Column Assay Research Articles

Valorization of organic residues for lettuce production minimizes nitrogen loss in soil in comparison to mineral fertilization

The agri-food industry, produces organic residues that could be reused as fertilizer, thereby allowing the recycling of nutrients for crops and the replacement of mineral fertilizers. In this context, here we examined the capacity of anaerobic digestates and mixed residues (digestates plus spent mushroom substrates) to supply nutrients to lettuce (Lactuca sativa L.) grown in a greenhouse experiment. Moreover, the potential leaching of nutrients into the soil was evaluated in a column assay. The results obtained from organic residues treatments were then compared to those achieved in soil treated with mineral fertilizer and in untreated soil. Digestates treatments minimized the potential risk of nitrate (NO3 −) leaching by allowing the slow release of NO3 − maintaining crop yield. Given our observations, this type of organic residues emerges as an alternative to mineral fertilizer. The loss of NO3 −-N from the total amount of N applied reached ∼3% in the mixed residues treatments, 36–46% in the digestate treatments, and 68% in the mineral fertilizer treatment. The mixed treatments showed low available N content as a result of immobilization in the soil, which is a limiting factor for crop production. Moreover, the organic residues treatments had no negative effect on lettuce seed germination or plant growth with respect to parameters such as chlorophyll, total polyphenols, and heavy metals in aerial biomass. The findings reported herein contribute to a better understanding of nutrient availability provided by organic residues materials and the capacity of these amendments to replace mineral fertilizers and avoid nutrient loss by leaching.

Chitinase from the Latex of Ficus benjamina L. Displays Antifungal Activity by Inducing ROS Generation and Structural Damage to the Fungal Cell Wall and Plasma Membrane.

Chitinases are plant defense-related proteins with a high biotechnological potential to be applied in agriculture. This study aimed to purify a chitinase from the latex of Ficus benjamina. An antifungal class I chitinase, named FbLx-Chi-1, was purified from the latex of Ficus benjamina after precipitation with 30-60% ammonium sulfate and affinity chromatography on a chitin column and antifungal potential assay against phytopathogenic fungi important to agriculture. FbLx-Chi-1 has 30 kDa molecular mass, as estimated by SDS-PAGE and the optimal pH and temperature for full chitinolytic activity were 5.5 and 60ºC, respectively. FbLx-Chi-1 is a high pH-, ion-tolerant and thermostable protein. Importantly, FbLx-Chi-1 hindered the growth of the phytopathogenic fungi Colletotrichum gloeosporioides, Fusarium pallidoroseum, and Fusarium oxysporum. The action mode of FbLx-Chi-1 to hamper F. pallidoroseum growth seems to be correlated with alterations in the morphology of the hyphal cell wall, increased plasma membrane permeability, and overproduction of reactive oxygen species. These findings highlight the biotechnological potential of FbLx-Chi-1 to control important phytopathogenic fungi in agriculture. In addition, FbLx-Chi-1 could be further explored to be used in industrial processes such as the large-scale environmentally friendly enzymatic hydrolysis of chitin to produce its monomer N-acetyl-β-D-glucosamine, which is employed for bioethanol production, in cosmetics, in medicine, and for other multiple applications.

Microbial Enhanced Oil Recovery (MEOR) Surfactant from Pseudomonas aeruginosa isolated from Automobile Garage soil.

Abstract: Microbial enhanced oil recovery (MEOR), is gaining attention today for being environmentally friendly, economically attractive, demonstrating improvement in recovery of oil entrapped in porous media. It is considered to be more efficient than other EOR methods when applied to carbonate oil reservoirs. In the current study oil degrading bacteria was isolated from soil of automobile garage. The biosurfactant producing organism was screened and characterized. 16SrRNA sequence of the most potent bacterial strain suggests it to be belonging to the genus Pseudomonas and species aeruginosa. The biosurfactant produced by the bacteria was detected as rhamnolipid with emulsification index of 69%, foaming of 57.69% and interfacial surface tension of 0.6 mN/m. The MEOR column assay revealed that the additional oil recovery for sand1 and sand2 was 62.142% and 52.173 respectively.

Fluoxetine Removal from Aqueous Solutions Using a Lignocellulosic Substrate Colonized by the White-Rot Fungus Pleurotus ostreatus.

