Whatman Grade Research Articles

An ultrasensitive paper-based SERS sensor for detection of nucleolin using silver-nanostars, plastic antibodies and natural antibodies

A state-of-the-art, ultrasensitive, paper-based SERS sensor has been developed using silver nanostars (AgNSs) in combination with synthetic and natural antibodies. A key component of this innovative sensor is the plastic antibody, which was synthesized using molecularly imprinted polymer (MIP) technology. This ground-breaking combination of paper substrates/MIPs with AgNSs, which is similar to a sandwich immunoassay, is used for the first time with the aim of SERS detection and specifically targets nucleolin (NCL), a cancer biomarker. The sensor device was carefully fabricated by synthesizing a polyacrylamide-based MIP on cellulose paper (Whatman Grade 1 filter) by photopolymerization. The binding of NCL to the MIP was then confirmed by natural antibody binding using a sandwich assay for quantitative SERS analysis. To facilitate the detection of NCL, antibodies were pre-bound to AgNSs with a Raman tag so that the SERS signal could indicate the presence of NCL. The composition of the sensory layers/materials was meticulously optimized. The intensity of the Raman signal at ∼1078 cm−1 showed a linear trend that correlated with increasing concentrations of NCL, ranging from 0.1 to 1000 nmol L−1, with a limit of detection down to 0.068 nmol L−1 in human serum. The selectivity of the sensor was confirmed by testing its analytical response in the presence of cystatin C and lysozyme. The paper-based SERS detection system for NCL is characterized by its simplicity, sustainability, high sensitivity and stability and thus embodies essential properties for point-of-care applications. This approach is promising for expansion to other biomarkers in various fields, depending on the availability of synthetic and natural antibodies.

Fluid Flow Dynamics in Partially Saturated Paper.

This study presents an integrated approach to understanding fluid dynamics in Microfluidic Paper-Based Analytical Devices (µPADs), combining empirical investigations with advanced numerical modeling. Paper-based devices are recognized for their low cost, portability, and simplicity and are increasingly applied in health, environmental monitoring, and food quality analysis. However, challenges such as lack of flow control and the need for advanced detection methods have limited their widespread adoption. To address these challenges, our study introduces a novel numerical model that incorporates factors such as pore size, fiber orientation, and porosity, thus providing a comprehensive understanding of fluid dynamics across various saturation levels of paper. Empirical results focused on observing the wetted length in saturated paper substrates. The numerical model, integrating the Highly Simplified Marker and Cell (HSMAC) method and the High Order accuracy scheme Reducing Numerical Error Terms (HORNET) scheme, successfully predicts fluid flow in scenarios challenging for empirical observation, especially at high saturation levels. The model effectively mimicked the Lucas-Washburn relation for dry paper and demonstrated the increasing time requirement for fluid movement with rising saturation levels. It also accurately predicted faster fluid flow in Whatman Grade 4 filter paper compared with Grade 41 due to its larger pore size and forecasted an increased flow rate in the machine direction fiber orientation of Whatman Grade 4. These findings have significant implications for the design and application of µPADs, emphasizing the need for precise control of fluid flow and the consideration of substrate microstructural properties. The study's combination of empirical data and advanced numerical modeling marks a considerable advancement in paper-based microfluidics, offering robust frameworks for future development and optimization of paper-based assays.

Development of a biodegradable microfluidic paper-based device for blood-plasma separation integrated with non-enzymatic electrochemical detection of ascorbic acid

In the present article, we developed an electrochemical microfluidic paper-based device (EμPAD) for the non-enzymatic detection of Ascorbic Acid (AA) concentration in plasma using whole human blood. We combined LF1 blood plasma separation membrane and Whatman grade 1 filter paper to separate plasma from whole blood through wax printing. A screen-printed electrode (SPE) was modified with spherical-shaped MgFe2O4 nanomaterial (n-MgF) to improve the catalytic properties of SPE. The n-MgF was prepared via hydrothermal method, and its material phase and morphology were confirmed via XRD, FTIR, TEM, SEM, and AFM analysis. The fabricated n-MgF/SPE/EμPAD exhibited detection of AA ranging from 0 to 80 μM. The obtained value of the detection limit, limit of quantification, sensitivity, and response time are 2.44 μM, 8.135 μM, 5.71 × 10−3 mA μM−1 cm−2, and 10 s, respectively. Our developed n-MgF/SPE/EμPAD shows marginal interference with the common analytes present in plasma, such as uric acid, glutamic acid, glucose, urea, lactic acid, and their mixtures. Overall, our low-cost, portable device with its user-friendly design and efficient plasma separation capability offers a practical and effective solution for estimating AA concentration from whole human blood in a single step.

