Detergent Sodium Dodecyl Sulfate Research Articles

Comparison of three techniques for decellularization of porcine bone-tendon-bone grafts

ABSTRACT Anterior cruciate ligament injuries are frequent afflictions related to sports or physical trauma. Autograft reconstruction strategies cause secondary injury to the patient. One alternative, supported by clinical evidence, is porcine xenografts. For clinical use, xenografts must be conditioned to avoid immune rejection. The most widely accepted procedure is tissue decellularization. We analyzed three decellularization strategies: the application of the anionic detergent sodium dodecyl sulfate (SDS), sonication, and freezing and thawing cycles. The treated tissues were evaluated histologically using H&E, Masson’s trichrome, Verhoeff-van Gieson staining, and DAPI for fluorescent staining of nuclei. Finally, collagen fiber preservation was evaluated by quantifying this protein by colorimetry. The most efficient decellularization techniques were sonication and SDS. Collagen fibers were preserved in all experimental conditions.

Bioengineering of vascularized porcine flaps using perfusion-recellularization

Large volume soft tissue defects greatly impact patient quality of life and function while suitable repair options remain a challenge in reconstructive surgery. Engineered flaps could represent a clinically translatable option that may circumvent issues related to donor site morbidity and tissue availability. Herein, we describe the regeneration of vascularized porcine flaps, specifically of the omentum and tensor fascia lata (TFL) flaps, using a tissue engineering perfusion-decellularization and recellularization approach. Flaps were decellularized using a low concentration sodium dodecyl sulfate (SDS) detergent perfusion to generate an acellular scaffold with retained extracellular matrix (ECM) components while removing underlying cellular and nuclear contents. A perfusion-recellularization strategy allowed for seeding of acellular flaps with a co-culture of human umbilical vein endothelial cell (HUVEC) and mesenchymal stromal cells (MSC) onto the decellularized omentum and TFL flaps. Our recellularization technique demonstrated evidence of intravascular cell attachment, as well as markers of endothelial and mesenchymal phenotype. Altogether, our findings support the potential of using bioengineered porcine flaps as a novel, clinically-translatable strategy for future application in reconstructive surgery.

Polycarbonate Ultracentrifuge Tube Re-use in Proteomic Analyses of Extracellular Vesicles.

Single-use laboratory plastics exacerbate the pollution crisis and contribute to consumable costs. In extracellular vesicle (EV) isolation, polycarbonate ultracentrifuge (UC) tubes are used to endure the associated high centrifugal forces. EV proteomics is an advancing field and validated re-use protocols for these tubes are lacking. Re-using consumables for low-yield protein isolation protocols and downstream proteomics requires reagent compatibility with mass spectroscopy acquisitions, such as the absence of centrifuge tube-derived synthetic polymer contamination, and sufficient removal of residual proteins. This protocol describes and validates a method for cleaning polycarbonate UC tubes for re-use in EV proteomics experiments. The cleaning process involves immediate submersion of UC tubes in H2O to prevent protein drying, washing in 0.1% sodium dodecyl sulfate (SDS) detergent, rinsing in hot tap water, demineralized water, and 70% ethanol. To validate the UC tube re-use protocol for downstream EV proteomics, used tubes were obtained following an experiment isolating EVs from cardiovascular tissue using differential UC and density gradient separation. Tubes were cleaned and the experimental process was repeated without EV samples comparing blank never-used UC tubes to cleaned UC tubes. The pseudo-EV pellets obtained from the isolation procedures were lysed and prepared for liquid chromatography-tandem mass spectrometry using a commercial protein sample preparation kit with modifications for low-abundance protein samples. Following cleaning, the number of identified proteins was reduced by 98% in the pseudo-pellet versus the previous EV isolation sample from the same tube. Comparing a cleaned tube against a blank tube, both samples contained a very small number of proteins (≤20) with 86% similarity. The absence of polymer peaks in the chromatograms of the cleaned tubes was confirmed. Ultimately, the validation of a UC tube cleaning protocol suitable for the enrichment of EVs will reduce the waste produced by EV laboratories and lower the experimental costs.

