Novel fluorescent sensor using molecularly imprinted silica microsphere‐coated CdSe@CdS quantum dots and its application in the detection of 2,4,6‐trichlorophenol from environmental water samples

In this study, a high fluorescence sensitivity and selectivity, molecularly imprinted nanofluorescent polymer sensor (MIP@SiO₂@QDs) was prepared using a reverse microemulsion method. 2,4,6‐Trichlorophenol (2,4,6‐TCP) was detected using fluorescence quenching. Tetraethyl orthosilicate (TEOS), quantum dots (QDs) and 3‐aminopropyltriethoxysilane (APTS) were used as cross‐linker, signal sources and functional monomer respectively. The sensor (MIP@SiO₂@QDs) and the non‐imprinted polymer sensor (NIP@SiO₂@QDs) were characterized using infra‐red (IR) analysis, X‐ray diffraction (XRD), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The selectivity of MIP@SiO₂@QDs was examined by comparing 2,4,6‐TCP with other similar functional substances including 2,4‐dichlorophenol (2,4‐DCP), 2,6‐dichlorophenol (2,6‐DCP) and 4‐chlorophenol (4‐CP). Results showed that MIP@SiO₂@QDs had better selectivity for 2,4,6‐TCP than the other compounds. Fluorescence quenching efficiency displayed a good linear response at the 2,4,6‐TCP concentration range 5–1000 μmol/L. The limit of detection (LOD) was 0.9 μmol/L (3σ, n = 9). This method was equally applicable for testing actual samples with a recovery rate of 98.0–105.8%. The sensor had advantages of simple pretreatment, good sensitivity and selectivity, and wide linear range and could be applied for the rapid detection of 2,4,6‐TCP in actual samples.     

The dipeptidyl carboxypeptidase of <Emphasis Type="Italic">Escherichia coli</Emphasis> novablue: overproduction and molecular characterization of the recombinant enzyme

To express Escherichia coli novablue dipeptidyl carboxypeptidase (EcDCP), the gene was amplified by PCR and cloned into the expression plasmid pQE-31 to yield pQE-EcDCP. His₆-tagged EcDCP (His₆-EcDCP) was over-expressed in E. coli M15 (pQE-EcDCP) as a soluble and active form under 0.05 mM IPTG induction at 26°C for 12 h. The recombinant enzyme was purified to homogeneity by Ni²⁺-NTA resin and had a molecular mass of approximately 75 kDa. The temperature and pH optima for His₆-EcDCP were 37°C and 7.0, respectively. In the presence of 200 mM NaCl, His₆-EcDCP was stimulated by 1.5 fold. The K _M and k _cat values of the enzyme for N-benzoyl-l-glycyl-l-histidyl-l-leucine were 1.83 mM and 168.3 s⁻¹, respectively. His₆-EcDCP activity was dramatically inhibited by 10 mM EDTA, 0.25 mM 1.10-phenanthroline, and 2.5 mM DEPC, but it was not affected by Ser, Asp, Lys, and Trp protease inhibitors. Analysis of His₆-EcDCP by circular dichroism revealed that the secondary structures of the enzyme in 30 mM universal buffer (pH 7.0) were 17% α-helix, 35% β-sheet and 47% random coil. Mid point of thermal transition was calculated to be 55°C for the recombinant enzyme.     

New vanadium-based magnetic resonance imaging probes: clinical potential for early detection of cancer

We have developed a magnetic resonance imaging (MRI) method for improved detection of cancer with a new class of cancer-specific contrast agents, containing vanadyl (VO²⁺)-chelated organic ligands, specifically bis(acetylacetonato)oxovanadium(IV) [VO(acac)₂]. Vanadyl compounds have been found to accumulate within cells, where they interact with intracellular glycolytic enzymes. Aggressive cancers are metabolically active and highly glycolytic; an MRI contrast agent that enters cells with high glycolytic activity could provide high-resolution functional images of tumor boundaries and internal structure, which cannot be achieved by conventional contrast agents. The present work demonstrates properties of VO(acac)₂ that may give it excellent specificity for cancer detection. A high dose of VO(acac)₂ did not cause any acute or short-term adverse reactions in murine subjects. Calorimetry and spectrofluorometric methods demonstrate that VO(acac)₂ is a blood pool agent that binds to serum albumin with a dissociation constant K _d ~ 2.5 ± 0.7 × 10⁻⁷ M and a binding stoichiometry n = 1.03 ± 0.04. Owing to its prolonged blood half-life and selective leakage from hyperpermeable tumor vasculature, a low dose of VO(acac)₂ (0.15 mmol/kg) selectively enhanced in vivo magnetic resonance images of tumors, providing high-resolution images of their interior structure. The kinetics of uptake and washout are consistent with the hypothesis that VO(acac)₂ preferentially accumulates in cancer cells. Although VO(acac)₂ has a lower relaxivity than gadolinium-based MRI contrast agents, its specificity for highly glycolytic cells may lead to an innovative approach to cancer detection since it has the potential to produce MRI contrast agents that are nontoxic and highly sensitive to cancer metabolism.     

