Quenching of Fluorescence Caused by Graphene Oxide as an Immunosensing Platform in a Microwell Plate Format

Immunoassays are, at present, an important tool for diagnostics, drug development, and environmental monitoring. However, most immunoassays involve procedures that require many elements for their development. We introduce a novel biosensing platform based on fluorescence quenching caused by graphene oxide (GO) for the detection of Human-IgG and Prostate-Specific Antigen (PSA). We employ a single antibody for the capture and detection processes, avoiding washing steps. FITC fluorophore was conjugated with antibodies for H-IgG detection, whereas quantum dots were conjugated with antibodies for PSA detection. The simple biosensing platform consists of covering a 96-well microplate (with a polystyrene bottom) with GO. The graphene oxide adhesion is possible by way of electrostatic interactions between the plate surface modified with amino groups (positively charged) and the graphene oxide (negatively charged). This proposal showed an excellent response for the detection of Human-IgG, with acceptable precision (from 0.27% to 5%). The limit of detection reached for H-IgG was 3.35 ng mL-1. In the same manner, for PSA detection, the limit of detection reached was 0.02 ng mL-1 and the precision range was from 0.7% to 15.2%. Furthermore, this biosensing platform was demonstrated to operate with real samples of human urine doped with different concentrations of prostate-specific antigen.

Development of 96-microwell plate assay with Fluorescence Reader and HPLC method with Fluorescence Detection for High-throughput Analysis of Linifanib in its Bulk and Dosage Forms

Background: Linifanib (LFB) is a tyrosine kinase inhibitor with antineoplastic activity. The existing methods for analysis of LFB in bulk and dosage forms do not meet the requirements of quality control (QC) analysis. Objective: The present study was devoted to the development of two methods with high throughputs for determination of LFB. These methods are 96-microwell plate assay with microplate fluorescence reader (MWP-FR) and high-performance liquid chromatography with fluorescence detection (HPLC-FD). Methods: The MWP-FR assay was carried out in white opaque 96-well assay plates and the native fluorescence signals of LFB were measured at 360 nm for excitation and 500 nm for emission. In the HPLC-FD, the chromatographic separation of LFB and quinine sulphate (QS) as internal standard (IS) was performed on μ-Bondapack CN HPLC column using a mobile phase consisting of acetonitrile:water (60:40, v/v) pumped at a flow rate of 1 ml/min in an isocratic mode. The fluorescence detector was set at 350 nm for excitation and 454 nm for emission. Results: The linear ranges of the MWP-FR and HPLC-FD were 1 – 12 μg/well and 10 – 500 ng/ml, respectively. The limits of quantitation were 0.85 μg/well and 8.24 ng/ml for MWP-FR and HPLC-FD, respectively. Both MWP-FR and HPLC-FL methods were successfully applied for the determination of LFB in both bulk and tablets. Conclusion: Both methods have high analytical throughputs, they are suitable for use in QC laboratories for analysis of large numbers of LFB samples, and are environmentally friendly as they consume low volumes of chemicals and solvents.

AKTIVITAS ANTIBAKTERI EKSTRAK KULTUR JAMUR ENDOFIT Fusarium sp. CSP-4 YANG DIISOLASI DARI Curcuma sumatrana Miq.

Endophytic fungus Fusarium sp. CSP-4 was isolated from the rhizome of Curcuma sumatrana Miq. The ethyl acetate extract of CSP-4 was fractionated by chromatographic column with silica as a stationary phase. The fractions were tested for antibacterial activity against S.aureus and E.coli by Thin Layer Chromatography (TLC)- bioautography. Determination of Minimum Inhibitory Concentration (MIC) was carried out by the microdilution method on a 96-microwell plate with the concentrations from 2 to 256 µg/ml. The results showed that fraction 15 and 11 were able to inhibit bacterial growth with the MIC values of 256 and> 256 μg / mL against S.aureus and>256 μg / mL against E.coli, respectively.The antibacterial activity of fractions 15 and 11 was weaker compared to the positive control of chloramphenicol with the MIC value of 4 µg/mL against S.aureus and E.coli. Based on the results obtained showed that the fractions of the methanol extract of endophytic fungus Fusarium sp. CSP-4 have weak antibacterial activity.