In Vitro Cytocompatibility Assessment of Novel 3D Chitin/Glucan- and Cellulose-Based Decellularized Scaffolds for Skin Tissue Engineering

Background: Naturally derived sustainable biomaterials with high flexibility, mechanical properties, biocompatibility, and the ability to manipulate surface chemistry, providing a natural cellular environment, can be used for tissue engineering applications. However, only a few researchers have demonstrated the exploitation of natural architectures for constructing three-dimensional scaffolds. The chemical decellularization technique for fabricating natural scaffolds and their cytocompatibility assessment for tissue engineering applications need to be thoroughly explored and evaluated. Methods: Decellularization of natural scaffolds has been performed via a chemical method using anionic detergent sodium dodecyl sulfate (SDS) which was used for the in vitro culturing of murine embryonic NIH/3T3 fibroblasts. Techniques such as field-emission scanning electron microscopy (FE-SEM), compressive testing and swelling ratio, and biodegradation were performed to characterize the properties of fabricated decellularized natural scaffolds. Nucleic acid quantification, DAPI, and H&E staining were performed to confirm the removal of nuclear components. In vitro cytocompatibility and live/dead staining assays were performed to evaluate cultured fibroblasts’ metabolic activity and qualitative visualization. Results: 3D chitin/glucan- and cellulose-based scaffolds from edible mushroom (stem) (DMS) and unripe jujube fruit tissue (DUJF) were fabricated using the chemical decellularization technique. FE-SEM shows anisotropic microchannels of highly microporous structures for DMS and isotropic and uniformly arranged microporous structures with shallow cell cavities for DUJF. Both scaffolds exhibited good mechanical properties for skin tissue engineering and DUJF showed a higher compressive strength (200 kPa) than DMS (88.3 kPa). It was shown that the DUJF scaffold had a greater swelling capacity than the DMS scaffold under physiological conditions. At 28 days of incubation, DUJF and DMS displayed approximately 14.97 and 15.06% biodegradation, respectively. In addition, DUJF had greater compressive strength than DMS. Compared to DMS scaffolds, which had a compressive stress of 0.088 MPa at a 74.2% strain, the DUJF scaffolds had a greater compressive strength of 0.203 MPa at a 73.6% strain. The removal of nuclear DNA in the decellularized scaffolds was confirmed via nucleic acid quantification, DAPI, and H&E staining. Furthermore, both of these scaffolds showed good adherence, proliferation, and migration of fibroblasts. DMS showed better biocompatibility and high viability of cells than DUJF. Conclusions: This sustainable scaffold fabrication strategy is an alternative to conventional synthetic approaches for the in vitro 3D culture of mammalian cells for various tissue engineering and cultured meat applications.

Anionic surfactant causes dual conformational changes in insulin

Amyloid fibrillation is a process by which proteins aggregate and form insoluble fibrils that are implicated in several neurodegenerative diseases. In n this study, we aimed to investigate the impact of the negatively charged detergent sodium dodecyl sulfate (SDS) on insulin amyloid fibrillation at pH 7.4 and 2.0, as SDS has been linked to the acceleration of amyloid fibrillation in vitro, but the underlying molecular mechanism is not fully understood. Our findings show that insulin forms amyloid-like aggregates in the presence of SDS at concentrations ranging from 0.05 to 1.8 mM at pH 2.0, while no aggregates were observed at SDS concentrations greater than 1.8 mM, and insulin remained soluble. However, at pH 7.4, insulin remained soluble regardless of the concentration of SDS. Interestingly, the aggregated insulin had a cross-β sheet secondary structure, and when incubated with higher SDS concentrations, it gained more alpha-helix. The electrostatics and hydrophobic interaction of SDS and insulin may contribute to amyloid induction. Moreover, the SDS-induced aggregation was not affected by the presence of salts. Furthermore, as the concentration of SDS increased, the preformed insulin amyloid induced by SDS began to disintegrate. Overall, our study sheds light on the mechanism of surfactant-induced amyloid fibrillation in insulin protein.

