Concentration of foot-and-mouth disease virus by ultrafiltration

Stirred cell, flat sheet and spiral configurations were used to concentrate foot-and-mouth disease virus fermentation broth. The initial screening was carried out in a stirred cell using 50–300 k MWCO ultrafiltration membranes made of different polymers. Acrylate membrane showed highest recovery. No significant effect of MWCO and polymer material was observed on operating fluxes. Flat sheet configuration studies indicated as the CFR increases the recovery increased. No significant change was observed in operating fluxes over seven successive concentration and cleaning cycles. 97% recovery was obtained with the spiral module. Negligible effect on operating fluxes was observed over six successive cycles. A ∼30% saving in process time was observed in comparison with the conventional sedimentation process.     

Rapid microchip-based electrophoretic immunoassays for the detection of swine influenza virus

Towards developing rapid and portable diagnostics for detecting zoonotic diseases, we have developed microchip-based electrophoretic immunoassays for sensitive and rapid detection of viruses. Two types of microchip-based electrophoretic immunoassays were developed. The initial assay used open channel electrophoresis and laser-induced fluorescence detection with a labeled antibody to detect influenza virus. However, this assay did not have adequate sensitivity to detect viruses at relevant concentrations for diagnostic applications. Hence, a novel assay was developed that allows simultaneous concentration and detection of viruses using a microfluidic chip with an integrated nanoporous membrane. The size-exclusion properties of the in situ polymerized polyacrylamide membrane are exploited to simultaneously concentrate viral particles and separate the virus/fluorescent antibody complex from the unbound antibody. The assay is performed in two simple steps--addition of fluorescently labeled antibodies to the sample, followed by concentration of antibody-virus complexes on a porous membrane. Excess antibodies are removed by electrophoresis through the membrane and the complex is then detected downstream of the membrane. This new assay detected inactivated swine influenza virus at a concentration four times lower than that of the open-channel electrophoresis assay. The total assay time, including device regeneration, is six minutes and requires <50 microl of sample. The filtration effect of the polymer membrane eliminates the need for washing, commonly required with surface-based immunoassays, increasing the speed of the assay. This assay is intended to form the core of a portable device for the diagnosis of high-consequence animal pathogens such as foot-and-mouth disease. The electrophoretic immunoassay format is rapid and simple while providing the necessary sensitivity for diagnosis of the illness state. This would allow the development of a portable, cost-effective, on-site diagnostic system for rapid screening of large populations of livestock, including sheep, pigs, cattle, and potentially birds.     

Rapid screening of phenylketonuria using a CD microfluidic device

We herein present a compact disc (CD) microfluidic chip based hybridization assay for phenylketonuria (PKU) screening. This CD chip is composed of a polydimethylsiloxane (PDMS) top layer containing 12 DNA hybridization microchannels, and a glass bottom layer with hydrogel pad conjugated DNA oligonucleotides. Reciprocating flow was generated on the CD chip through a simple rotation-pause operation to facilitate rapid DNA hybridization. When rotated the CD chip, the sample solution was driven into the hybridization channel by centrifugal force. When stopped the CD chip, the sample plug was pulled backward through the channel by capillary force. The hybridization assay was firstly validated with control samples and was then used to analyze 30 clinical samples from pregnant women with suspected PKU fetus. The on-chip DNA hybridization was completed in 15 min with a sample consumption as low as 1.5μL, and the limit-of-detection (LOD) of DNA template was 0.7ng/μL. Among the 30 samples tested, V245V mutation was identified in 4 cases while R243Q mutation was detected in one case. Results of the hybridization assay were confirmed by DNA sequencing. This CD-chip based hybridization assay features short analysis time, simple operation and low cost, thus has the potential to serve as the tool for PKU screening.     

