Immobilization Buffer Research Articles

Tuning almond lipase features by the buffer used during immobilization: The apparent biocatalysts stability depends on the immobilization and inactivation buffers and the substrate utilized

The lipase from Prunus dulcis almonds was inactivated under different conditions. At pH 5 and 9, enzyme stability remained similar under the different studied buffers. However, when the inactivation was performed at pH 7, there were some clear differences on enzyme stability depending on the buffer used. The enzyme was more stable in Gly than when Tris was employed for inactivation. Then, the enzyme was immobilized on methacrylate beads coated with octadecyl groups at pH 7 in the presence of Gly, Tris, phosphate and HEPES. Its activity was assayed versus triacetin and S-methyl mandelate. The biocatalyst prepared in phosphate was more active versus S-methyl mandelate, while the other ones were more active versus triacetin. The immobilized enzyme stability at pH 7 depends on the buffer used for enzyme immobilization. The buffer used in the inactivation and the substrate used determined the activity. For example, glycine was the buffer that promoted the lowest or the highest stabilities depending on the substrate used to quantify the activities.

Electrochemical Aptasensing Platform for the Detection of Retinol Binding Protein-4.

Here, we present the results of our the electrochemical aptasensing strategy for retinol binding protein-4 (RBP-4) detection based on a thiolated aptamer against RBP-4 and 6-mercaptohexanol (MCH) directly immobilized on a gold electrode surface. The most important parameters affecting the magnitude of the analytical signal generated were optimized: (i) the presence of magnesium ions in the immobilization and measurement buffer, (ii) the concentration of aptamer in the immobilization solution and (iii) its folding procedure. In this work, a systematic assessment of the electrochemical parameters related to the optimization of the sensing layer of the aptasensor was carried out (electron transfer coefficients (α), electron transfer rate constants (k0) and surface coverage of the thiolated aptamer probe (ΓApt)). Then, under the optimized conditions, the analytical response towards RBP-4 protein, in the presence of an Fe(CN)63-/4- redox couple in the supporting solution was assessed. The proposed electrochemical strategy allowed for RBP-4 detection in the concentration range between 100 and 1000 ng/mL with a limit of detection equal to 44 ng/mL based on electrochemical impedance spectroscopy (EIS). The specificity studies against other diabetes biomarkers, including vaspin and adiponectin, proved the selectivity of the proposed platform. These preliminary results will be used in the next step to miniaturize and test the sensor in real samples.

Bound Ca2+ moves faster and farther from single open channels than free Ca2.

A concept of Ca2+ nanodomains established in the cytoplasm after opening single-calcium channels helps mechanistically understand the physiological mechanisms of Ca2+ signaling. It predicts standing gradients of cytoplasmic free Ca2+ around single channels in the plasma membrane. The fate of bound Ca2+ attracted much less attention. This study aimed to examine the profiles of Ca2+ bound to low-mobility buffers such as bulky Ca2+-binding proteins. The solution of non-linear PDEs for an immobile buffer predicts fast decay of free [Ca2+] from the channel lumen and the traveling wave for bound Ca2+. For low-mobility buffers like calmodulin, the calculated profiles of free and bound Ca2+ are similar. Theoretical predictions are tested by imaging 1D profiles of Ca2+ bound to low-mobility fluo-4-dextran. The traveling waves of bound Ca2+ are observed that develop during the opening of single channels. The findings tempt to propose that Ca2+ signaling may not be solely related by the absolute free [Ca2+] at the sensor location, which is extremely localized, but determined by the time when a wave of bound Ca2+ reaches a threshold needed for sensor activation.