Denaturation and Digestion Increase the Antioxidant Capacity of Proteins

It has been estimated and demonstrated that the antioxidant capacity of proteins is increased as a result of digestion in the gastrointestinal tract, which can be contributed by denaturation and digestion. This study aimed to evaluate the effect of denaturation and proteolytic digestion on the antioxidant activity of bovine serum albumin (BSA) and chicken egg white proteins in model systems. Denaturation with an anionic detergent (sodium dodecyl sulfate) and digestion with papain and trypsin increased the antioxidant activity/capacity of the proteins, apparently due to the increased exposure of amino acid residues responsible for the antioxidant activity of proteins (tyrosine, tryptophan, cysteine, histidine, arginine, and cystine in the ABTS● decolorization assay; cysteine, tryptophan, tyrosine, and cystine in the FRAP assay). As the increase in the protein antioxidant activity/capacity was limited in extent, it does not invalidate the use of the antioxidant capacity of proteins to be consumed as a rough measure of their antioxidant capacity after modifications in the gastrointestinal tract.

Comparison of Sodium Lauryl Ether Sulfate and Sodium Dodecyl Sulfate in the Intestinal Decellularization of Rats Using a Histological Method and Confocal Raman Microscopy

Background: Sodium dodecyl sulfate (SDS) detergent is widely used in tissue decellularization to produce scaffolds for tissue engineering. Despite its strong decellularization, this substance has relatively high toxicity and causes changes in tissue composition. Sodium lauryl ether sulfate (SLES) is a new poly anionic detergent that is less toxic than SDS but weaker than it. The present study aimed to decellularize the intestinal tissue using SDS and SLES solutions, forming a cell scaffold, and examining scaffolds obtained from this tissue. Methods: Eighteen male Sprague-Dawley rats were divided into three groups. The intestines of all rats were removed after anesthesia. In the first group (controls), rats’ intestines were placed in a 10% formalin solution. In the second group, intestines were decellularized using an SLES solution. In the third group animals’ intestines were decellularized using an SDS solution. To evaluate decellularization, samples were stained with hematoxylin-eosin staining and Alcian blue staining for glycosaminoglycans (GAGs), and Masson’s trichrome for collagen fibers. A confocal Raman microscope was used to compare collagen, lipid, GAG, and genetic content. Results: Hematoxylin-eosin staining showed that the nucleus and DNA were removed in the decellularized scaffolds by SDS or SLES. The SLES group, compared to the SDS group, showed fewer changes in the epithelial tissue, and muscle layers in both scaffolds were well preserved. The results of confocal Raman microscopy showed that tryptophan, lipid, glycogen, and protein were broken down by both detergents; however, the residual amount of glycogen was the same in both substances, but disulfide bonds of proteins, hydroxyproline, and lipids in the decellularized intestine with SLES were mostly preserved. Conclusion: Both substances were suitable for intestinal decellularization and removed the overall structure of intestinal tissue, but SLES retained collagen and GAG content better than SDS.

Oral detergent exposure alters the esophageal microbiome and induces eosinophilic inflammation

Detergent exposure is a possible environmental factor recently implicated in the pathogenesis of eosinophilic esophagitis (EoE). We have shown the detergent sodium dodecyl sulfate (SDS) induces epithelial barrier dysfunction and eosinophilic inflammation in mice. We aimed to examine changes in the mouse esophageal microbiome following SDS exposure.

Effects of Chemical Additives in Refolding Buffer on Recombinant Human BMP-2 Dimerization and the Bioactivity on SaOS-2 Osteoblasts.

