Molecules In Complex Matrices Research Articles

Determination of Aminophylline in Human Serum Using Hydrogel Microspheres for Coupled Surface-Enhanced Raman Spectroscopy (SERS) and Solid-Phase Extraction.

Aminophylline (AMP) is a bronchodilator. The therapeutic and toxic doses are very close. Therefore, therapeutic drug monitoring (TDM) of AMP is essential in clinical practice. Microgels were synthesized by free radical precipitation polymerization. Silver@poly(N-isopropyl acrylamide) (Ag@PNIPAM) hybrid microgels were obtained by loading silver (Ag) nanoparticles into the three-dimensional network of the microgels by in situ reduction. The microgel is a three-dimensional reticular structure with tunable pore size, large specific surface area, and good biocompatibility, which can be used as a sorbent for solid-phase extraction (SPE) of target molecules in complex matrices and as a surface-enhanced Raman spectroscopy (SERS) substrate. We optimized the conditions affecting SERS enhancement, such as silver nitrate (AgNO3) concentration and SPE time, according to the SERS strategy of Ag@PNIPAM hybrid microgels to achieve label-free TDM for trace AMP in human serum. The results showed good linearity between the logarithmic concentration of AMP and its SERS intensity in the range of 1-1.1 × 102 µg/mL, with a correlation coefficient (R2) of 0.9947 and a low detection limit of 0.61 µg/mL. The assay accuracy was demonstrated by spiking experiments, with recoveries ranging from 93.0 to 101.8%. The method is rapid, sensitive, reproducible, requires simple sample pretreatment, and has good potential for use in clinical treatment drug monitoring.

DNA Chimeras as Electrochemical Biosensors for Host-Guest Measurements in Blood.

Few sensing platforms have become ubiquitous to enable rapid and convenient measurements at the point-of-care. Those, however, are "one-off" technologies, meaning that they can only detect a single target and are hardly adaptable. In response, we plan to develop a sensing platform that can be extended to detect other classes of molecules and that affords rapid, convenient, continuous measurements directly in undiluted complex matrices. For this, we decided to rely on a host molecule that presents reversible interactions toward specific guest molecules to develop a new class of sensors that we coined "Electrochemical DNA-host chimeras". As a proof-of-concept for our sensor, we decided to use cyclobis(paraquat-p-phenylene) ("blue box") that we attached on an electrode-bound DNA to allow measurements of electron-rich guests such as dopamine and aspirin. Doing so allows to promote host-guest complex that could be quantified using blue box's electrochemistry. Because of this unique sensor architecture, we achieve, to our knowledge, the first reagentless, continuous and rapid (< 5 min) host-guest measurements in undiluted whole blood. We envision that given the library of electroactive host molecules that this will allow the development of a sensing platform for measurements of several classes of molecules in complex matrices at the point-of-care.

A core-satellite self-assembled SERS aptasensor containing a “biological-silent region” Raman tag for the accurate and ultrasensitive detection of histamine

Herein, a novel interference-free surface-enhanced Raman spectroscopy (SERS) strategy based on magnetic nanoparticles (MNPs) and aptamer-driven assemblies was proposed for the ultrasensitive detection of histamine. A core-satellite SERS aptasensor was constructed by combining aptamer-decorated Fe3O4@Au MNPs (as the recognize probe for histamine) and complementary DNA-modified silver nanoparticles carrying 4-mercaptobenzonitrile (4-MBN) (Ag@4-MBN@Ag-c-DNA) as the SERS signal probe for the indirect detection of histamine. Under an applied magnetic field in the absence of histamine, the assembly gave an intense Raman signal at “Raman biological-silent” region due to 4-MBN. In the presence of histamine, the Ag@4-MBN@Ag-c-DNA SERS-tag was released from the Fe3O4@Au MNPs, thus decreasing the SERS signal. Under optimal conditions, an ultra-low limit of detection of 0.65 × 10-3 ng/mL and a linear range 10-2–105 ng/mL on the SERS aptasensor were obtained. The histamine content in four food samples were analyzed using the SERS aptasensor, with the results consistent with those determined by high performance liquid chromatography. The present work highlights the merits of indirect strategies for the ultrasensitive and highly selective SERS detection of small biological molecules in complex matrices.