Comparative study of codon substitution patterns in foot-and-mouth disease virus (serotype O)

We compared genetic variations in the VP1 gene of foot-and-mouth disease viruses (FMDVs) isolated since 2000 from various region of the world. We analyzed relative synonymous codon usage (RSCU) and phylogenetic relationship between geographical regions, and calculated the genetic substitution patterns between Korean isolate and those from other countries. We calculated the ratios of synonymously substituted codons (SSC) to all observed substitutions and developed a new analytical parameter, EMC (the ratio of exact matching codons within each synonymous substitution group) to investigate more detailed substitution patterns within each synonymous codon group. We observed that FMDVs showed distinct RSCU patterns according to phylogenetic relationships in the same serotype (serotype O). Moreover, while the SSC and EMC values of FMDVs decreased according to phylogenetic distance, G + C composition at the third codon position was strictly conserved. Although there was little variation among the SSC values of 18 amino acids, more dynamic differences were observed in EMC values. The EMC values of 4- and 6-fold degenerate amino acids showed significantly lower values while most 2-fold degenerate amino acids showed no significant difference. Our findings suggest that different EMC patterns among the 18 amino acids might be an important factor in determining the direction of evolution in FMDV.     

Rapid screening of antibiotic toxicity in an automated microdroplet system

We report an automated microfluidic platform for 'digitally' screening the composition space of droplets containing cocktails of small molecules and demonstrate the features of this system by studying epistatic interactions between antibiotics and Escherichia coli ATCC 25922. This system has several key characteristics: (i) it uses small (<100 μL) samples of liquids and suspensions of bacteria that are introduced directly into the chip; (ii) it generates a sequence of droplets with compositions, including reagents and bacterial cell suspensions that are programmed by the user; (iii) it exports the sequence of droplets to an external segment of tubing that is subsequently disconnected for incubation and storage; and (iv) after incubation of bacteria in droplets, the droplets are injected into a second device equipped with an in-line fiber optic spectrophotometer that measures cell growth. The system generates and fuses droplets with precise (<1% in standard deviation) control of liquid volumes and of the concentrations of input substrates. We demonstrate the application of this technology in determining the minimum inhibitory concentration and pair-wise interactions of ampicillin, tetracycline, and chloramphenicol against E. coli. The experiments consumed small volumes of reagents and required minutes to create the droplets and several hours for their incubation and analysis.     

Rapid, practical and predictive excipient compatibility screening using isothermal microcalorimetry

Conditions for conducting excipient compatibility studies via isothermal microcalorimetry were explored using model reactions. The resulting recommended procedure for rapid and practical screening consisted of using binary mixtures (100 mg of each component), the addition of 20% (w/w) water, and monitoring the mixture at 50°C for 3 days using an isothermal microcalorimeter. The correlation between calorimetric excipient compatibility results and formulation stability was investigated for two developmental drugs. A comparison of calorimetric results to actual formulation stability suggested that it was possible to predict relative stability within functional classes. However, caution should be exercised in such predictions, because apparent reaction enthalpies were found to vary three-fold among excipients in the same functional class. Based on these observations, a two-step procedure is suggested for efficient development of stable formulations. First, excipient compatibility screening should be conducted using a rapid calorimetric technique. The calorimetric results are then used to evaluate relative risk of incompatibility for each excipient within a particular functional class. The calorimetric data and the functional requirements of the dosage form are then integrated in developing a limited number of model formulations that are likely to succeed from both a performance and a stability perspective. The second step of the process is to conduct traditional HPLC-based accelerated stability studies on the limited number of model formulations.     

Rapid determination of microorganisms using a flow-injection system

An automated method for the rapid determination of microorganisms using a flow-injection system is presented. Electrochemical measurement of a mediator reduced by microbial metabolism allowed the determination of fungi and bacteria in a few minutes. The lowest detection limit was 5 × 10⁶ colony-forming units (cfu) ml^?1 for Escherichia coli. Correlation between the flow-injection method and standard microbiological methods was excellent (r = 0.997, n = 4 for Beauveria bassiana; r = 0.997, n = 7 for E. coli). The flow-injection system was applied to the on-line control of an E. coli cultivation.     