Enhancing biotechnological applications of l-asparaginase: Immobilization on amino-epoxy-agarose for improved catalytic efficiency and stability

l-asparaginase has been shown to be effective in reducing acrylamide formation in food and in treating certain cancers, such as acute lymphoblastic leukemia. This study aimed to improve the application of l-asparaginase in these areas, through its immobilization in two distinct supports: epoxy-agarose and amino-epoxy-agarose. Different immobilization conditions were investigated, including immobilization pH and buffer concentration. The best activity results were obtained employing amino-epoxy-agarose, being the 0.1 M sodium carbonate buffer pH 10 the conditions that offered the highest immobilization yield (76%), immobilization efficiency (89.8%) and relative activity (68.2%). Immobilization of l-asparaginase in amino-epoxy-agarose improved its catalytic efficiency and stability, including at pH 4 and 60 °C. All immobilized biocatalysts preserved more than 70% of their activity after 7 cycles of reuse, and maintained about 82% of their activity after 60 days of storage at 4°C. These results show the efficacy of immobilized biocatalysts in the preservation of enzymatic activity and highlight their potential for biotechnological applications.

Effects of Lipase Immobilization Conditions and Support Materials for the Production of Structured Triacylglycerols

AbstractStructured triacylglycerols (STAG) with desired properties can be synthesized by transesterification using immobilized lipases. Herein, the effect of immobilization conditions and support material on the immobilization yield, specific activity and regioselectivity of lipases from Rhizomucor miehei (RML) and Rhizopus oryzae (ROL) in the production of STAG, are evaluated. Four different support materials utilizing adsorption and one with covalent binding are investigated. Ammonium sulfate is found to significantly increase the activity‐based immobilization yield (12% and 38% for RML and ROL, respectively) and specific activity on Accurel MP1000 (MP1000) when used as the immobilization buffer. Furthermore, the immobilization principle and support material influenced both the activity and regiospecificity. Immobilization by adsorption is found to result in higher catalytic activity, while covalent binding resulted in lipase inactivation. For RML, the highest specific activity of 43 µmol STAG min−1 g−1 (U) is obtained on MP1000, while ROL, which exhibited higher activities in general, results in a maximum activity of 120 U on Lifetech ECR8806. The obtained specific activites are comparable to the commercial preparations Novozym 40086 (45 U) and Lipase DF “Amano” IM (147 U) while the regiospecificity of the developed preparations is even higher, forming at least 64% less byproduct.Practical applications: There is an increasing need for lipids with specific nutritional and physical properties in health, nutrition, and food applications. In this context, STAG are highly promising products due to the possibility to tailor the composition to obtain the desired properties. Immobilized lipases are the catalyst of choice for the production of STAG, in which activity and regioselectivity are particularly important parameters for the process performance. In this study, it is shown that these parameters can be affected by the immobilization conditions and support material. Immobilized preparations with high activity and excellent regiospecificity are created on commercially available supports. This shows the possibility of STAG synthesis with high purity and beneficial properties.

Uniqueness and global stability of V-shaped fronts for the buffered bistable system in [formula omitted

This paper is devoted to V-shaped calcium concentration waves of the buffered bistable system in R2. We prove that multiple immobile buffers (where all buffers do not diffuse) do not affect the global asymptotic stability and uniqueness of V-shaped calcium concentration waves. The existence of such curved fronts has been proved in our previous work (Jia et al., 2022). However, the study of its stability is difficult since the buffers do not diffuse. We first apply the sliding method to construct a crucial sub-solution. Then, based on the super- and sub-solution method, and comparison principle, we achieve the global asymptotic stability of the V-shaped fronts.

Immunoglobulin-Immobilized Quartz Crystal Microbalance for Staphylococcus Aureus Real-Time Detection