A molecular networking-assisted automatic database screening strategy for comprehensive annotation of small molecules in complex matrices

Liquid chromatography-tandem with high-resolution mass spectrometry (LCHRMS) has proven challenging for annotating multiple small molecules within complex matrices due to the complexities of chemical structure and raw LCHRMS data, as well as limitations in previous literatures and reference spectra related to those molecules. In this study, we developed a molecular networking assisted automatic database screening (MN/auto-DBS) strategy to examine the combined effect of MS1 exact mass screening and MS2 similarity analysis. We compiled all previously reported compounds from the relevant literatures. With the development of a Python software, the in-house database (DB) was created by automatically calculating the m/z and data from experimental MS1 hits were rapid screened with DB. We then performed a feature-based molecular network analysis on the auto-MS2 data for supplementary identification of unreported compounds, including clustered FBMN and annotated GNPS compounds. Finally, the results from both strategies were merged and manually curated for correct structural assignment. To demonstrate the applicability of MN/auto-DBS, we selected the Huangqi-Danshen herb pair (HD), commonly used in prescriptions or patent medicines to treat diabetic nephropathy and cerebrovascular disease. A total of 223 compounds were annotated, including 65 molecules not previously reported in HD, such as aromatic polyketides, coumarins, and diarylheptanoids. Using MN/auto-DBS, we can profile and mine a wide range of complex matrices for potentially new compounds.

Novel Insights on Pyoverdine: From Biosynthesis to Biotechnological Application.

Pyoverdines (PVDs) are a class of siderophores produced mostly by members of the genus Pseudomonas. Their primary function is to accumulate, mobilize, and transport iron necessary for cell metabolism. Moreover, PVDs also play a crucial role in microbes’ survival by mediating biofilm formation and virulence. In this review, we reorganize the information produced in recent years regarding PVDs biosynthesis and pathogenic mechanisms, since PVDs are extremely valuable compounds. Additionally, we summarize the therapeutic applications deriving from the PVDs’ use and focus on their role as therapeutic target themselves. We assess the current biotechnological applications of different sectors and evaluate the state-of-the-art technology relating to the use of synthetic biology tools for pathway engineering. Finally, we review the most recent methods and techniques capable of identifying such molecules in complex matrices for drug-discovery purposes.

A compact liquid chromatography-mass spectrometry instrument for the quantitation of immunosuppressants

Liquid chromatography tandem mass spectrometry (LC-MS/MS) plays an important role in clinical diagnostics. Although LC-MS/MS is superior in terms of accurately quantifying molecules in complex matrices, instrument footprint, operation and maintenance complexity also hinder its expansion as the analytical technique of choice. In this study, a compact LC-MS instrument was developed, in which an assembled liquid chromatograph was coupled with a miniature ion trap mass spectrometer. The overall instrument has a footprint of 69 cm × 31 cm × 31 cm, and it requires no gas supply as well as minimum maintenance. Furthermore, the use of LC-MS is in accord with conventional clinical diagnostic protocols, and the choice of ion trap offers tandem MS performance. The results showed that the use of LC could improve both mixture analysis capability and detection sensitivity of the miniature mass spectrometer. After optimization, feasibility of this instrument in clinical practice was demonstrated by the quantitation of four widely used immunosuppressants in blood samples. Relatively good linearities were obtained, which spanned the reference ranges of effective therapeutic concentrations of each immunosuppressant. Intra-day and inter-day accuracy and precision of analytical method were also assessed. This work showed that a compact LC-MS instrument could be used in clinical diagnosis, either to replace conventional lab-scale instruments or to be used in POCT applications.

Analysis of low-abundance molecules in complex matrices by quadrupole-linear ion trap mass spectrometry using a simultaneous fragmentation and accumulation strategy.

Fast and sensitive analysis of low-abundance molecules in complex matrices has always been a challenge in chemical and biological applications. Mass spectrometry (MS) has been widely used in the fields of chemical and biological analysis due to its unparalleled specificity and sensitivity. However, the MS signals consistently deteriorate in the presence of matrices. Demands for more sensitive and efficient methods to analyze those low-abundance molecules in chemical and biological systems are in urgent need. Based on a home-made quadrupole-linear ion trap (Q-LIT) mass spectrometer, a simultaneous fragmentation and accumulation strategy was developed to improve the sensitivity of the analysis for the low-abundance molecules in complex matrices. Ions were filtered by the quadrupole into the LIT. The precursor ions were fragmented and the product ions were isolated and accumulated in the LIT simultaneously. The fragmentation, isolation and accumulation processes were conducted at the same time. The accumulation time could be controlled to accumulate sufficient product ions. With this strategy, the signal intensity of targeted molecules could be increased by 2-8 times and by increasing the accumulation time, this could be further enhanced. Those interferences induced by isomers and matrices can be reduced by using our method. We further applied our method to the quantification and analysis of biological samples. Tryptic digested peptides of myoglobin (Mb) were successfully detected by our method. We have established a new method with great advantages in the detection of molecules in complex matrices. The application of this method promises better results in the bioanalytical area, especially for the analysis of substances in complex matrices in the future.