High-Throughput Optimization of Effective Microbes Nutrient Formulation via Automated Media Optimization System (AMOS)

Fortification of effective microbes (EM) such as Pediococcus acidilactici and photosynthetic bacteria in fertilizer and animal feed exhibiting effective outcomes in enhancing crops and livestock production as well as upholds sustainable agriculture concept. However, the productivity of the EM biomass stocks is unsurpassed to the demand due to non-optimized media. In this study, two sets of nutrient pair comprising of beef extract-manganese and yeast extract-glucose for P. acidilactici and photosynthetic bacteria respectively were studied. The optimum media formulated was based on the highest growth rate in 96-microwell plate system were subsequently tested in 1 L cultures volume for EM biomass production. Results showed that growth rate and dry weight of P. acidilactici in media supplemented with 64 g/L beef extract (0.145 h-1, 0.243 g) and 0.28 g/L manganese (0.1134 h-1, 0.32 g) were higher compared to control medium with an increment of 17.39 % and 54.59 % biomass productivity as compared to MRS medium respectively. Meanwhile, the growth rate and dry weight for Photosynthetic bacteria in media supplemented with 6.63 g/L glucose (0.2538 h-1, 0.167 g) and 0.44 g/L yeast extract (0.2733 h-1, 0.163 g) recorded 11.3 % and 8.73 % biomass productivity increment in comparison to the HCH medium.

Drop-In Biofuel production by using fatty acid photodecarboxylase from Chlorella variabilis in the oleaginous yeast Yarrowia lipolytica

BackgroundOleaginous yeasts are potent hosts for the renewable production of lipids and harbor great potential for derived products, such as biofuels. Several promising processes have been described that produce hydrocarbon drop-in biofuels based on fatty acid decarboxylation and fatty aldehyde decarbonylation. Unfortunately, besides fatty aldehyde toxicity and high reactivity, the most investigated enzyme, aldehyde-deformylating oxygenase, shows unfavorable catalytic properties which hindered high yields in previous metabolic engineering approaches.ResultsTo demonstrate an alternative alkane production pathway for oleaginous yeasts, we describe the production of diesel-like, odd-chain alkanes and alkenes, by heterologously expressing a recently discovered light-driven oxidase from Chlorella variabilis (CvFAP) in Yarrowia lipolytica. Initial experiments showed that only strains engineered to have an increased pool of free fatty acids showed to be susceptible to sufficient decarboxylation. Providing these strains with glucose and light in a synthetic medium resulted in titers of 10.9 mg/L of hydrocarbons. Using custom 3D printed labware for lighting bioreactors, and an automated pulsed glycerol fed-batch strategy, intracellular titers of 58.7 mg/L were achieved.ConclusionsOleaginous yeasts such as Yarrowia lipolytica can transform renewable resources such as glycerol into fatty acids and lipids. By heterologously expressing a fatty acid photodecarboxylase from the algae Chlorella variabilis hydrocarbons were produced in several scales from microwell plate to 400 ml bioreactors. The developed bioprocess shows a route to the renewable production of hydrocarbons for a variety of applications ranging from representing a substrate for further enzymatic or chemical modification or as a drop-in biofuel blend.Short abstractOleaginous yeasts are potent hosts for the renewable production of lipids, fatty acids and derived products such as biofuels. Here, we describe, the production of odd-numbered alkanes and alkenes with a length of 17 and 15 carbons by expression of a fatty acid photodecarboxylase (CvFAP) from Chlorella variabilis in different Yarrowia lipolytica strains under different regimes of blue light exposure in several scales from microwell plate to 400 ml bioreactors.