Bone morphogenetic protein-2 (BMP-2) is a promising osteogenic agent in tissue engineering. BMP-2 is usually expressed in Escherichia coli owing to the high yield and low cost, but the protein is expressed as inclusion bodies. Thus, the bottleneck for BMP-2 production in E. coli is the refolding process. Here, we explored the effects of the refolding buffer composition on BMP-2 refolding. The BMP-2 inclusion body was solubilized in urea and subjected to refolding by the dilution method. Various additives were investigated to improve the BMP-2 refolding yield. Nonreducing SDS-PAGE showed that BMP-2 dimers, the presumably biologically active form, were detected at approximately 25 kDa. The highest yield of the BMP-2 dimers was observed in the refolding buffer that contained ionic detergents (sarkosyl and cetylpyridinium chloride) followed by zwitterionic and nonionic detergents (NDSB-195, NP-40, and Tween 80). In addition, sugars (glucose, sorbitol, and sucrose) in combination with anionic detergents (sodium dodecyl sulfate and sarkosyl) reduced BMP-2 oligomers and increased the BMP-2 dimer yield. Subsequently, the refolded BMP-2s were tested for their bioactivity using the alkaline phosphatase assay in osteogenic cells (SaOS-2), as well as the luciferase reporter assay and the calcium assays. The refolded BMP-2 showed the activities in the calcium deposition assay and the luciferase reporter assay but not in the alkaline phosphatase activity assay or the intracellular calcium assay even though the dimers were clearly detected. Therefore, the detection of the disulfide-linked dimeric BMP-2 in nonreducing SDS-PAGE is an inadequate proxy for the bioactivity of BMP-2.

Study of Generation of Singlet Oxygen in Human Saliva <i>in vitro</i> Under the Action of Nanosecond Pulsed Laser Radiation

Background. The development of new laser devices for use in the treatment of complex dental pathology and microsurgery of the oral cavity with unique parameters with the ability to generate nanosecond pulsed radiation in a quasi-monochromatic radiation band is necessary. The high peak output power per pulse allows laser light to penetrate deeper into biological media without significant heating. The possibility of excitation of singlet oxygen without the use of endogenous photosensitizers is an indisputable advantage of such laser generators.
 Aims study of singlet oxygen generation in human oral fluid in vitro depending on the parameters of nanosecond pulsed laser radiation with a wavelength of ~1265 nm.
 Methods. We used a laser device with a main infrared (IR) emitter with a wavelength approximately corresponding to the oxygen absorption maximum (~1265 nm) with the generation of nanosecond pulsed radiation. A study was carried out to study the generation of singlet oxygen in the human oral fluid and the washing out of the oral fluid by the fading of the trap in solutions under the action of laser radiation before irradiation, after 30 and 60 min. The laser radiation parameters were set as follows: power 1 W, nanosecond pulsed radiation with a pulse duration of 400 ns and a frequency of 0.5, 1.0, 1.5 and 2.0 MHz in comparison with the continuous radiation mode.
 Results. It has been established that nanosecond laser radiation leads to the oxidation of a chemical singlet oxygen trap solubilized with sodium dodecyl sulfate (0.05 M SDS) detergent in the oral fluid and saliva flushing from the oral cavity. In pulsed nanosecond modes, laser radiation is more efficient than in continuous mode. With an increase in the repetition frequency, an increase in the effect by an average of 10% compared to a lower frequency was observed and the effect with a maximum frequency of 2.0 MHz was almost 50% higher than when using continuous radiation in all studies. A decrease in optical density at 414 nm was reliably observed in samples with oral fluid washing, the effect was 0.07 0.01 for 60 min of exposure. In the oral fluid, the effect with the same parameters was slightly lower and amounted to 0.05 0.03.
 Conclusions. The rate of fading of the trap in the saliva wash turned out to be 1.3 times faster than in water with detergent, which may indicate the activation of photoacceptors and their oversaturation with oxygen when using aqueous dilution of the oral fluid. The maximum effect was observed when using laser radiation with a pulse repetition rate of 2.0 MHz.

Rapid and reliable inactivation protocols for the diagnostics of emerging viruses: The example of SARS-CoV-2 and monkeypox virus.