Quartz crystal microbalance (QCM) sensor is still a high-precision surface sensing technique. Here, we described an immunoglobulin G (IgG) biofunctionalized QCM sensor for <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">Staphylococcus aureus</i> (S. aureus) sensing application. In the case of the QCM sensor, a gold-titanium (Au/Ti) electrode layer was fabricated by magnetron sputtering and sequentially immobilized with thoil self-assembled monolayer (SAM) and IgG gripper. The selectivity test showed that the rabbit IgG-based QCM sensor exhibited a good sensitivity performance. IgG immobilization optimal experiment was conducted by adjusting parameters including immobilization time, temperature, buffer pH and IgG concentration. The lower limit of detection and sensitivity of the IgG-based QCM biosensor toward <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">S. aureus</i> were suggested to be <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$1.0\times {10}^{{4}}$ </tex-math></inline-formula> CFU/mL and 562.5 Hz/ng, respectively. Furthermore, the real-time monitoring and frequency response of IgG immobilization process and its sensitive adsorption behavior to <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">S. aureus</i> were performed in droplet environment. By comparison, the average sensitivity of IgG-immobilized QCM sensor in liquid phase is 2-3.5 times higher than that in air. Overall, it is indicative that IgG-based QCM sensors do have great potential for biological monitoring applications.

Synthesis of activated carbon from hydrothermally carbonized tamarind seeds for lipase immobilization: characterization and application in aroma ester synthesis

AbstractBACKGROUNDThis study aimed to evaluate the effect of hydrothermal carbonization on the activated carbons properties of tamarind seeds, which were used as a support in porcine pancreatic lipase (PPL) immobilization, and to evaluate the biocatalyst in the butyl butyrate synthesis.RESULTSThe activated carbon from hydrochar (ACHT) had oxygen functional groups content about 32.5% higher and a lower point of zero charge (4.42) when compared to those obtained from crude bran (ACT). Differences in textural properties were also observed, with an average pore diameter of 3.20 and 6.46 nm for ACHT and ACT, respectively. The optimal immobilization pH was 5.0, resulting in enzyme loading of 71.8 ± 1.42 mg.g1 and 53.03 ± 0.31 mg·g−1, and in a hydrolytic activity of 222.47 ± 2.80 U·g−1 and 257.18 ± 7.31 U·g−1 for ACT and ACHT, respectively. The addition of Triton X‐100 0.025 w·v−1 to the immobilization buffer led to an increase in ~7% of the hydrolytic activity. The PPL immobilized in pH 5.0 was used in the butyl butyrate synthesis, using a mixture of butanol (240 mmol L−1) and butyric acid (120 mmol L−1), with an esterification yield of ±83% after 4 h of reaction at 40 °C.CONCLUSIONThe PPL immobilized on ACHT showed a small decrease in activity over time, with a residual esterification yield of 84% after 4 cycles of reuse. The present results suggest that the supported biocatalyst has the potential to be used in aqueous and organic media, such as hydrolytic lipid reactions and aroma synthesis mostly used in the food industry. © 2021 Society of Chemical Industry (SCI).

Measuring and Controlling the Local Environment of Surface-Bound DNA in Self-Assembled Monolayers on Gold When Prepared Using Potential-Assisted Deposition

DNA self-assembled monolayers (SAMs) were prepared using potential-assisted deposition on clean gold single-crystal bead electrodes under a number of conditions (constant or square-wave potential perturbations in TRIS or phosphate immobilization buffers with and without Cl-). The local environment around the fluorophore-labeled DNA tethered to the electrode surface was characterized using in situ fluorescence microscopy during electrochemical measurements as a function of the underlying surface crystallography. Potential-assisted deposition from a TRIS buffer containing Cl- created DNA SAMs that were uniformly distributed on the surface with little preference to the underlying crystallography. A constant (+0.4 V/SCE) or a square-wave potential perturbation (+0.4 to -0.3 V/SCE, 50 Hz) resulted in similar DNA-modified surfaces in TRIS immobilization buffer. Deposition using a square-wave potential without Cl- resulted in lower DNA surface coverage. Despite this, the local environment around the DNA in the SAM appears to be densely packed. This implies the formation of clusters of densely packed DNA in the SAM. This effect was also demonstrated when depositing from a phosphate buffer. DNA clusters were significantly reduced when Cl- was present in the buffer. Clusters were most prevalent on the low-index plane surfaces (e.g., {111} and {100}) and less on the higher-index planes (e.g., {210} or {311}). A mechanism is proposed to rationalize the formation of DNA-clustered regions for deposition using a square-wave potential perturbation. The conditions for creating clusters of DNA in a SAM or for preventing these clusters from forming provide an approach for tailoring the surfaces used for biosensing.