Blocking-Free ELISA Using a Gold Nanoparticle Layer Coated Commercial Microwell Plate

Enzyme-linked immunosorbent assays (ELISA) show extensive application in immunoassays, to detect and monitor protein biomarkers in clinical diagnosis. Nevertheless, the time required and its multiple steps limit its application. We take advantage of a polyethyleneimine (PEI) gold nanoparticle (GNP) coated microwell plate to perform blocking-free ELISA, in which no nonspecific protein adsorption appears on the GNP layer. If the PEI-GNP coated microwell plate and immobilization of captured antibodies on the plate are prepared in advance, such as using an ELISA kit, the whole ELISA process can be finished in less than 2 h. Meanwhile, we have ensured that the GNP layer can preserve the precision and good linearity of ELISA without causing negative effects on the plate.

Isolasi dan Identifikasi Bakteri Pembentuk Biofilm dari Tambak Udang Balai Besar Pengembangan Budidaya Air Payau Jepara untuk Menghilangkan Amoniak

Brackish water shrimp aquaculture activities often result in organic waste from excess of unconsumed foodstuff and biological waste from shrimp biological waste. The high organic contents increase the levels of ammonia, which is toxic to shrimp and many other aqua lives. One of the most widely used organic material biodegradation system as biofilters, biofilm has not yet seen many uses in shrimp aquafarm waste management. This study aims to isolate and screen biofilm-forming primary bacteria with abilities to degrade ammoniacal nitrogen compounds. The processes involved in this study are location survey, wooden and fiber panel installation, planting of panel in the ponds, isolation of bacteria by dispersion method, purification of primary bacteria by scratch method. Ammoniacal nitrogen degradation test was performed by Microwell Plate Chromatogram Assay and UV-Vis Spectrophotometry. The analysis of the bacteria isolates found 66 primary bacteria with biofilm formation abilities. Based on qualitative analysis, 20 isolates displayed potential in degrading ammoniacal nitrogen compound and 7 isolates showed low (<10%) capacity in degrading ammoniacal nitrogen. Key words: biofilm-forming primary bacteria, ammoniacal nitrogen, degradation Kegiatan budidaya udang di tambak akan menghasilkan limbah organik yang berasal dari sisa pakan yang tidak termakan maupun kotoran udang. Kandungan bahan organik yang tinggi akan meningkatkan kandungan amonia yang bersifat toksik bagi udang dan biota air lainnya. Salah satu sistem biodegradasi bahan organik yang telah banyak digunakan sebagai biofilter namun belum dimanfaatkan dalam pengolahan limbah organik tambak udang adalah biofilm. Tujuan dari penelitian ini adalah mengisolasi dan skrining bakteri primer pembentuk biofilm yang mampu mendegradasi senyawa amonia nitrogen. Untuk mencapai tujuan tersebut, maka beberapa tahap penelitian yang telah dilakukan adalah survei lokasi tambak udang, pemasangan panel bahan kayu dan fiber, penanaman panel dalam badan air tambak, mengisolasi bakteri dengan metode sebaran, purifikasi bakteri primer pembentuk biofilm dengan metode goresan. Uji oksidasi amonium nitrogen dilakukan secara kualitatif dan kuantitatif dengan metode Micro well plate chromatogram assay dan UV-Vis Spektrofotometer. Hasil penelitian menunjukkan bahwa hasil isolasi diperoleh sebanyak 66 isolat bakteri primer pembentuk biofilm. Berdasarkan uji kualitatif diperoleh 20 isolat yang memiliki potensi mendegradasi senyawa amoniurn nitrogen. Namun hasil uji kuantitatif bakteri seleksi pendegradasi amonium nitrogen menunjukkan 7 isolat yang memiliki kemampuan rendah (< 10%) mendegradasi amonium nitrogen. Kata kunci: bakteri primer pembentuk biofilm, amonia nitrogen, degradasi