Ultrasensitive biogenic amine sensor using an enhanced multiple nanoarray chip based on competitive reactions in an evanescent field

Considering the importance of biogenic amines (BAs), such as histamine, tyramine, and putrescine, as biomarkers and the challenges in their simultaneous detection at low concentrations, a novel enhanced multiple nanoarray BA sensor was developed using a competitive reaction in an evanescent field (EF). BAs were bound to specific antibodies on different gold nanoarray islands on a multiple nanoarray sensor chip. The electromagnetic field in the EF produced fluorescence signals of BA-conjugated quantum dots with an excellent signal-to-noise ratio on the islands. Compared to conventional fluorescent dyes or plasmonic nanoparticles as detection probes, quantum dots showed improved detection sensitivity with excellent photostability. The competitive reaction in EF was simultaneously used to detect three BA molecules on the multiple nanoarray. The multiple BA sensor exhibited an excellent detection limit of 63 aM with a wide dynamic range of 100 aM–10 nM, which had a 1,000,000-fold higher sensitivity than that of previously reported methods. The proposed BA sensor was validated by high-performance liquid chromatography and enzyme-linked immunosorbent assay and was applied to identify BAs in wine samples. The results agreed with those of standard methods, showing 95 % confidence level. The combined competitive reaction and simultaneous detection of BA-conjugated quantum dots in EF on the multiple nanoarray sensor exhibited excellent enhancement of detection sensitivity. This high-throughput screening method shows highly specific labeling using promiscuous antibodies for detecting small molecules in complex matrices.

Molecularly Imprinted Polymer Micro- and Nano-Particles. A review.

In recent years, molecularly imprinted polymers (MIPs) have become an excellent solution to the selective and sensitive determination of target molecules in complex matrices where other similar and relative structural compounds could coexist. Although MIPs show the inherent properties of the polymers, including stability, robustness, and easy/cheap synthesis, some of their characteristics can be enhanced, or new functionalities can be obtained when nanoparticles are incorporated in their polymeric structure. The great variety of nanoparticles available significantly increase the possibility of finding the adequate design of nanostructured MIP for each analytical problem. Moreover, different structures (i.e., monolithic solids or MIPs micro/nanoparticles) can be produced depending on the used synthesis approach. This review aims to summarize and describe the most recent and innovative strategies since 2015, based on the combination of MIPs with nanoparticles. The role of the nanoparticles in the polymerization, as well as in the imprinting and adsorption efficiency, is also discussed through the review.

Ultrasensitive and rapid detection of T-2 toxin using a target-responsive DNA hydrogel

The T-2 toxin is generated as a secondary metabolite by various species of Fusarium that are found extensively in barley, wheat, maize, and oats; consequently, the development of methods that can rapidly and efficiently detect this toxin is vital. Herein, we report on a novel and highly sensitive method based on DNA hydrogels and etched gold nanorods (AuNRs) to detect the T-2 toxin. The T-2 toxin aptamer acts as a cross-linking agent for a hydrogel in which horseradish peroxidase (HRP) is uniformly embedded. The target T-2 toxin binds to the aptamer, causing the hydrogel to collapse and release the embedded HRP, which catalyzes the reaction of H2O2 with KI to produce I2 that etches the AuNRs. The UV spectrum of the AuNRs is blue-shifted following etching; this shift is proportional to the T-2-toxin content. This method offers a low limit of detection (0.87 pg mL−1), low limit of quantification (2.90 pg mL−1), and wide detection range (0.01–10000 ng mL−1). Moreover, coffee, corn, and soybean were assayed using the optimized method. The approach offers good recoveries and relative standard deviations of 93.72–103.07 % and 3.87–7.07 %, respectively. The total assay time was 1.5 h. Thus, the new method is promising as an ultrasensitive assay for detecting various small molecules in complex matrices.