Isolation of an acidic phospholipase A2 from the venom of the snake Bothrops asper of Costa Rica: Biochemical and toxicological characterization

Phospholipases A₂ (PLA₂) are major components of snake venoms, exerting a variety of relevant toxic actions such as neurotoxicity and myotoxicity, among others. Since the majority of toxic PLA₂s are basic proteins, acidic isoforms and their possible roles in venoms are less understood. In this study, an acidic enzyme (BaspPLA₂-II) was isolated from the venom of Bothrops asper (Pacific region of Costa Rica) and characterized. BaspPLA₂-II is monomeric, with a mass of 14,212 ± 6 Da and a pI of 4.9. Its complete sequence of 124 amino acids was deduced through cDNA and protein sequencing, showing that it belongs to the Asp49 group of catalytically active enzymes. In vivo and in vitro assays demonstrated that BaspPLA₂-II, in contrast to the basic Asp49 counterparts present in the same venom, lacks myotoxic, cytotoxic, and anticoagulant activities. BaspPLA₂-II also differed from other acidic PLA₂s described in Bothrops spp. venoms, as it did not show hypotensive and anti-platelet aggregation activities. Furthermore, this enzyme was not lethal to mice at intravenous doses up to 100 μg (5.9 μg/g), indicating its lack of neurotoxic activity. The only toxic effect recorded in vivo was a moderate induction of local edema. Therefore, the toxicological characteristics of BaspPLA₂-II suggest that it does not play a key role in the pathophysiology of envenomings by B. asper, and that its purpose might be restricted to digestive functions. Immunochemical analyses using antibodies raised against BaspPLA₂-II revealed that acidic and basic PLA₂s form two different antigenic groups in B. asper venom.     

In vitro study of the trypanocidal activity of anilinophenanthrolines against Trypanosoma cruzi

Chagas disease is present in Latin America, North America, Europe, and Asia, where between 6 and 7 million people are infected. This illness is transmitted mainly by the insect vector during blood feeding and by oral transmission. Chagas disease is treated with benznidazole and its effectiveness depends on which phase of the disease the treatment starts. Therefore, the identification of new compounds with anti-Chagas activities is important. Protozoan parasites present cysteine proteases, important for host cell infection and differentiation, which have been explored as valid targets against pathogenic parasites. In the present study, the effects of 10 new 1,10-phenanthroline derivatives were evaluated on T. cruzi. Three of them were effective against amastigotes (IC₅₀ from 0.5 to 3 μM), epimastigotes (IC₅₀ from 0.5 to at least 10 μM) and trypomastigotes (and LD₅₀ from 1 to 10 μM), and they were not toxic to mammalian cells (CC₅₀ ≥ 20 μM). These compounds also promoted the formation of autophagosomes, alter the level of heterochromatin condensation, caused the loss of kDNA topology, and the elongated cell body shape. Apart from ultrastructural alterations, an increased generation of ROS and decreased mitochondrial membrane potential were observed. Therefore, these drugs revealed potential trypanocidal effects and warrant further antiparasitic studies against Chagas disease.     

2,4-Dichlorophenol (DCP) containing wastewater treatment using a hybrid-loop bioreactor

Synthetic wastewater containing 2,4-dichlorophenol (DCP) was biologically treated using a hybrid-loop bioreactor system consisting of a packed column biofilm reactor (PCBR) and an aerated tank with effluent recycle. Effects of the feed DCP concentration on COD, DCP and toxicity removals were investigated. Biomass concentration in the packed column and in the aeration tank decreased with increasing feed DCP content due to toxic effects of DCP on the microorganisms. Low biomass concentrations at high DCP contents resulted in low COD, DCP and toxicity removals. Therefore, percent DCP, COD and toxicity removals decreased with increasing feed DCP content. Nearly 70% COD removal was achieved with a feed DCP content of 380 mg L(-1). The system should be operated with the feed DCP lower than 100 mg L(-1) in order to obtain DCP, COD and toxicity removals above 90%.     