Modeling of Paper-Based Bi-Material Cantilever Actuator for Microfluidic Biosensors.

This research explores the dynamics of a fluidically loaded Bi-Material cantilever (B-MaC), a critical component of μPADs (microfluidic paper-based analytical devices) used in point-of-care diagnostics. Constructed from Scotch Tape and Whatman Grade 41 filter paper strips, the B-MaC's behavior under fluid imbibition is examined. A capillary fluid flow model is formulated for the B-MaC, adhering to the Lucas-Washburn (LW) equation, and supported by empirical data. This paper further investigates the stress-strain relationship to estimate the modulus of the B-MaC at various saturation levels and to predict the behavior of the fluidically loaded cantilever. The study shows that the Young's modulus of Whatman Grade 41 filter paper drastically decreases to approximately 20 MPa (about 7% of its dry-state value) upon full saturation. This significant decrease in flexural rigidity, in conjunction with the hygroexpansive strain and coefficient of hygroexpansion (empirically deduced to be 0.008), is essential in determining the B-MaC's deflection. The proposed moderate deflection formulation effectively predicts the B-MaC's behavior under fluidic loading, emphasizing the measurement of maximum (tip) deflection using interfacial boundary conditions for the B-MaC's wet and dry regions. This knowledge of tip deflection will prove instrumental in optimizing the design parameters of B-MaCs.

A controlled evaluation of filter paper use during staining of sputum smears for tuberculosis microscopy.

Background: Some sputum smear microscopy protocols recommend placing filter paper over sputum smears during staining for Mycobacterium tuberculosis (TB) . We found no published evidence assessing whether this is beneficial. We aimed to evaluate the effect of filter paper on sputum smear microscopy results. Methods: Sputum samples were collected from 30 patients with confirmed pulmonary TB and 4 healthy control participants. From each sputum sample, six smears (204 smears in total) were prepared for staining with Ziehl-Neelsen (ZN), auramine or viability staining with fluorescein diacetate (FDA). Half of the slides subjected to each staining protocol were randomly selected to have Whatman grade 3 filter paper placed over the dried smears prior to stain application and removed prior to stain washing. The counts of acid-fast bacilli (AFB) and precipitates per 100 high-power microscopy fields of view, and the proportion of smear that appeared to have been washed away were recorded. Statistical analysis used a linear regression model adjusted by staining technique with a random effects term to correct for between-sample variability. Results: The inclusion of filter paper in the staining protocol significantly decreased microscopy positivity independent of staining with ZN, auramine or FDA (p=0.01). Consistent with this finding, there were lower smear grades in slides stained using filter paper versus without (p=0.04), and filter paper use reduced AFB counts by 0.28 logarithms (95% confidence intervals, CI=0.018, 0.54, p=0.04) independent of staining technique. In all analyses, auramine was consistently more sensitive with higher AFB counts versus ZN (p=0.001), whereas FDA had lower sensitivity and lower AFB counts (p<0.0001). Filter paper use was not associated with the presence of any precipitate (p=0.5) or the probability of any smear washing away (p=0.6) during the staining process. Conclusions: Filter paper reduced the sensitivity of AFB microscopy and had no detectable beneficial effects so is not recommended.