One of the main challenges in both the design of new wastewater treatment plants and the expansion and improvement of existing ones is the removal of emerging pollutants. Therefore, the search for economic and sustainable treatments is needed to enhance the removal of pharmaceuticals. The potential of a lignocellulosic substrate colonized by Pleurotus ostreatus, a waste from mushroom production, to remove fluoxetine from aqueous solutions was studied. Batch assays were performed to remove 600 µg∙L−1 fluoxetine from aqueous solutions using the colonized mushroom substrate (CMS) and crude enzyme extracts. The removal efficiencies achieved were, respectively, ≥83.1% and 19.6% in 10 min. Batch assays with sterilized CMS and 1-aminobenzotriazole (to inhibit cytochrome P450 enzymes) showed that the higher removal efficiencies achieved in the CMS assays may be attributed to the synergistic contribution of biosorption onto the CMS and lignin modifying enzymes activity, namely laccase activity. A column assay was performed with the CMS, fed with 750 µg∙L−1 fluoxetine aqueous solution. The removal efficiency was 100% during 30 min, decreasing to a final value of 70% after 8 h of operation. The results suggested that CMS can be a promising eco-friendly alternative to remove fluoxetine from aqueous solutions.

Aromatic compounds that attract Meloidogyne species second-stage juveniles in soil.

Nematode attractants could serve in nematode control strategies by combining with chemical or biological nematicides or by interrupting the nematodes' host-finding process. The attractiveness of some benzenoid aromatic compounds, mainly benzoic acids, alcohols, aldehydes and phenols, to second-stage juveniles (J2) of four Meloidogyne species (M. hapla, M. incognita, M. javanica and M. marylandi) was evaluated by using trap tubes and balls filled with washed dune sand buried in nematode-inoculated sand in Petri dishes. Two-methoxybenzaldehyde, 2-methoxycinnamaldehyde, 2-hydroxybenzoic acid (salicylic acid), 2-hydroxy-3-methoxybenzaldehyde (o-vanillin), 3-methoxybenzoic acid, 4-methoxybenzoic acid and trans-cinnamic acid effectively attracted J2 of all or most of the four Meloidogyne species to trap tubes in a one-compound assay. When nematodes were exposed to three different compounds simultaneously in the three-compound assay, J2 of all Meloidogyne species were attracted mainly to 2-methoxycinnamaldehyde, salicylic acid and 4-methoxybenzoic acid. Exceptions were M. hapla J2, which were not attracted to salicylic acid. In the soil column assay, M. javanica and M. incognita J2 were attracted upward to 4-methoxybenzoic acid in a trap ball located 4 or 8cm above the inoculation point, whereas salicylic acid and 3-methoxybenzoic acid demonstrated slight, if any attraction. Although some of the tested compounds exist in root exudates, it is not clear whether they are involved in the nematode host-searching process in nature. The attractants found in the study have potential for use in Meloidogyne species control, probably as a nematode trap constituent or as compounds that disrupt the nematodes' host-finding process. © 2021 Society of Chemical Industry.

Flipped Quick-Response Code Enables Reliable Blood Grouping.

Accurate and rapid blood typing plays a vital role in a variety of biomedical and forensic scenarios, but recognizing weak agglutination remains challenging. Herein, we demonstrated a flipping identification with a prompt error-discrimination (FLIPPED) platform for automatic blood group readouts. Bromocresol green dye was exploited as a characteristic chromatography indicator for the differentiation of plasma from whole blood by presenting a teal color against a brown color. After integrating these color changes into a quick-response (QR) code, prompt typing of ABO and Rhesus groups was automatically achieved and data could be uploaded wirelessly within 30 s using a commercially available smartphone to facilitate blood cross-matching. We further designed a color correction model and algorithm to remove potential errors from scanning angles and ambient light intensities, by which weak agglutination could be accurately recognized. With comparable accuracy and repeatability to classical column assay in grouping 450 blood samples, the proposed approach further demonstrates to be a versatile sample-to-result platform for clinical diagnostics, food safety, and environmental monitoring.