Electro-optical behaviour of CuFe2O4@rGO nanocomposite for nonenzymatic detection of uric acid via the electrochemical method

Uric acid (UA) is a blood and urine component obtained as a metabolic by-product of purine nucleotides. Abnormalities in UA metabolism cause crystal deposition as monosodium urate and lead to various diseases such as gout, hyperuricemia, Lesch–Nyhan syndrome, etc. Monitoring these diseases requires a rapid, sensitive, selective, and portable detection approach. Therefore, this study demonstrates the hydrothermal synthesis of CuFe₂O₄/reduced graphene oxide (rGO) nanocomposite for selective detection of UA. After the nanocomposite synthesis, characterization was performed by X-ray diffraction spectroscopy, Fourier transform infrared spectroscopy, Raman spectroscopy, X-ray photoelectron spectroscopy, UV–visible spectrometry, atomic force spectroscopy, scanning electron microscopy, and electrochemical analysis. Furthermore, from the electrochemical analysis using cyclic voltammetry (CV), kinetic studies were carried out by varying the scan rate to obtain the diffusion coefficient, surface concentration, and rate of charge transfer to achieve a calibration curve that indicates the quasi reversible nature of the fabricated electrode with a linear regression coefficient of oxidation (R²: 0.9992) and reduction (R²: 0.9971) peaks. Moreover, the fabricated nonenzymatic amperometric sensor to detect UA with a linearity (R²: 0.9989) of 1–400 μM was highly sensitive (2.75 × 10⁻⁴ mAμM⁻¹ cm⁻²) and had a lower limit of detection (0.01231 μM) at pH 7.5 in phosphate-buffered saline solution. Therefore, the CuFe₂O₄/rGO/ITO-based nonenzymatic sensor could detect interfering agents and spiked real bovine serum samples with higher sensitivity and selectivity for UA detection.     

Glucose biosensor based on nano-SiO2 and “unprotected” Pt nanoclusters

The “unprotected” Pt nanoclusters (average size 2 nm) mixed with the nanoscale SiO₂ particles (average size 13 nm) were used as a glucose oxidase immobilization carrier to fabricate the amperometric glucose biosensor. The bioactivity of glucose oxidase (GOx) immobilized on the composite was maintained and the as-prepared biosensor demonstrated high sensitivity (3.85 μA mM⁻¹) and good stability in glucose solution. The Pt–SiO₂ biosensor showed a detection limit of 1.5 μM with a linear range from 0.27 to 4.08 mM. In addition, the biosensor can be operated under wide pH range (pH 4.9–7.5) without great changes in its sensitivity. Cyclic voltammetry measurements showed a mixed controlled electrode reaction.     

Polymeric micelles of PEG-PE as carriers of all-trans retinoic acid for stability improvement

The topical application of all-trans retinoic acid (ATRA) is an effective treatment for several skin disorders, including photo-aging. Unfortunately, ATRA is susceptible to light, heat, and oxidizing agents. Thus, this study aimed to investigate the ability of polymeric micelles prepared from polyethylene glycol conjugated phosphatidylethanolamine (PEG-PE) to stabilize ATRA under various storage conditions. ATRA entrapped in polymeric micelles with various PEG and PE structures was prepared. The critical micelle concentrations were 97–243 μM, depending on the structures of the PEG and PE molecules. All of the micelles had particle diameters of 6–20 nm and neutral charges. The highest entrapment efficiency (82.7%) of the tested micelles was exhibited by ATRA in PEG with a molecular weight of 750 Da conjugated to dipalmitoyl phosphatidylethanolamine (PEG₇₅₀-DPPE) micelles. The PEG₇₅₀-DPPE micelle could significantly retard ATRA oxidation compared to ATRA in 75% methanol/HBS solution. Up to 87% of ATRA remained in the PEG₇₅₀-DPPE micelle solution after storage in ambient air for 28 days. This result suggests that PEG₇₅₀-DPPE micelle can improve ATRA stability. Therefore, ATRA in PEG₇₅₀-DPPE micelle is an interesting alternative structure for the development of cosmeceutical formulations.     