Rapid screening of multiple antibiotic residues in milk using disposable amperometric magnetosensors

Disposable amperometric magnetosensors, involving a mixture of modified-magnetic beads (MBs), for the multiplex screening of cephalosporins (CPHs), sulfonamides (SAs) and tetracyclines (TCs) antibiotic residues in milk are reported for the first time in this work. The multiplexed detection relies on the use of a mixture of target specific modified magnetic beads (MBs) and application of direct competitive assays using horseradish peroxidase (HRP)-labeled tracers. The amperometric responses measured at −0.20 V vs. the Ag pseudo-reference electrode of screen-printed carbon electrodes (SPCE) upon the addition of H₂O₂ in the presence of hydroquinone (HQ) as redox mediator, were used to monitor the extent of the different affinity reactions. The developed methodology, involving a simple and short pretreatment, allowed discrimination between no contaminated UHT and raw milk samples and samples containing antibiotic residues at the maximum residue limits (MRLs). The usefulness of the multiplexed magnetosensor was demonstrated by analyzing spiked milk samples in only 5 min. The results demonstrated that a clear discrimination of milk samples contaminated with antibiotics at their MRL level or their mixtures, allowing the identification of milk not complying with current legislation. These features make the developed methodology a promising alternative in the development of user-friendly devices for on-site analysis to ensure quality control for dairy products.     

Rapid screening of bioactive components from Zingiber cassumunar using elution-extrusion counter-current chromatography

Elution-extrusion counter-current chromatography (EECCC) takes full advantages of the liquid nature of the stationary phase. It effectively extends the solute hydrophobicity window that can be studied and renders the CCC technique particularly suitable for rapid analysis of complex samples. In this paper, EECCC was used to screen the crude ethanol extract of Zingiber cassumunar and to isolate milligram-amounts of bioactive components. The two column volume (2V(C)) EECCC method was applied to rapidly optimize the composition of the biphasic liquid system in both reversed- and normal-phase separation mode. With the n-hexane/ethyl acetate/methanol/water 1/1/1/1 (v/v) system, 100mg of crude Z. cassumunar extract were fractionated on a 140 mL-capacity semi-preparative hydrodynamic CCC column and 0.5 g on a 1600 mL column for large-scale preparation. Satisfactory separation efficiency was achieved in both cases, producing milligram-amounts of four phenylbutenoids over 90% pure and of a mixture of diastereoisomers (phenylbutenoid dimers). However, the global throughputs of the two columns were 8 and 11 mg/h, not very different. This is due to the fact that the 1600 mL column could not retain the liquid stationary phase as well as the smaller 140 mL column. It was necessary to work at much lower flow rate than calculated. Methanol was added as a post-column clarifying reagent for stable continuous UV detection. A lipophilic biphasic liquid system composed of n-hexane/acetonitrile/water (5/3/2, v/v) allowed to resolve the pair of diastereoisomers with the larger preparative instrument producing 35 mg of the (+/-)-trans form 99.1% pure and 28 mg of the (+/-)-cis isomer 98.1% pure. Compared with classical elution, the EECCC approach exhibits strong separation efficiency and great potential to be a high-throughput separation technique in the case of complex samples.     

Rapid screening for ethyl carbamate in stone-fruit spirits using FTIR spectroscopy and chemometrics

Ethyl carbamate (EC, urethane, C₂H₅OCONH₂) is a known genotoxic carcinogen of widespread occurrence in fermented food and beverages with the highest concentrations being found in stone-fruit spirits. Time-consuming procedures requiring extraction and gas chromatographic–mass spectrometric determination are regarded as reference procedures for the analysis of EC in alcoholic beverages. In this study, the rapid method of Fourier transform infrared (FTIR) spectroscopy in combination with partial least-squares (PLS) regression using selected wavelength bands is applied for the first time to the screening analysis of EC in stone fruit spirits (analysis time only 2 min). Apart from the actual content of EC in the sample, additional information was available from the FTIR spectra. This included data concerning the EC precursor hydrocyanic acid (HCN) and the maximum EC concentration which could be formed during storage. The PLS procedure was validated using an independent set of samples (Q² = 0.71–0.76, SEP = 0.42–0.67). The method was found to lack the accuracy required for a quantitative determination; it could only be used semi-quantitatively in the context of a screening analysis. If a rejection level of 0.8 mg L^–1 is applied as cut-off, overall correct classification rates of 85–91% for the calibration set and 77–85% for the validation set were achieved. False negative results can be avoided by lowering the cut-off to 0.6 mg L^–1. Through use of FTIR screening, 60–70% of all samples can be classified as negative and removed, leaving only conspicuous analysis results exceeding cut-off to be confirmed by complex and labour-intensive reference analyses.     