Development and application of a novel immunoaffinity test column assay for visual detection of cloxacillin residues in animal-derived food samples

Nowadays, the abuse of cloxacillin (CLOX) in livestock has generated considerable problems for food safety. In this study, a rapid and visual immunoaffinity test column (IATC) assay was developed for the detection of CLOX residues in animal derived food. The novel IATC assay was used by traditional immune competition and gel separation methods. Traditional immune competition could detect CLOX, and gel separation could reduce the effect of matrix in the samples. The established IATC assay was successfully applied to detect CLOX residue in food samples, with a visual limit of detection of 25 ng/g. The results obtained with the proposed IATC assay agreed well with those obtained by liquid chromatography–tandem mass spectrometry. It indicated that the developed method could be an effective analytical tool to monitor CLOX residues in food products.

Development of a Rapid and Versatile Method of Enzyme-Linked Immunoassay Combined with Immunoaffinity Column for Aflatoxin Analysis

We combined immunoaffinity column (IAC) and enzyme-linked immunosorbent assay (ELISA) methods to develop a rapid method to analyze total aflatoxin (AF) in foods, using a large number of samples. Using newly developed monoclonal antibodies, with high cross-reactivity and high organic solvent tolerance, we developed the IAC-ELISA method. Our IAC-ELISA method showed a good correlation with the high-performance liquid chromatography method for corn samples spiked with total AF. Certain food samples, such as chili powder, chocolate, green coffee beans, and roasted coffee beans, are difficult to measure owing to their matrices, which affect ELISA adversely. Our IAC-ELISA method clearly improved the recovery rates (79 to 109%) compared with the ELISA method (97 to 164%) for four food samples. Our method is simple and quick; thus, it may be ideal for routine inspections.

Modified stall-side crossmatch for transfusions in horses.

BackgroundAfter‐hours or out‐of‐clinic crossmatches are often limited by the lack of access to specialized material and technical expertise.Hypothesis/ObjectivesThe goal was to adapt a stall‐side crossmatch test for pretransfusion evaluation in horses.AnimalsTwelve healthy mares (plasma and blood donors, teaching mares).MethodsIn a prospective study, blood from 12 mares was used to compare the results of 132 crossmatches performed with a rapid gel assay to crossmatches performed with a microgel column assay, and with predicted compatibilities based on blood types and detection of antibodies at a reference laboratory (microplate assay). The rapid gel assay protocol for dogs was adapted to decrease the formation of rouleaux that initially precluded equine erythrocytes migration through the gel.ResultsThere was a good agreement between the rapid gel assay and the microgel assay as well as with the predicted compatibilities (κ > .6 for both). Agreement was higher between the microgel assay and the predicted compatibilities (κ = .8). The rapid gel assay failed to detect 6 predicted Aa incompatibilities (agglutinins‐related), 3 of which were also not detected with the microgel assay.Conclusions and Clinical ImportanceBased on these results, the modified rapid gel assay could be useful in settings when access to the microgel assay is not available. Discrepancies between both gel techniques and predicted compatibilities were most often low‐grade agglutination, which warrants further investigation to assess their clinical importance.

Combining an effective immuno-affinity column with ELISA for reliable and visual detection of furaltadone metabolites in aquatic products

In this study, a reliable and visual immunoaffinity test column (IATC) assay was developed for the detection of furaltadone metabolites in aquatic products based on the immuno-affinity column with ELISA.

Fluorescence Quenching Immunoaffinity Test Column with Quantum Dots as Fluorescence Donors for the Quick Detection of Malachite Green and Crystal Violet in Aquatic Products

A visual fluorescence quenching immunoaffinity test column (FQ-ITC) assay was developed for the simultaneous detection of malachite green (MG) and crystal violet (CV) residues in aquatic products. The proposed FQ-ITC assay was based on fluorescence resonance energy transfer from the fluorescence donor (quantum dot) to the fluorescence acceptor (MG or CV) and the specific identification between the analytes and their polyclonal antibodies. The process can be completed within 10 min, and the results can be observed by the naked eye. After the evaluation of the specificity and sample matrix effect, the established FQ-ITC was found to be successfully applied in the detection of MG or CV in grass carp, striped bass, carp, and shrimp, with a visual limit of detection of 2 μg/kg. The accuracy of the FQ-ITC assay was verified by comparison with liquid chromatography-tandem mass spectrometry. The simple, time-saving, sensitive, and visual observation characteristics make the established FQ-ITC assay an effective analytical tool for the simultaneous detection of MG and CV in aquatic products.