The emergence and sustained transmission of novel pathogens are exerting an increasing demand on the diagnostics sector worldwide, as seen with the ongoing severe acute respiratory coronavirus 2 (SARS‐CoV‐2) pandemic and the more recent public health concern of monkeypox virus (MPXV) since May 2022. Appropriate and reliable viral inactivation measures are needed to ensure the safety of personnel handling these infectious samples. In the present study, seven commercialized diagnosis buffers, heat (56°C and 60°C), and sodium dodecyl sulfate detergent (2.0%, 1.0%, and 0.5% final concentrations) were tested against infectious SARS‐CoV‐2 and MPXV culture isolates on Vero cell culture. Cytopathic effects were observed up to 7 days postinoculation and viral load evolution was measured by semiquantitative polymerase chain reaction. The World Health Organization recommends an infectious titer reduction of at least 4 log10. As such, the data show efficacious SARS‐CoV‐2 inactivation by all investigated methods, with >6.0 log10 reduction. MPXV inactivation was also validated with all investigated methods with 6.9 log10 reductions, although some commercial buffers required a longer incubation period to yield complete inactivation. These results are valuable for facilities, notably those without biosafety level‐3 capabilities, that need to implement rapid and reliable protocols common against both SARS‐CoV‐2 and MPXV.

Efficacy of Nerve-Derived Hydrogels to Promote Axon Regeneration Is Influenced by the Method of Tissue Decellularization.

Injuries to large peripheral nerves are often associated with tissue defects and require reconstruction using autologous nerve grafts, which have limited availability and result in donor site morbidity. Peripheral nerve-derived hydrogels could potentially supplement or even replace these grafts. In this study, three decellularization protocols based on the ionic detergents sodium dodecyl sulfate (P1) and sodium deoxycholate (P2), or the organic solvent tri-n-butyl phosphate (P3), were used to prepare hydrogels. All protocols resulted in significantly decreased amounts of genomic DNA, but the P2 hydrogel showed the best preservation of extracellular matrix proteins, cytokines, and chemokines, and reduced levels of sulfated glycosaminoglycans. In vitro P1 and P2 hydrogels supported Schwann cell viability, secretion of VEGF, and neurite outgrowth. Surgical repair of a 10 mm-long rat sciatic nerve gap was performed by implantation of tubular polycaprolactone conduits filled with hydrogels followed by analyses using diffusion tensor imaging and immunostaining for neuronal and glial markers. The results demonstrated that the P2 hydrogel considerably increased the number of axons and the distance of regeneration into the distal nerve stump. In summary, the method used to decellularize nerve tissue affects the efficacy of the resulting hydrogels to support regeneration after nerve injury.

Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor.

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient's quality of life. Although surgical reconstruction can be performed using autologous free flap transfer or vascularized composite allotransplantation (VCA), such methods also have disadvantages. Issues such as donor site morbidity and tissue availability limit autologous free flap transfer, while immunosuppression is a significant limitation of VCA. Engineered tissues in reconstructive surgery using decellularization/recellularization methods represent a possible solution. Decellularized tissues are generated using methods that remove native cellular material while preserving the underlying extracellular matrix (ECM) microarchitecture. These acellular scaffolds can then be subsequently recellularized with recipient-specific cells. This protocol details the procurement and decellularization methods used to achieve acellular scaffolds in a pig model. In addition, it also provides a description of the perfusion bioreactor design and setup. The flaps include the porcine omentum, tensor fascia lata, and the radial forearm. Decellularization is performed via ex vivo perfusion of low concentration sodium dodecyl sulfate (SDS) detergent followed by DNase enzyme treatment and peracetic acid sterilization in a customized perfusion bioreactor. Successful tissue decellularization is characterized by a white-opaque appearance of flaps macroscopically. Acellular flaps show the absence of nuclei on histological staining and a significant reduction in DNA content. This protocol can be used efficiently to generate decellularized soft tissue scaffolds with preserved ECM and vascular microarchitecture. Such scaffolds can be used in subsequent recellularization studies and have the potential for clinical translation in reconstructive surgery.

Human Claudin-7 cis-Interactions Are Not Crucial for Membrane-Membrane (Trans-) Interactions.

