSPR) of gold nanorod for detection of CRP.

C-reactive protein (CRP) is a plasma protein that is one of the most expressed proteins in acute phase inflammation cases. It is a well-known biomarker for inflammatory disorders. There is a significant correlation between increasing CRP concentration and the risk of being exposed to cardiovascular diseases (CVD) and sepsis; thus, monitoring and quantifying CRP levels in a simple, inexpensive, and quick manner can improve clinical diagnostics and help prevent major inflammatory conditions. Here a nanobiosensor was developed, benefiting from the LSPR property of gold-nanorod (GNR) to measure CRP concentration. Nanorods were fabricated using One-pot synthesis by trimethyl ammonium bromide (CTAB) as a surfactant. This method provides the advantage of both step and time reduction in synthesis and decreases the contamination probability of nanorods as the products. The nanorods were characterized using TEM with an average size of (24 ± 1 nm) × (5 ± 1 nm) and a typical aspect ratio of ∼4.9. The surface of the rods was modified with a specific aptamer for the target protein, and the LSPR shifts due to the gold nanorod's refractive index change as the result of protein interaction with the biosensor investigated using a 100-900 nm UV absorption device. The results indicated that the nanobiosensor could respond to different CRP concentrations within 30 min. The selectivity test has shown nonresponsive results of nanobiosensor to BSA and TNF-α proteins which are used to evaluate the biosensor behavior in non-target proteins. The detection limit was evaluated at 2 nM, and the sensor's linear response ranged between 2 - 20 nM.