Visual Non-Instrumental On-Site Detection of Fumonisin B1, B2, and B3 in Cereal Samples Using a Clean-Up Combined with Gel-Based Immunoaffinity Test Column Assay

A visual immunoaffinity test column (IATC) assay was developed to detect fumonisins in cereal samples for spot tests without the need for special instruments. The developed IATC assay had equivalent recognition capability for fumonisin B1 (FB1), fumonisin B2 (FB2), or fumonisin B3 (FB3), and exhibited no cross-reactivity with aflatoxin B1, ochratoxin A, zearalenone, or the T-2 toxin. The sample pretreatment was accomplished more rapidly and with greater ease, the entire assay procedure was completed in approximately 10 min, including sample pretreatment and testing. The limits of detection (LODs) of the IATC assay to detect fumonisins in the maize, barley, oat, and millet samples were 20 μg kg−1. The results of the spiked maize, barley, oat, and millet and real maize samples by the IATC assay agreed well with the results obtained by the commercial fumonisin enzyme-linked immunosorbent assay (ELISA) test kit and liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively. The developed IATC assay can serve as a useful screening tool for the rapid, qualitative, and semi-quantitative detection of the total content of fumonisins (sum of FB1, FB2, and FB3) in cereal samples on-site.

Capture of red blood cells onto optical sensor for rapid ABO blood group typing and erythrocyte counting

ABO blood group typing and erythrocyte counting are essential for blood transfusion especially in the vital lifesaving procedures. Here, we developed a simple, rapid and reliable assay for ABO blood group typing and erythrocyte counting based on localized surface plasmon resonance biosensor. The optical biosensors were constructed by the deposition of gold nanoprisms (GNPs) on glass substrates, and the immobilization of antibodies on the GNPs surface. The sensors were further integrated into a microfluidic chip containing two independent sample cells with anti-isoagglutinins A and anti-B antibodies modified at the bottoms, respectively. The sensing chips coupled with ultraviolet-visible (UV–vis) spectrometer were applied for the identification of blood type A, B, AB and O, respectively; meanwhile, a simple proof-of-principle strategy for quantifying red blood cells (RBCs) through the optical biosensors were demonstrated with series of RBCs dilution, and observed by UV–vis spectra. The limit of detection for erythrocyte counting was as low as ∼104 cells/mL (R2 = 0.99). Additionally, we compared our sensing results with those from conventional gel column assay on 7 different blood samples, in which the accuracy of blood typing was 100%. The RBC concentrations estimated by our sensor chips were between 3.9–5.0 × 109 cells/mL, which locates in the normal range of RBCs concentration of human. The LSPR biosensors that we developed are capable of qualitative ABO blood typing and quantitative erythrocyte counting simultaneously, which have great potential to develop a preliminary and lab-training-free biomedical device for rapid clinical diagnosis.

Copper deficiency can limit nitrification in biological rapid sand filters for drinking water production

Incomplete nitrification in biological filters during drinking water treatment is problematic, as it compromises drinking water quality. Nitrification problems can be caused by a lack of nutrients for the nitrifying microorganisms. Since copper is an important element in one of the essential enzymes in nitrification, we investigated the effect of copper dosing on nitrification in different biological rapid sand filters treating groundwater. A lab-scale column assay with filter material from a water works demonstrated that addition of a trace metal mixture, including copper, increased ammonium removal compared to a control without addition. Subsequently, another water works was investigated in full-scale, where copper influent concentrations were below 0.05 μg Cu L−1 and nitrification was incomplete. Copper dosing of less than 5 μg Cu L−1 to a full-scale filter stimulated ammonium removal within one day, and doubled the filter's removal from 0.22 to 0.46 g NH4-N m−3 filter material h−1 within 20 days. The location of ammonium and nitrite oxidation shifted upwards in the filter, with an almost 14-fold increase in ammonium removal rate in the filter's top 10 cm, within 57 days of dosing. To study the persistence of the stimulation, copper was dosed to another filter at the water works for 42 days. After dosing was stopped, nitrification remained complete for at least 238 days. Filter effluent concentrations of up to 1.3 μg Cu L−1 confirmed that copper fully penetrated the filters, and determination of copper content on filter media revealed a buildup of copper during dosing. The amount of copper stored on filter material gradually decreased after dosing stopped; however at a slower rate than it accumulated. Continuous detection of copper in the filter effluent confirmed a release of copper to the bulk phase. Overall, copper dosing to poorly performing biological rapid sand filters increased ammonium removal rates significantly, achieving effluent concentrations of below 0.01 mg NH4-N L−1, and had a long-term effect on nitrification performance.