Preparing DNA SAM Electrochemical Sensors Using Potential Assisted Deposition Methods. Controlling the Coverage and Local Organization of the DNA in the SAM.

Typically, DNA SAMs used for electrochemical based sensing are prepared without controlling the gold electrode potential[1]. Applying a potential during DNA deposition enables control over the coverage and DNA organization[2-4]. These characteristics are determined by the composition of the immobilization buffer, the immersion time, the DNA concentration, the extent of backfilling with a small alkylthiol and whether deposition occurs on a bare or alkythiol coverage gold surface[3-4]. Few studies have explored the influence of the underlying surface crystallography. Here, we show the possibilities of using electrochemical potential control to manipulate the characteristics of the DNA SAM. Moreover, the use of a single crystal gold bead electrode, coupled with the use of a fluorophore tagged DNA and fluorescence microscopy, enables examination of the influence of the underlying surface crystallography on the DNA SAM prepared. Various constant potentials were used as well as using a square-wave potential perturbation during deposition. There are significant differences in the DNA SAM produced under these conditions. In some cases, the DNA coverage was low on the <111> surfaces, higher on more open or atomically rough regions. In other cases, the coverage was higher on the <100> surfaces as compared to the other regions. The use of constant positive or negative potentials (vs SCE) resulted in significant differences in the relative coverages among regions of different atomic arrangements (Fig 1). In addition, a significant difference was observed between immobilization buffers that contain Cl and those that are Cl free. Also, the use of constant or square-wave potential perturbations resulted in significantly different local organizations of DNA in the SAM. Examples of these types of DNA SAM surfaces will be presented with some conclusions on the implications for use on polycrystalline planar gold surfaces.

Covalent immobilization of Aspergillus niger amyloglucosidase (ANAG) with ethylenediamine-functionalized and glutaraldehyde-activated active carbon (EFGAAC) obtained from sesame seed shell

This study was aimed the covalently immobilization of Aspergillus niger amyloglucosidase (ANAG) onto activated carbon (AC) obtained from sesame seed shell. AC was firstly functionalized with ethylenediamine, and after then activated with glutaraldehyde. 99.80% immobilization yield and 99.83% activity yield were obtained as the result of optimization of immobilization conditions (pH and molarity of immobilization buffer, AC amount, and reaction time). The optimum pH (5.5) and the optimum temperature range (55–60 °C) for ANAG were not affected by immobilization. After immobilization, Vmax value decreased from 1464.1 μmol D-glucose/L.min to 1342.3 μmol D-glucose/L.min, while Km value decreased from 116.3 g maltodextrin/L to 109.9 g maltodextrin/L. The immobilized enzyme retained 99.30% and 98.30% of its initial activity, respectively after twenty repeated uses and after twenty days of storage in 5 mL sodium phosphate buffer (0.1 M, pH 5.5) at +4 °C in a refrigerator. Finally, glucose syrup was produced from maltodextrin solution having 1% (w/v) concentration by using the immobilized ANAG. Maltodextrin was completely converted to glucose after four hours. Consequently, it can be said that the immobilized ANAG obtained in this study can be used in the industrial production of glucose syrup.

Modulating the properties of the lipase from Thermomyces lanuginosus immobilized on octyl agarose beads by altering the immobilization conditions