Feed digestion,rumen fermentation and blood biochemical constituents in Malpura rams fed a complete feed-block diet with the inclusion of tree leaves

This experiment assayed the influence of the inclusion of dried Azardirachta indica, Albizzia lebbek or Ailanthus excelsa leaves in pearl millet stover-based complete feed block diets on feed intake, nutrient utilization, rumen fermentation characteristics, ciliate protozoa population and blood biochemical constituents in adult Malpura sheep. Complete feed blocks were formulated to have roughage-to-concentrate ratio of 70:30. Pearl millet stover (PMS) was used as basal roughage; 30 parts of pearl millet stover was replaced with dried leaves either of Azardirachta indica (NL), Albizzia lebbek (SL) or Ailanthus excelsa (AL). Twelve hogget Malpura rams, divided into four equal groups, were offered one of the four dietary treatments. A feeding-cum-metabolic trial was conducted to assess nutrient utilization. Rumen liquor samples were collected at 0, 3, 6, 12, 18 and 24 h post-feeding to assess rumen fermentation pattern and ciliate protozoa population. Inclusion of dried leaves in PMS-based diets improved CP and DCP content. Dietary DCP was low (P < 0.01) in PMS (8.52%) compared to tree leaves (9.77–11.59%) diets. AL and NL diets had higher (P < 0.05) DCP than the SL diet. The inclusion of tree leaves did not influence organic matter, crude protein or cellulose digestibility, but depressed dry matter, NDF, ADF and energy digestibility. DE content was also lower in tree leave diets. Inclusion of tree leaves improved CP and DCP intake, but DE intake and nitrogen utilization did not change. The pH of rumen liquor (SRL) was low (6.99, P < 0.05), but total nitrogen (52.9 mg/dl SRL) and NH₃-nitrogen (9.34 mg/dl SRL) concentrations were higher (P < 0.01) in the AL diet. TVFA concentrations and ciliate protozoa population were similar on the four diets. Animals in the four groups had the desired concentration of rumen metabolites required for fibrous diets. Complete feed-block feeding provided a constant nutrient supply to rumen microbes that optimise rumen fermentation. Blood biochemical constituents did not change due to the inclusion of tree leaves. Therefore, tree leaves can be included with roughage-based feeding to improve the protein nutrition status of ruminants. Further studies are required to assess the negative influence of tree leaves on digestibility.     

The allelopathy and allelopathic mechanism of phenolic acids on toxic <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis>

Cyanobacterial contamination of water has been a serious problem in recent years. Thus, the effective control of undesired cyanobacteria has become an urgent issue. We studied therefore the effects of ρ-coumaric acid and vanillic acid on toxic Microcystis aeruginosa and the allelopathic mechanisms. The results showed that the growth of toxic M. aeruginosa was significantly inhibited by ρ-coumaric acid and vanillic acid, with an EC₅₀ of 0.26 ± 0.07 and 0.34 ± 0.05 mmol L⁻¹, respectively. Our data also demonstrated that both ρ-coumaric acid and vanillic acid triggered the generation of superoxide anion radicals (O₂ ^•−). The O₂ ^•− might induce a lipid peroxidation which may change cell membrane penetrability, thereby leading to the eventual death of M. aeruginosa. Our current studies further provide evidence that some phenolic acids such as ρ-coumaric acid and vanillic acid may be a potential effective solution for aquatic management.     

Establishment of an EC50 database of pesticides using a Vibrio fischeri bioluminescence method

An EC₅₀ database was established to assess the acute toxicity of 16 PESTANAL pesticide standards and of seven pesticide commercial formulations using a Vibrio fischeri bioluminescence method. Half maximal effective concentration ( EC₅₀) is defined as the concentration of pollutant (in this case, pesticide) destroying 50% of the bacteria population and causing 50% bioluminescence inhibition, after a specified exposure time. Linear curves of bioluminescence inhibition versus pesticide concentration and EC₅₀ values were obtained for exposure times (t) of 5 or 15 min for these pesticides. The EC₅₀ values ranged from 6.90 × 10^?4 to 0.83 mg/ml (t = 5 min), and from 9.00 × 10^?4 to 0.37 mg/ml (t = 15 min) for pesticide standards, plus from 0.0077 to 0.74 mg/ml (t = 5 min), and from 0.0076 and 0.57 mg/ml (t = 15 min) for pesticide commercial formulations. The EC₅₀ database allowed classification of the pesticides under study into three categories according to their toxicity: very toxic, toxic and moderately toxic. These results demonstrated that the establishment of an EC₅₀ database and of linear curves of bioluminescence inhibition versus the pesticide concentration resulted in very important and irreplaceable tools to estimate the global and individual toxicity of pesticides present in environmental samples.     