Development of a quantitative immuno-affinity test column assay for on-site screening of clindamycin residues in milk

A sensitive and specific monoclonal antibody against clindamycin (CLIN) was produced and used to develop an immuno-affinity test column (IATC) assay for on-site screening of CLIN residues in milk. The qualitative limit of detection of the IATC assay, estimated as 1.0 μg L−1 by visual detection, was sufficient to measure maximum residue levels for lincosamide antibiotics in milk. The quantitative IATC assay resulted in a lower detection limit (0.11 μg L−1) by evaluating the colour intensity of the test layers, which was approximately 9 times greater sensitivity than visual detection. During the spike and recovery test, the average recoveries ranged from 76% to 114% at different spiked levels, and the intra-/interday coefficients of variation were in the range 9.6–16.7%. The detection time was shortened to 20 min, whereas the sensitivity was comparable with a traditional ELISA. The IATC assay shows promise for on-site screening of CLIN residues in milk.

A novel bench-scale column assay to investigate site-specific nitrification biokinetics in biological rapid sand filters

A bench-scale assay was developed to obtain site-specific nitrification biokinetic information from biological rapid sand filters employed in groundwater treatment. The experimental set-up uses granular material subsampled from a full-scale filter, packed in a column, and operated with controlled and continuous hydraulic and ammonium loading. Flowrates and flow recirculation around the column are chosen to mimic full-scale hydrodynamic conditions, and minimize axial gradients. A reference ammonium loading rate is calculated based on the average loading experienced in the active zone of the full-scale filter. Effluent concentrations of ammonium are analyzed when the bench-scale column is subject to reference loading, from which removal rates are calculated. Subsequently, removal rates above the reference loading are measured by imposing short-term loading variations. A critical loading rate corresponding to the maximum removal rate can be inferred. The assay was successfully applied to characterize biokinetic behavior from a test rapid sand filter; removal rates at reference loading matched those observed from full-scale observations, while a maximum removal capacity of 6.9 g NH4+–N/m3 packed sand/h could easily be determined at 7.5 g NH4+–N/m3 packed sand/h. This assay, with conditions reflecting full-scale observations, and where the biological activity is subject to minimal physical disturbance, provides a simple and fast, yet powerful tool to gain insight in nitrification kinetics in rapid sand filters.

Biosorption of heavy metals by lactic acid bacteria and identification of mercury binding protein

Heavy metals cause various health hazards. Using lactic acid bacteria (LAB), we tested the biosorption of heavy metals e.g. cadmium (Cd) (II), lead (Pb) (II), arsenic (As) (III), and mercury (Hg) (II). Cd (II) sorption was tested in 103 strains using atomic absorption spectrophotometery (AAS). Weissella viridescens MYU 205 (1 × 108 cells/ml) decreased Cd (II) levels in citrate buffer (pH 6.0) from one ppm to 0.459 ± 0.016 ppm, corresponding to 10.46 μg of Cd (II). After screening, 11 LAB strains were tested using various pH (pH 4.0, 5.0, 6.0, 7.0) showing the sorption was acid sensitive; and was cell concentration dependent, where the Cd (II) concentration decreased from one ppm to 0.042 (max)/0.255 (min) ppm at 1 × 1010 cells/ml. Additionally, the biosorption of Pb (II), As (III), and Hg (II) were tested using an inductively coupled plasma mass spectrometer (ICP-MS). The Hg (II) concentration was reduced the most followed by Pb (II) and As (III). Many of the bacterial cell surface proteins of W. viridescens MYU 205 showed binding to Hg (II) using the Hg (II) column assay. Having a CXXC motif, a ∼14 kDa protein may be one of the Hg (II) binding proteins. LAB biosorption may aid the detoxification of people exposed to heavy metals.