Rapid screening of alkylphenol exposure in fish bile using an enzymatic peroxidase biosensor

This article deals with the development and novel application of an amperometric peroxidase biosensor for monitoring fish exposure to petroleum-related discharges, in particular, to alkyl phenols (AP) using fish bile as the main sample material. The biosensor consisted of a screen-printed electrode coupled with peroxidase immobilized by glutaraldehyde cross-linking. The sensor was optimized with regards to factors such as immobilization procedures, substrate selectivity, and matrix effects. The biosensor was used for the analysis of fish bile samples from Atlantic cod (Gadus morhua L.) exposed in the laboratory during a 2 week period to different petroleum related compounds. The biosensor could distinguish between bile samples of fish exposed to water containing high concentrations (a mixture of C₄–C₇) to moderate levels (mainly C₀–C₅) of alkylphenols and that of the control group.     

Rapid and label-free screening of enzyme inhibitors using segmented flow electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) is an attractive analytical tool for high-throughput screening because of its rapid scan time and ability to detect compounds without need for labels. Impediments to the use of ESI-MS for screening have been the relatively large sample consumed and slow sample introduction rates associated with commonly used flow injection analysis. We have previously shown that by segmenting nanoliter plugs of sample with air, an array of discrete samples can be delivered to a platinum-coated emitter tip for ESI-MS analysis with throughput as high as 0.8 Hz and carry-over between samples less than 0.1%. This method was applied to screening for inhibitors of acetylcholinesterase as a demonstration of the potential of segmented flow ESI-MS for such applications. Each enzyme assay consumed 10 nL of sample. At 1 μL/min infusion rate, 102 samples were analyzed, corresponding to a 0.65 Hz sample analysis rate. Linear quantification of choline was achieved from 200 μM to 10 mM using this method and Z′ values were over 0.8 for the assay. Detailed pharmacologic dose-response curves of selected inhibitors were also measured in high-throughput fashion to validate the method.     

Rapid detection of dengue virus in serum using magnetic separation and fluorescence detection

A magnetophoretic fluorescence sensor (MFS) has been developed to rapidly detect dengue virus in serum at a sensitivity that was approximately three orders of magnitude higher than conventional solid phase immunoassays. UV inactivated type 2 dengue virus was first reacted with a mixture of superparamagnetic and fluorescent microparticles functionalised with an anti-type 2 dengue virus monoclonal antibody in 10% fetal calf serum. The magnetic particles were separated from the serum based on their magnetophoretic mobility, and dengue virus was detected by the co-localization of magnetic and fluorescent particles at a specific point in the flow chamber. The MFS was capable of detecting dengue-2 virus at 10 PFU ml(-1) with a reaction time of 15 min. The MFS demonstrated a high specificity in the presence of yellow fever virus, a closely related flavivirus, which also did not produce any detectable increase in background signal. The improved performance of this technique appears to result from the rapid kinetics of the microparticle reaction, improved signal-to-noise ratio resulting from magnetophoretic separation, and rapid fluorescent particle detection. These results suggest that the MFS may be useful in early stage diagnosis of dengue infections, as well as other diseases.     