Does the ion mobility resolving power as provided by commercially available ion mobility quadrupole time-of-flight mass spectrometry instruments permit the unambiguous identification of small molecules in complex matrices?

Quadrupole based mass spectrometry based detection has experienced enormous improvements in terms of sensitivity over the last centuries. This development has not been equally matched with improvements in selectivity. Hence, the use of unit mass based MS/MS transitions or high resolution (HRMS) based extracted ion chromatograms is gradually becoming insufficient in the field of high sensitivity multi-residue analysis (e.g. pesticides in food). As a consequence, commercial instruments hyphenating ion mobility (IMS) with low or high resolution mass spectrometry based detection have appeared. The use of such an additional (frequently claimed to be orthogonal) dimension is intended to increase selectivity. In addition, IMS derived collision cross section (CCS) has been proposed to be used as an additional identification point for the unambiguous identification of trace compounds in complex matrices.It is the topic of this paper to investigate the benefit of using such a hyphenated technique for trace analysis of small molecules in complex matrices. The potential of CCS to serve as additional identification point has been critically evaluated. Discussed are the effect of CCS data on false detects and missing detects of analytes present at trace levels. This involves the investigation of the physical resolving power provided by HRMS, IMS and chromatography as well as the correlation among these parameters (orthogonality).It is the conclusion that currently commercially available travelling wave and linear drift tube based IMS devices with a resolving power of up to 50 permit a reduction of false detects, yet this comes at the price of a higher likelihood of missing detects. The reduction of missing detects and the use of CCS as potential confirmatory information would require IMS resolving powers above 100.

Hunting Molecules in Complex Matrices with SPME Arrows: A Review

Thirty years since the invention and public disclosure of solid phase microextraction (SPME), the technology continues evolving and inspiring several other green extraction technologies amenable for the collection of small molecules present in complex matrices. In this manuscript, we review the fundamental and operational aspects of a novel SPME geometry that can be used to “hunt” target molecules in complex matrices: the SPME Arrow. In addition, a series of applications in environmental, food, cannabis and forensic analysis are succinctly covered. Finally, special emphasis is placed on novel interfaces to analytical instrumentation, as well as recent developments in coating materials for the SPME Arrow.

Utilizing Drift Tube Ion Mobility Spectrometry for the Evaluation of Metabolites and Xenobiotics.

Metabolites and xenobiotics are small molecules with a molecular weight that often falls below 600Da. Over the last few decades, multiple small molecule databases have been curated listing structures, masses, and fragmentation spectra possible in metabolomic and exposomic measurements. To date only a small portion of the spectra in these databases are experimentally derived due to the high expense of obtaining, synthesizing, and analyzing standards. A vast majority of spectra have thus been created using theoretical programs to fit the available experimental data. The errors associated with theoretical data have however caused problems with current small molecule identifications, and accurate quantitation as searching the databases using just one or two analysis dimensions (i.e., chromatography retention times and mass spectrometry (MS) m/z values) results in numerous annotations for each experimental feature. Additional analysis dimensions are therefore needed to better annotate and identify small molecules. Drift tube ion mobility spectrometry coupled with MS (DTIMS-MS) is a promising technique to address this challenge as it is able to perform rapid structural evaluations of small molecules in complex matrices by assessing the collision cross section values for each in addition to their m/z values. The use of IMS in conjunction with other separation techniques such as gas or liquid chromatography and MS has therefore enabled more accurate identifications for the small molecules present in complex biological and environmental samples. Here, we present a review of relevant parameter considerations for DTIMS application with emphasis on xenobiotics and metabolomics isomer separations.

Combination of liquid-based column separations with surface-enhanced Raman spectroscopy.

Surface-enhanced Raman spectroscopy is a constantly developing analytical method providing not only high-sensitive quantitative but also qualitative information on an analyte. Thus, it is reasonable that it has been tested as a promising detection method in column separations. Although its implementation in analytical separations is not widespread, some surprising results, like enormous signal enhancement and demonstrations of single-molecule identifications, proved in only a few special examples, indicate the potential of the method. The high detection sensitivity and selectivity would be of paramount importance in trace analyses of biologically relevant molecules in complex matrices. However, the combination of surface-enhanced Raman spectroscopy with column separation methods brings two principal issues. Interactions of analytes with metal substrates can cause deteriorations of separations and the detection can be affected by background electrolytes or elution agents. Thus, in principle, this review is on the experimental and methodological solutions to these problems. First, theoretical and practical aspects of Raman scattering, and excitation of surface plasmon in colloid suspensions of nanoparticles and on planar nanostructured substrates are briefly explained. Advances in experimental arrangements of on-line and at-line couplings with column liquid phase separation methods, including microfluidic devices, are described together with chosen analytical applications.