Human Claudin-7 (Cldn7) is a member of the Claudin (Cldn) superfamily. In vivo, these proteins form tight junctions, which establish constricted connections between cells. Cldns oligomerize within the membrane plane (= cis-interaction), and also interact with Cldns from adjacent cells (= trans-interaction). Interactions of Cldns are typically studied in vivo and structural analyses of isolated Cldns are limited. Here, we describe heterologous expression in E. coli and purification of human Cldn7, enabling in vitro analyses of the isolated protein using detergent and model membrane systems. Cldn7 exists as a monomer, hexamer, and various higher oligomers in micelles. While only limited unfolding of the protein was observed in the presence of the anionic detergent sodium dodecyl sulfate, decreased ionic strength did affect Cldn7 cis-interactions. Furthermore, we identified two amino acids which mediate electrostatic cis-interactions and analyzed the impact of disturbed cis-interaction on trans-contacts via atomic force microscopy and monitoring Förster resonance energy transfer between fluorescently labeled Cldn7-containing proteoliposomes. Our results indicate that Cldn7 cis-oligomerization might not be a prerequisite for establishing trans-contacts.

Membrane Ultrafiltration-Based Sample Preparation Method and Sheath-Flow CZE-MS/MS for Top-Down Proteomics.

Mass spectrometry (MS)-based denaturing top-down proteomics (dTDP) identify proteoforms without pretreatment of enzyme proteolysis. A universal sample preparation method that can efficiently extract protein, reduce sample loss, maintain protein solubility, and be compatible with following up liquid-phase separation, MS, and tandem MS (MS/MS) is vital for large-scale proteoform characterization. Membrane ultrafiltration (MU) was employed here for buffer exchange to efficiently remove the sodium dodecyl sulfate (SDS) detergent in protein samples used for protein extraction and solubilization, followed by capillary zone electrophoresis (CZE)-MS/MS analysis. The MU method showed good protein recovery, minimum protein bias, and nice compatibility with CZE-MS/MS. Single-shot CZE-MS/MS analysis of an Escherichia coli sample prepared by the MU method identified over 800 proteoforms.

Decellularization of porcine kidney with submicellar concentrations of SDS results in the retention of ECM proteins required for the adhesion and maintenance of human adult renal epithelial cells.

When decellularizing kidneys, it is important to maintain the integrity of the acellular extracellular matrix (ECM), including associated adhesion proteins and growth factors that allow recellularized cells to adhere and migrate according to ECM specificity. Kidney decellularization requires the ionic detergent sodium dodecyl sulfate (SDS); however, this results in a loss of ECM proteins important for cell adherence, migration, and growth, particularly glycosaminoglycan (GAG)-associated proteins. Here, we demonstrate that using submicellar concentrations of SDS results in a greater retention of structural proteins, GAGs, growth factors, and cytokines. When porcine kidney ECM scaffolds were recellularized using human adult primary renal epithelial cells (RECs), the ECM promoted cell survival and the uniform distribution of cells throughout the ECM. Cells maintained the expression of mature renal epithelial markers but did not organize on the ECM, indicating that mature cells are unable to migrate to specific locations on ECM scaffolds.

Total RNA Extraction from Saccharomyces cerevisiae Using Hot Acid Phenol.

Isolation of RNA from yeast is complicated by the need to first break the thick, rigid cell wall. The protocol provided here uses a cycle of heating and freezing of cells in the presence of phenol and the detergent sodium dodecyl sulfate (SDS). The extraction is performed in the presence of low salt so that, following separation of the aqueous and phenol phases by centrifugation, DNA can be collected from the interface while RNA remains in the aqueous phase. This protocol should yield ∼50-250 µg of RNA from 10 mL of culture. The RNA isolated using this approach is suitable for most follow-up applications such as northern blot hybridization, reverse transcriptase-polymerase chain reaction (RT-PCR), and cDNA construction.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis of Proteins.