The lipase from Thermomyces lanuginosus (TLL) has been immobilized on octyl-agarose beads via interfacial activation under 16 different conditions (changing the immobilization pH, the ionic strength, the presence of additives like calcium, phosphate or glycerol) and using a low loading (1 mg/g support). Then, the properties of the different biocatalysts have been evaluated: stability at pH 7.0 and 70 °C and activity versus p-nitro phenyl propionate, triacetin and R- and S- methyl mandelate. Results clearly indicate that the immobilization conditions determine the final enzyme properties, altering enzyme stability (by 10 folds), activity (by 8 folds using R- methyl mandelate) and specificity (VR/VS changed from 0.7 to 2.3 using mandelate esters). For instance, the enzymes immobilized at pH 7.0 using 5 mM buffer were the most stable preparations, while the presence of 250 mM sodium phosphate greatly decreased the final enzyme stability. The biocatalyst stability of TLL increased with increasing NaCl in the immobilization buffer at pH 5. Fluorescence studies confirmed that the conformation of the different immobilized enzymes were different, despite being a physical and reversible immobilization method. Thus, the immobilization of TLL on octyl agarose beads under different conditions produced biocatalysts with different properties, the optimal condition depends on the studied reaction and condition.

Multiplexed in-gel microfluidic immunoassays: characterizing protein target loss during reprobing of benzophenone-modified hydrogels

From whole tissues to single-cell lysate, heterogeneous immunoassays are widely utilized for analysis of protein targets in complex biospecimens. Recently, benzophenone-functionalized hydrogel scaffolds have been used to immobilize target protein for immunoassay detection with fluorescent antibody probes. In benzophenone-functionalized hydrogels, multiplex target detection occurs via serial rounds of chemical stripping (incubation with sodium-dodecyl-sulfate (SDS) and β-mercaptoethanol at 50–60 °C for ≥1 h), followed by reprobing (interrogation with additional antibody probes). Although benzophenone facilitates covalent immobilization of proteins to the hydrogel, we observe 50% immunoassay signal loss of immobilized protein targets during stripping rounds. Here, we identify and characterize signal loss mechanisms during stripping and reprobing. We posit that loss of immobilized target is responsible for ≥50% of immunoassay signal loss, and that target loss is attributable to disruption of protein immobilization by denaturing detergents (SDS) and incubation at elevated temperatures. Furthermore, our study suggests that protein losses under non-denaturing conditions are more sensitive to protein structure (i.e., hydrodynamic radius), than to molecular mass (size). We formulate design guidance for multiplexed in-gel immunoassays, including that low-abundance proteins be immunoprobed first, even when targets are covalently immobilized to the gel. We also recommend careful scrutiny of the order of proteins targets detected via multiple immunoprobing cycles, based on the protein immobilization buffer composition.

Enhancing the Microarray Signal Detection of Single Nucleotide Polymorphisms (SNPs) by Using Homemade Immobilisation Buffers

Our research is focused on improving the detection capabilities of single nucleotide polymorphisms (SNPs) with pathological significance within breast cancer 1, early onset gene (BRCA1). We employed the BRCA1 canonical probe sequence containing the common C allele and probes including the mismatches caused by C replacement with any of the other nucleotides (A, G or T). Two immobilization buffers with distinct pH and salt concentration were involved in the experimental and statistical analyses in order to assess the detection specificity and stability over time of the tethered probes in oligonucleotide microarrays. A high spreading of data points (standard deviation 0.16) was acquired from the fluorescent hybridization signal of the DNA probes diluted in Phosphate Buffered Saline (PBS), while the Disodium Phosphate (Na2HPO4) buffer was recommended by a standard deviation of 0.08 for further investigations. The high specificity of mismatch detection of DNA probes diluted in Na2HPO4 buffer was established by Student�s t test, revealing the statistical reliability of the DNA-printed slides after 1, 4, 8 and 21 days.

Highly efficient production of chiral amines in batch and continuous flow by immobilized ω-transaminases on controlled porosity glass metal-ion affinity carrier