Rapid screening and characterization of drug metabolites using a new quadrupole-linear ion trap mass spectrometer

The application of a new hybrid RF/DC quadrupole-linear ion trap mass spectrometer to support drug metabolism and pharmacokinetic studies is described. The instrument is based on a quadrupole ion path and is capable of conventional tandem mass spectrometry (MS/MS) as well as several high-sensitivity ion trap MS scans using the final quadrupole as a linear ion trap. Several pharmaceutical compounds, including trocade, remikiren and tolcapone, were used to evaluate the capabilities of the system with positive and negative turbo ionspray, using either information-dependent data acquisition (IDA) or targeted analysis for the screening, identification and quantification of metabolites. Owing to the MS/MS in-space configuration, quadrupole-like CID spectra with ion trap sensitivity can be obtained without the classical low mass cutoff of 3D ion traps. The system also has MS(3) capability which allows fragmentation cascades to be followed. The combination of constant neutral loss or precursor ion scan with the enhanced product ion scan was found to be very selective for identifying metabolites at the picogram level in very complex matrices. Owing to the very high cycle time and, depending on the mass range, up to eight different MS experiments could be performed simultaneously without compromising chromatographic performance. Targeted product ion analysis was found to be complementary to IDA, in particular for very low concentrations. Comparable sensitivity was found in enhanced product ion scan and selected reaction monitoring modes. The instrument is particularly suitable for both qualitative and quantitative analysis.     

Rapid screening for bisbibenzyls in bryophyte crude extracts using liquid chromatography/tandem mass spectrometry

A simple and rapid qualitative liquid chromatography-diode-array detection/tandem mass spectrometry (LC-DAD/MS/MS) method was developed and validated for screening bisbibenzyl compounds in bryophyte crude extracts at sub-ppm levels. After simple extraction with ethanol and analyte concentration with diethyl ether, the extracts were subjected to LC-DAD/MS/MS analysis. The overall instrument turnaround time was 50 min to obtain baseline separation of bisbibenzyl isomers in bryophytes. MS full scan, MS/MS precursor ion scan and MS/MS product ion scan modes were used for the screening. The bisbibenzyl standards studied gave limits of detection (LODs) at or below 10 ng/mL. The results also indicated that the method had acceptable precision to be used on a day-to-day basis for qualitative identification. The bisbibenzyl types, i.e. one biphenyl ether bond (A-type), two biphenyl ether bonds (B-type), one biphenyl ether and one biphenyl bond (C-type), or other biphenyl types can be differentiated by their ESI-MS/MS product profiles, and the number of alkoxyl substituents can also be identified. The linkage sites of biphenyl and biphenyl ether bonds cannot be identified for an unknown bisbibenzyl solely from its mass spectra. This system was used to support three screening assays of bryophytes including Marchantia polymorpha L., Ptagiochasm intermedium L. and Asterella angusta, which were collected from different places in China. From them, 7/12, 8/5 and 8/9 confirmed/unconfirmed bisbibenzyls were identified, respectively, based on their MS/MS data, UV spectra and the retention behavior. The screening method considerably reduced the time and the cost for the qualitative analyses, and the structure-fragmentation-UV relationships will facilitate the high-throughput screening (HTS) of bisbibenzyl compounds in bryophytes. It is also intended as a simple and convenient way for the determination of other structural families of natural products.     

Rapid on-site radionuclide screening of aqueous waste streams using dip-stick technologies and liquid scintillation counting

Journal of Radioanalytical and Nuclear Chemistry - This paper describes an early-stage evaluation of a purpose-designed extraction/detection system that can be deployed by non-specialists either...     

Rapid and efficient screening of adsorbent for oligopeptide using molecular docking and isothermal titration calorimetry

A rapid and efficient method based on molecular docking and isothermal titration calorimetry (ITC) was developed to identify effective adsorbents for the target peptide Ser‐Glu‐Ala‐Asp‐His (SEADH). Preliminary screening of five candidate adsorbents using molecular docking revealed that three suitable structures (A1, A2, and A3) either with or without coordination of Zn²⁺ should be effective. The three selected structures were then prepared and further screened by evaluating their affinities for the peptide SEADH using ITC. The screening results revealed that the adsorbent A2 coordinated with Zn²⁺ was the best adsorbent, and subsequent static adsorption experiments confirmed the reliability of the screening method. Further ITC analysis, combined with molecular docking, was performed to provide the possible adsorption mechanism.