Ultrasensitive detection of T-2 toxin in food based on bio-barcode and rolling circle amplification

A novel and highly sensitive method based on bio-barcode with rolling circle amplification (RCA) was developed for the detection of T-2 toxin. Gold nanoparticles (AuNPs) were modified with anti-T-2 monoclonal antibody and single-stranded thiol-oligonucleotides (SH-ssDNAs) and magnetic microparticles (MMPs) coated with T-2 antigen. The T-2 toxin competes with the antigen on MMPs for the anti-T-2 antibody on AuNPs. Then, the isolating complex system was separated by a magnetic field, and the DNA of the probes was released after washing in dithiothreitol solution. The barcode DNA via RCA and products were stained by SYBR Green I and then detected by fluorescence spectrophotometry. The optimized method was performed on oats, millet, flour, and other substances. This method exhibits a low limit of detection (0.26 pg mL−1) and linear range of 0.002–200 ng mL−1. Moreover, the approach offers good recovery and relative standard deviations ranging from 88.65% to 10.04% and 0.6%–13.1%, respectively. In conclusion, this method exhibits potential for use as an ultrasensitive assay for the detection of a variety of small molecules in complex matrices.

Adsorption of Phthalates on Activated Carbons in Monosolute Solution and in Mix Within Complex Matrices

Phthalates are considered as dangerous priority pollutants, several effects being attributed to them: foetal deformations, cancers, and endocrine disruptions. Activated carbons are highly efficient materials for the adsorption of numerous organic molecules. Before their use, it is important first to determine both textural and chemical properties and to study kinetics and thermodynamics adsorption, to understand and to optimize the interactions between material and molecules. The aim of this work was to study the kinetics and the adsorption isotherms of three phthalates (dimethylphthalate, diethylphthalate, and diethylhexylphthalate) currently found in industrial effluents, on two different activated carbons. The co-adsorption of these molecules in a synthetic mix and in complex matrices was modeled. The kinetic study and adsorption isotherms of dimethylphthalate and diethylphthalate in monosolute and bisolute were first investigated, followed by a similar study with a mix of the three molecules in complex matrices (surface water (Loire and Loiret Rivers near Orleans city) and municipal wastewater treatment plant outflow). The pseudo-second-order kinetic model was used to determine the kinetic adsorption parameters. The Langmuir equation was used to calculate the surface occupied. Results showed that non-electrostatic interactions are predominant in phthalate adsorption in complex matrices, mainly due to dispersion forces and hydrophobic interactions.

Development and application of a new in-line coupling of a miniaturized boronate affinity monolithic column with capillary zone electrophoresis for the selective enrichment and analysis of cis-diol-containing compounds

An integrated, miniaturized and fully automated system was developed for the analysis (preconcentration/purification, separation and detection) of cis-diol containing molecules in complex matrices. This innovative in-line coupling system was achieved via the in-situ and localized synthesis of a short segment of silica-based monolith at the inlet of a 75-μm inner diameter fused silica capillary. The monolithic segment was locally functionalized with an acrylamide derivative of phenylboronic acid by free radical photopolymerization within 10min of irradiation time. Efficiency of the photopolymerization reaction was followed by frontal affinity chromatography of 1,2-dihydroxybenzene (catechol) as cis-diol model solute. An active-site amount of 0.43nmolcm−1 (9.8nmolμL−1) of phenylboronic acid moieties was obtained, with a Kd value of about 290μM close to reported value for the phenyl boronate-catechol complex. The optimal conditions of use of the miniaturized boronate affinity monolithic column (μBAMC) were determined and adapted to the in-line coupling with capillary electrophoresis. Catechol was specifically preconcentrated in a pH 8.5 phosphate buffer/MeOH (80/20, v/v) mixture. A volume up to 20 times the monolith volume can be percolated with a quantitative recovery yield. Three catecholamines were purified, preconcentrated and in-line separated. Elution from the μBAMC was performed with a small plug of acidic solution, allowing field amplified sample stacking of solutes within the plug before their in-line electrophoretic separation at pH 8.75. This unique in-line coupling was successfully used for the fully automated analysis of catecholamines neurotransmitters in urine samples, highlighting the purification efficiency of the μBAMC and the potential of such a fully integrated approach. In addition to the low sample volume required (less than 2μL), the limits of detection (LOD) accomplished with this coupling were estimated at 9.0, 9.5 and 4.8ngmL−1 for dopamine, adrenaline and noradrenaline respectively, which improves the LOD of theses solutes compared to other CE methods.