In this study, two stereocomplementary ω-transaminases from Arthrobacter sp. (AsR-ωTA) and Chromobacterium violaceum (Cv-ωTA) were immobilized via iron cation affinity binding onto polymer-coated controlled porosity glass beads (EziG™). The immobilization procedure was studied with different types of carrier materials and immobilization buffers of varying compositions, concentrations, pHs and cofactor (PLP) concentrations. Notably, concentrations of PLP above 0.1 mM were correlated with a dramatic decrease of the immobilization yield. The highest catalytic activity, along with quantitative immobilization, was obtained in MOPS buffer (100 mM, pH 8.0, PLP 0.1 mM, incubation time 2 h). Leaching of the immobilized enzyme was not observed within 3 days of incubation. EziG-immobilized AsR-ωTA and Cv-ωTA retained elevated activity when tested for the kinetic resolution of rac-α-methylbenzylamine (rac-α-MBA) in single batch experiments. Recycling studies demonstrated that immobilized EziG3-AsR-ωTA could be recycled for at least 16 consecutive cycles (15 min per cycle) and always affording quantitative conversion (TON ca. 14,400). Finally, the kinetic resolution of rac-α-MBA with EziG3-AsR-ωTA was tested in a continuous flow packed-bed reactor (157 μL reactor volume), which produced more than 5 g of (S)-α-MBA (>49% conversion, >99% ee) in 96 h with no detectable loss of catalytic activity. The calculated TON was more than 110,000 along with a space-time yield of 335 g L−1 h−1.

Immobilized cutinases: Preparation, solvent tolerance and thermal stability

Developing active immobilized enzymes and characterization of their use conditions is critically important prior to initiating studies of enzyme selectivity and substrate specificity in organic media. To this end, physical immobilization by hydrophobic interactions was performed with three well-characterized cutinases (Aspergillus oryzae Cutinase (AoC), Humicola insolens Cutinase (HiC), and Thielavia terrestris Cutinase (TtC)) using Lewatit VP OC 1600 as the macroporous support. We found that immobilization yields >98% were achieved for all three cutinases under the following immobilization conditions: 100 mg/g loading ratio, immobilization buffers of 100 mM phosphate pH 8 (AoC and HiC) and 100 mM acetate pH 5 (TtC), mixing at 150 rpm and 30 °C for 24 h. Among the three cutinases, HiC has the highest tolerance towards solvents of increased polarity while TtC has the highest thermal stability (up to 80 °C) in a bulk reaction system that consists of the reactants butanol and lauric acid. In nonane, these cutinases retain >64% of their activity at 90 °C. Furthermore, kinetic stability (residual activity as a function of time) analysis reveals that the cutinases retain >75% residual activity at 70 °C in 3 h. Moreover, at 80 °C, the kinetic stability of TtC is higher than that of HiC and AoC. Collectively, the results herein set the stage for the in-depth evaluation of these catalysts for selective transformations in organic media.

Feruloyl esterase immobilization in mesoporous silica particles and characterization in hydrolysis and transesterification

BackgroundEnzymes display high reactivity and selectivity under natural conditions, but may suffer from decreased efficiency in industrial applications. A strategy to address this limitation is to immobilize the enzyme. Mesoporous silica materials offer unique properties as an immobilization support, such as high surface area and tunable pore size.ResultsThe performance of a commercially available feruloyl esterase, E-FAERU, immobilized on mesoporous silica by physical adsorption was evaluated for its transesterification ability. We optimized the immobilization conditions by varying the support pore size, the immobilization buffer and its pH. Maximum loading and maximum activity were achieved at different pHs (4.0 and 6.0 respectively). Selectivity, shown by the transesterification/hydrolysis products molar ratio, varied more than 3-fold depending on the reaction buffer used and its pH. Under all conditions studied, hydrolysis was the dominant activity of the enzyme. pH and water content had the greatest influence on the enzyme selectivity and activity. Determined kinetic parameters of the enzyme were obtained and showed that Km was not affected by the immobilization but kcat was reduced 10-fold when comparing the free and immobilized enzymes. Thermal and pH stabilities as well as the reusability were investigated. The immobilized biocatalyst retained more than 20% of its activity after ten cycles of transesterification reaction.ConclusionsThese results indicate that this enzyme is more suited for hydrolysis reactions than transesterification despite good reusability. Furthermore, it was found that the immobilization conditions are crucial for optimal enzyme activity as they can alter the enzyme performance.