Most analytical electrophoreses of proteins are achieved by separation in polyacrylamide gels under conditions that ensure dissociation of proteins into individual polypeptide subunits and minimize aggregation. Most commonly, the anionic detergent sodium dodecyl sulfate (SDS) is used in combination with a reducing agent (β-mercaptoethanol or dithiothreitol) and with heating to dissociate proteins before loading onto the gel. SDS binding denatures the polypeptides and imparts a negative charge that masks their intrinsic charge. The amount of SDS bound is generally sequence-independent and proportional to molecular weight; at saturation, approximately one SDS molecule is bound per two amino acids, or ∼1.4 g of SDS per gram of polypeptide. Therefore, the migration of SDS-polypeptide complexes in an electric field is proportional to the relative size of the polypeptide chain, and its molecular weight can be estimated by comparison to protein markers of known molecular weight. However, hydrophobicity, highly charged sequences, and certain posttranslational modifications such as glycosylation or phosphorylation may also influence migration. Thus, the apparent molecular weight of modified proteins does not always accurately reflect the mass of the polypeptide chain. This protocol describes preparation and running of SDS-PAGE gels, followed by staining to detect proteins using Coomassie Brilliant Blue. Finally, the stained SDS-PAGE gel may be scanned to an image or preserved by drying.

Development of a Cell-Based Biosensor for Residual Detergent Detection in Decellularized Scaffolds.

Ex vivo engineering of organs that uses decellularized whole organs as a scaffold with autologous stem cells is a potential alternative to traditional transplantation. However, one of the main challenges in this approach is preparing cytocompatible scaffolds. So far, high-precision and specific evaluation methods have not been developed for this purpose. Cell-based biosensors (CBBs) are promising tools to measure analytes with high sensitivity and specificity in a cost-effective and noninvasive manner. In this paper, using the NF-κB inducible promoter we developed a CBB for residual detergent detection. Proximal and core sections of the inducible promoter, containing NF-κB binding sequence, are designed and cloned upstream of the reporter gene (secreted alkaline phosphatase (SEAP)). After transfection into HEK293 cells, stable and reliable clones were selected. After confirmation of induction of this gene construct by sodium dodecyl sulfate (SDS), the stability and function of cells treated by qPCR and SEAP activity were measured. This biosensor was also used to evaluate the cytocompatibility of decellularized tissue. Results showed that the developed biosensor could detect very small amounts of SDS detergent (3.467 pM). It has the best performance 8 h after exposure to detergent, and its stability in high passage numbers was not significantly reduced. Applying this biosensor on decellularized tissues showed that SEAP activity higher than 4.36 (U/L) would lead to a viability reduction of transplanted cells below 70%. This paper presents a novel method to evaluate the cytocompatibility of decellularized tissues. The developed CBB can detect residual detergents (such as SDS) in tissues with high sensitivity and efficiency.

Effects of Microenvironmental Changes on the Fluorescence Signal of Alternariol: Magnesium Induces Strong Enhancement in the Fluorescence of the Mycotoxin.

Alternariol (AOH) is an emerging mycotoxin produced by Alternaria molds. It occurs as a contaminant e.g., in oilseeds, cereals, grapes, and tomatoes. Chronic exposure to AOH may cause genotoxic and endocrine disruptor effects. Our recent studies demonstrated that the fluorescence signal of AOH can be strongly affected by the environmental pH as well as by the presence of serum albumin or cyclodextrins. In the current study, we aimed to characterize the most optimal circumstances regarding the highly sensitive fluorescent detection of AOH. Therefore, the further detailed investigation of the microenvironment on the fluorescence signal of the mycotoxin has been performed, including the effects of different buffers, organic solvents, detergents, and cations. Organic solvents (acetonitrile and methanol) caused only slight increase in the emission signal of AOH, while detergents (sodium dodecyl sulfate and Triton-X100) and Ca2+ induced considerably higher enhancement in the fluorescence of the mycotoxin. In addition, Mg2+ proved to be a superior fluorescence enhancer of the AOH. Spectroscopic and modeling studies suggest the formation of low-affinity AOH-Mg2+ complexes. The effect of Mg2+ was also tested in two HPLC assays: Our results show that Mg2+ can considerably increase the fluorescence signal of AOH even in a chromatographic system.