The paradoxical effects of progesterone on the eggshell quality of laying hens

This study demonstrates the effects of progesterone on eggshell quality and ultrastructure by injecting progesterone into laying hens 2 and 5 h post-oviposition, respectively. Progesterone injected 2 h post-oviposition (P₄-2 h) improved eggshell quality with a significant decrease (P < 0.01) in the thickness of the mammillary layer and a significant increase (P < 0.01) in the thickness of the effective layer in the eggshell ultrastructure compared to the control. Progesterone injected 5 h post-oviposition (P₄-5 h) damaged the eggshell quality by significantly reducing (P < 0.01) the effective layer thickness. Progesterone injected delayed obviously (P < 0.01) the following oviposition. Moreover, the concentrations of Thr, Cys, Leu, Lys, and His in the eggshell membranes were significantly higher (P < 0.05) in the P₄-2 h treated hens whereas Val and Lys were significantly lower (P < 0.05) in P₄-5 h treated hens compared to the control. Therefore, progesterone shows paradoxical effects on eggshell quality depending on the injection time-points post-oviposition, which could explain the contradictions in previous related reports. P₄ injected affected the content of amino acids in eggshell membranes, especially lysine which contributed to eggshell quality. In addition, P₄ injected 2 h after oviposition improved eggshell quality by promoting the premature fusion of mammillary knobs. This work contributed to a novel insight to understanding the mechanism of improving eggshell quality.     

Cryopreservation of Bufotes viridis embryos by vitrification

Loss of biodiversity among amphibians is a current concern. Our hypothesis is that the embryos of amphibian species at risk of extinction could be cryopreserved by vitrification, using methods which have proved successful with fish oocyte. To test this hypothesis, samples of four cryoprotectants - methanol (MeOH), dimethyl sulphoxide (Me2SO), propylene glycol (PG) and polyethylene glycol (PEG), some singly, some in combination, were plunged in liquid nitrogen for 5 min to find the best solution for vitrification. To find the least toxic of these solutions, blastulae and stage G17 embryos of Bufotes Viridis, a typical amphibian, were exposed to solutions at different concentrations (0.5–10 M) for different lengths of time (15–30 min), with and without their normal protective jelly coats. In each case the number of survivors, which reached stage G25 was counted. Finally a series of embryos was vitrified in liquid nitrogen using the most efficient and least toxic cryoprotectants.Propylene glycol had the best vitrification characteristics, but MeOH vitrified at higher concentrations. The optimum regime, with the least toxic ctyoprotectants, consisted of 1M Me2SO for 15 min and a combination of 15% PEG_(w/v) + 3M PG + 2M Me2SO for 3 min, with the jelly coat intact, followed by vitrification. This gave a survival percentage of 87.6% immediately after vitrification. Methods designed for cryopreservation of fish embryos make a good starting point for cryopreservation of the embryos of amphibian.     

BSA–AuNPs@Tb–AMP metal–organic frameworks for ratiometric fluorescence detection of DPA and Hg2+

An easy and effective strategy for synthesizing a ratiometric fluorescent nanosensor has been demonstrated in this work. Novel fluorescent BSA–AuNPs@Tb–AMP (BSA, bovine serum albumin; AMP, adenosine 5′‐monophosphate; AuNPs, Au nanoparticles) metal–organic framework (MOF) nanostructures were synthesized by encapsulating BSA–AuNPs into Tb–AMP MOFs for the detection of 2,6‐pyridinedicarboxylic acid (DPA) and Hg²⁺. DPA could strongly co‐ordinate with Tb³⁺ to replace water molecules from the Tb³⁺ center and accordingly enhanced the fluorescence of Tb–AMP MOFs. The fluorescence of BSA–AuNPs at 405 nm remained constant. While the fluorescence of BSA–AuNPs at 635 nm was quenched after Hg²⁺ was added, the fluorescence of Tb–AMP MOFs remained constant. Accordingly, a ratiometric fluorescence nanosensor was constructed for detection of DPA and Hg²⁺. The ratiometric nanosensor exhibited good selectivity to DPA over other substances. The F₅₄₅/F₄₀₅ linearly increased with increase of DPA concentration in the range of 50 nM to 10 μM with a detection limit as low as 17.4 nM. F₆₃₅/F₄₀₅ increased linearly with increase of Hg²⁺ concentration ranging from 50 nM to 1 μM with a detection limit as low as 20.9 nM. Additionally, the nanosensor could be successfully applied for the determination of DPA and Hg²⁺ in running water.     