Discovering the enzyme mimetic activity of metal-organic framework (MOF) for label-free and colorimetric sensing of biomolecules

A label-free sensing strategy based on the enzyme-mimicking activity of MOF was demonstrated for colorimetric detection of biomolecules. Firstly obvious blue color was observed due to the high efficiency of peroxidase-like catalytic activity of Fe-MIL-88A (an ion-based MOF material) toward 3,3′,5,5′-tetramethylbenzidine (TMB). Then in the presence of target biomolecule and corresponding aptamer, the mimetic activity of Fe-MIL-88A can be strongly inhibited and used directly to realize the colorimetric detection. On the basis of the interesting findings, we designed a straightforward, label-free and sensitive colorimetric method for biomolecule detection by using the enzyme mimetic property of MOF coupling with molecular recognition element. Compared with the existed publications, our work breaks the routine way by setting up an inorganic-organic MOF-aptamer hybrid platform for colorimetric determination of biomolecules, expanding the targets scope from H2O2 or glucose to biomolecules. As a proof of concept, thrombin and thrombin aptamer was used as a model analyte. The limit of detection of 10nM can be achieved with naked eyes and ultrahigh selectivity of thrombin toward numerous interfering substances with 10-fold concentration was demonstrated significantly. Of note, the method was further applied for the detection of thrombin in human serum samples, showing the results in agreement with those values obtained in an immobilization buffer by the colorimetric method. This inorganic-organic MOF-aptamer sensing strategy may in principle be universally applicable for the detection of a range of environmental or biomedical molecules of interests.

Real Colorimetric Thrombin Aptasensor by Masking Surfaces of Catalytically Active Gold Nanoparticles.

We presented a simple, cost-effective, and ultrasensitive colorimetric approach for visually detecting thrombin by the catalytic amplification of gold nanoparticles (AuNPs) and aptamer-thrombin recognition. Thrombin can be quantified in the presence of catalytic AuNP surface by using color-change time of 4-nitrophenol. Without thrombin, yellow 4-nitrophenol can freely access the surface of AuNP and becomes colorless 4-aminophenol. With the addition of thrombin, aptamer-thrombin with large size interaction masks the partial surfaces of AuNPs, and increases the reduction time of 4-nitrophenol to 4-aminophenol. The maximum number of bound thrombin fully mask the catalytic AuNP surface, and thus 4-nitrophenol cannot approach to AuNP surface, the color of the solution remains yellow. The limit of detection (LOD) of 0.1 nM can be achieved with naked eyes. Of note, the method was further applied for the detection of thrombin in human serum samples, showing the results in agreement with those values obtained in an immobilization buffer by the colorimetric method.

Buffer mobility and the regulation of neuronal calcium domains.

The diffusion of calcium inside neurons is determined in part by the intracellular calcium binding species that rapidly bind to free calcium ions upon entry. It has long been known that some portion of a neuron's intracellular calcium binding capacity must be fixed or poorly mobile, as calcium diffusion is strongly slowed in the intracellular environment relative to diffusion in cytosolic extract. The working assumption was that these immobile calcium binding sites are provided by structural proteins bound to the cytoskeleton or intracellular membranes and may thereby be relatively similar in composition and capacity across different cell types. However, recent evidence suggests that the immobile buffering capacity can vary greatly between cell types and that some mobile calcium binding proteins may alter their mobility upon binding calcium, thus blurring the line between mobile and immobile. The ways in which immobile buffering capacity might be relevant to different calcium domains within neurons has been explored primarily through modeling. In certain regimes, the presence of immobile buffers and the interaction between mobile and immobile buffers have been shown to result in complex spatiotemporal patterns of free calcium. In total, these experimental and modeling findings call for a more nuanced consideration of the local intracellular calcium microenvironment. In this review we focus on the different amounts, affinities, and mobilities of immobile calcium binding species; propose a new conceptual category of physically diffusible but functionally immobile buffers; and discuss how these buffers might interact with mobile calcium binding partners to generate characteristic calcium domains.