Proteoform-Specific Protein Binding of Small Molecules in Complex Matrices.

Characterizing the specific binding between protein targets and small molecules is critically important for drug discovery. Conventional assays require isolation and purification of small molecules from complex matrices through multistep chromatographic fractionation, which may alter their original bioactivity. Most proteins undergo posttranslational modification, and only certain proteoforms have the right conformation with accessible domains and available residues for small molecule binding. We developed a top-down mass spectrometry (MS) centric workflow for rapid evaluation of the bioactivity of crude botanical extracts after a one-step reaction. Our assay distinguished covalent from noncovalent binding and mapped the residue for covalent binding between bioactive constituents and specific proteoforms of the target protein. We augmented our approach with a nanoflow liquid chromatography-selected reaction monitoring (SRM)-MS assay for simultaneous identification and label-free multiplex quantitation of small molecules in the crude botanical extracts. Our assay was validated for various proteoforms of human serum albumin, which plays a key role in pharmacokinetics of small molecules in vivo. We demonstrated the utility of our proteoform-specific assay for evaluating thymoquinone in crude botanical extracts, studying its pharmacokinetics in human blood, and interpreting its toxicity to human breast cancer cells in tissue culture.

Post-column hydrogen–deuterium exchange technique to assist in the identification of small organic molecules by mass spectrometry

To improve the certainty that a specific small organic molecule has been detected in a given sample by high-resolution mass spectrometry, other techniques that give conclusive evidence about the chemical structure of a compound like nuclear magnetic resonance or complementary information on its composition such as hydrogen–deuterium exchange (HDX) are often necessary. This study presents a systematic investigation that aims to improve the applicability of post-column HDX for those purposes. Key parameters like mobile-phase flow rates, volume percentage of H2O in the mobile phase, and D2O addition flow rates were optimized to provide an isotopic pattern that allows the accurate determination of the number of exchangeable hydrogen atoms in small organic molecules. A loop injection setup was used to emulate chromatographic conditions in the optimization process, and trimethoprim, a widely used anti-infective, was used as test compounds for the experiments. As expected, results showed that deuteration percentage decreased with a higher mobile-phase flow rate and increased with higher D2O flow rate. The post-column HDX technique was then validated with extracts of samples of river water and plants separated by liquid chromatography in hydrophilic interaction or reversed-phase modes. Mass spectra showed a completely visible isotopic pattern that allowed assessing correctly and unambiguously the number of exchangeable hydrogens in the compounds of interest. This study shows that post-column HDX can be used as a complementary technique to identify unknown small organic molecules in complex matrices. The current paper proposes an efficient, cost-effective, versatile technique of HDX that is helpful to assign a unique structure to a given high-resolution mass spectrometry signal.

Diffusion Behavior of Microbial Transglutaminase to Induce Protein Crosslinking in Oil-in-Water Emulsions

Food-grade hydrogel particles composed of sodium alginate were used to investigate the diffusion behavior of microbial transglutaminase (mTG) to induce crosslinking of interfacially adsorbed protein. For this purpose, mTG-loaded hydrogel beads were mixed with caseinate-stabilized oil-in-water emulsions, whereas Bradford assay and ammonia measurements were utilized to monitor the enzyme-induced interfacial protein crosslinking. Different alginate (0.5–1.5%) and gelling concentrations (50–500 mM CaCl2) were used to modulate the hydrogel mesh size and number of junction zones. The results indicated that mTG was able to diffuse out of alginate beads. However, a decrease in NH3 concentration with increasing alginate and CaCl2 levels was observed due to the formation of tight and dense bead structures. These results illustrate that the spatial distribution of molecules in complex matrices plays a key role on the enzyme accessibility