Effect of sludge age on performance of an activated sludge unit treating 2,4 dichlorophenol-containing synthetic wastewater

Wastewaters containing chlorophenol compounds are difficult to treat by biological means because of toxic effects of chlorophenols on microorganisms. Synthetic wastewater containing 2,4 dichlorophenol (DCP) was biologically treated in an activated sludge unit at different sludge ages varying between 5 and 30 days while the feed COD, DCP contents and hydraulic residence time (HRT) were constant. Effects of sludge age on COD, DCP and toxicity removals were investigated. Increases in sludge age caused significant increases in biomass concentration in the aeration tank, which resulted in increases in percent COD, DCP and toxicity removals. COD removal increased from 58 to 90%, while DCP and toxicity removals increased from 15 to 100% and from 38 to 100%, respectively, when the sludge age was raised from 5 to 30 days. Resazurin method based on dehydrogenase activity was used for assessment of the feed and effluent wastewater toxicity. Sludge volume index (SVI) decreased with increasing sludge age indicating improved settling characteristics of the sludge at high sludge ages. Operation at a sludge age of 25 days resulted in more than 90% COD and nearly 100% DCP and toxicity removal with an SVI value of 108 ml g⁻¹ under the experimental conditions tested.     

Individual and Joint Toxicity Effects of Cu,Cr(III), and Cr(VI) on Pakchoi: A Comparison Between Solution and Soil Cultures

The single and joint toxicity effects of Cu, Cr(III), and Cr(VI) on the root elongation of pakchoi in solution and soil were investigated. The median effective concentration (EC₅₀) was determined to examine the toxic thresholds of the test elements. The results showed that individual contamination by Cu, Cr(III), or Cr(VI) can inhibit the root elongation of pakchoi. The EC₅₀ values of the test elements were 2.02 mg/L and 195.8 mg/kg, 62.2 mg/L and 1,773 mg/kg, and 6.88 mg/L and 8.08 mg/kg in solution and soil, respectively. Toxic unit (TU) was introduced to determine the outcome in combined tests, and different behaviors were observed in both solution and soil. The coexistence of Cu and Cr(III) in solution exhibited an antagonistic effect (EC_50mix = 1.76 TU_mix), whereas a synergistic effect was observed in soil (EC_50mix = 0.76 TU_mix). In contrast, combined Cu–Cr(VI) showed a less than additive toxicity both in solution and soil, with EC_50mix values of 3.31 and 1.24 TU_mix. In conclusion, the coexistence of toxicity in Cu–Cr(III) and Cu–Cr(VI) differs from the toxicity exhibited individually by Cu, Cr(III), and Cr(VI). Heavy metal interaction also changes depending on the medium.     

Cryoprotectant agent toxicity in porcine articular chondrocytes

Large articular cartilage defects have proven difficult to treat and often result in osteoarthritis of the affected joint. Cryopreservation of articular cartilage can provide an increased supply of tissues for osteochondral allograft but cryoprotective agents are required; however, few studies have been performed on the toxicity of these agents. This study was designed to determine the order of toxicity of five commonly used cryoprotectant agents as well as interactions that occur between them. Isolated porcine articular chondrocytes were exposed to individual cryoprotectant agents and combinations of these agents at 1 M and 3 M concentrations for 5 min and 120 min. Cell viability was determined using membrane integrity dyes and a metabolic activity assay. Subsequently, a regression analysis based study was undertaken to extract the maximum amount of information from this data. Results of this study demonstrated that all 1 M solutions were minimally toxic. The 3 M solutions demonstrated varying toxicity after 120 min. Ethylene glycol and glycerol were less toxic than propylene glycol, dimethyl sulfoxide, and formamide. Combinations of cryoprotectant agents were less toxic than single cryoprotectant agents at the same concentration. This is the most comprehensive study investigating cryoprotectant agent toxicity in articular chondrocytes and has resulted in important information regarding the order of toxicity and interactions that occur between these agents.