Efficacy of entomopathogenic nematodes against potato tuber moth, Phthorimaea operculella (Lepidoptera: Gelechiidae) under laboratory conditions

The susceptibility of potato tuber moth, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae) to native and commercial strains of entomopathogenic nematodes (EPNs) was studied under laboratory conditions. Native strains of EPNs were collected from northeastern Iran and characterised as Steinernema feltiae and Heterorhabditis bacteriophora (FUM 7) using classic methods as well as analysis of internal transcribed spacer (ITS) and D2/D3 sequences of 28S genes. Plate assays were performed to evaluate the efficiency of five EPN strains belonging to four species including Steinernema carpocapsae (commercial strain), S. feltiae, Steinernem glaseri and H. bacteriophora (FUM 7 and commercial strains). This initial assessment with 0, 75, 150, 250, 375 and 500 IJs/ml concentrations showed that S. carpocapsae and H. bacteriophora caused the highest mortality in both larval and prepupal stages of P. operculella, PTM. Thereafter, these three strains (i.e. S. carpocapsae, H. bacteriophora FUM 7 and the commercial strains) were selected for complementary assays to determine the effects of soil type (loamy, loamy–sandy and sandy) on the virulence of EPNs against the second (L2) and fourth instar (L4) larvae as well as prepupa. A soil column assay was conducted using 500 and 2000 IJs in 2-ml distilled water. Mortality in the L2 larvae was not affected by the EPN strain or soil type, while there was a significant interactive effect of nematode strains and soil type on larval mortality. The results also showed that EPN strains have higher efficiency in lighter soils and caused higher mortality on early larvae than that in loamy soil. In L4 larvae, mortality of PTM was significantly influenced by nematode strain and applied concentrations of infective juveniles. The larval mortality induced by S. carpocapsae was higher than those caused either by a commercial or the FUM 7 strain of H. bacteriophora. Prepupa were the most susceptible stage.

Batch and continuous flow studies of adsorptive removal of Cr(VI) by adapted bacterial consortia immobilized in alginate beads

The adsorptive removal of Cr(VI) by alginate beads containing Cr(VI)-adapted Acinetobacter junii, Escherichia coli and Bacillus subtilis in batch and continuous packed bed column reactors was investigated. Under optimized conditions (pH 3.0; contact time, 180min; 30°C; initial Cr(VI) concentration of 100mg/L), 65.86mg/g adsorption capacity was recorded in the batch study. When an adsorbent dosage of 1g/L, a flow rate of 5mL/min, a bed height of 20cm, an initial Cr(VI) concentration of 300mg/L was employed, a capacity of 657mg/g was noted for the continuous column assay. The batch sorption data followed the Langmuir isotherm and pseudo second order kinetics. Five sorption/desorption cycles yielded 100%, 99.63%, 95.31%, 80.7% and 74.22% regeneration, respectively. Cr(VI) adsorption studies using spiked ground water, freshwater and domestic wastewater in a packed bed reactor demonstrated Cr(VI) removals of 64.8%, 55.08%, 56.86% respectively. Cr(VI) sorption on immobilized bacteria was confirmed with Fourier-transform infrared and Energy dispersive X-ray spectroscopy.

Clinical application studies on AFP-L3 detected by micro-spin column method

To explore the clinical value of Lens culinaris agglutinin-reactive alpha-fetoprotein in the differentiation diagnosis between benign and malignant liver diseases, as well as the early warning of hepatocellular carcinoma. Alpha-fetoprotein variants from 300 patients with liver diseases were isolated with micro-spin column equipped lens culinaris agglutinin (LCA). The AFP and AFP-L3 were detected by the electrochemical luminescence (ECL) method, and the proportions of AFP-L3 were calculated. The positive rates of AFP-L3 of HCC patients and chronic liver disease patients were 95% and 64% respectively, there were significant difference in two groups (chi2 = 134.72, P < 0.01), the HCC incidence rates of AFP-L3 positive and negative chronic liver disease patients showed significant difference (chi2 = 80.158, P < 0.01). there were no correlations between the proportion of AFP-L3 and AFP consistency(r = 0.046, P > 0.05). The detection of AFP-L3 by micro-spin column assay show great clinical value in the differentiation diagnosis of benign and malignant liver diseases, as well as the early warning of hepatocellular carcinoma.