Determination of histamine levels in fresh fish using Near Infrared (NIR) technology

Fresh fish and fish products are highly perishable food. Development of fast, secure and non- destructive technique for estimation the presence and quantity of components which are related with safety of food products is of great interest for science but also and for the industry sector. Above-mentioned idea is the aim of this research work, precisely, determination of histamine levels in fresh fish with the use of different near infrared handheld devices. Histamine is one of biogenic amines among putrescine, cadaverine, tyramine which are non-volatile amines produced post mortem and are formed from decarboxylation of specific free amino acids in fish or shellfish tissue. Reason why we choose to developing technique for evaluation of histamine levels is EU Regulation 2073/2005 requires the determination of histamine at three different levels: 100, 200 and 400 mg/kg. For reaching the goal we decided to use paper-based technology for collecting samples for evaluation using Whatman grade 2 qualitative filter papers. In the first phase for validation of NIR devices specifications, and for creation of calibration curve we used histamine solution with 0.5, 1, 2.5 and 5 %. Results showed regression of R2 = 0.69 which indicates that NIR devices can be promising tools for spotting histamine beside small molecular weight.

Portable Rapid Paper-Based Device for Analysis of Carbamates and Organophosphates in Water Samples

Traditionally, the analysis of contaminants in water samples is performed using sophisticated techniques, namely HPLC, GC, and MS, requiring qualified personnel and laboratory infrastructure. Such methods are not feasible for the detection of contaminants in the field. In contrast, paper-based portable devices have been demonstrated as versatile, sustainable, and cheap solutions for such purposes. Paper consists of a pressed sheet of entangled hydrophilic cellulose fibres with excellent porosity. Such features enable the capillarity motion of liquids and the immobilization of active substances on the surface.Pesticides are critical pollutants in regions where agricultural activity is intense and represent a serious health risk for rural workers and their communities. These chemicals have been applied in enormous quantities to increase crop production in many countries. Carbamates (CBs) and organophosphates (OPs) are employed as active ingredients in industrial formulations of insecticides. They are considered highly toxic due to their inhibition action in the cholinergic transmission found in the nervous system of living organisms.This work aims to present a low-cost and simple operational paper-based testing device for the fast detection of CBs and OPs in water samples based on Ellman’s method of acetylcholinesterase (AChE) inhibition. We have named the proposed device “dip-and-fold” due to its how-to-use procedure: first, the device’s bottom edge is dipped in the water sample. The liquid moves up by capillarity towards the detection zone, in which the analytes find the AChE previously immobilized on the paper. The top edge of the paper is covered with a thin parafilm layer to avoid leaking.After a 10-minute incubation, the AChE is inhibited in the presence of CBs and/or OPs. Then, the device is folded to release both enzymatic substrate acetylthiocholine iodide (ACThI) and indicator 5,5′-Dithiobis (2-nitrobenzoic acid) (DTNB) from a secondary paper to the detection zone for the colorimetric reaction. The detection zone area remains white in the presence of CBs and/or OPs (inhibited AChE); otherwise, it becomes yellow in the absence of these pesticides (active AChE).It is worthy to emphasize that the dip-and-fold has all required ingredients immobilized onto the paper substrates prior to tests, which excludes the necessity of extra reagents and materials. Besides, the sample can be transferred to the device by capillarity motion when dipping it directly into the contaminated water, eliminating any risk of having contact with the pesticides. Experiment The dip-and-fold device was fabricated using two analytical papers and a backing card as a foldable scaffold. Blotting paper (Whatman GB003) was used as the main device’s component for the sample loading by capillarity as well as for the immobilization of 3 µL AChE (100 U/mL), using chitosan 1% (w/v, in acetic acid 2%), as a crosslinker. The secondary component was the filter paper (Whatman grade 4) in which the 3µL of the substrate ACThI (25mM) and 3 µL of DTNB (10mM) were impregnated on the surface. The detection tests were carried out using standard solutions of carbofuran, propoxur (both carbamates) and chlorpyriphos (an organophosphate) at concentrations between 0.1 and 0.0001 mM. Control samples of DI water were also tested. A minimum volume of methanol was required to dissolve the pesticides in water. Yellow-coloured results of the tests were evaluated by taking digital photos of the devices using a smartphone. The RGB channels of images were analyzed using ImageJ software. Results and Conclusion The result interpretation was performed by comparing the plotted curves of the blue channel (B) values obtained from the images of each tested device for the different analytes and their respective concentrations. The evaluation of the plotted curves confirmed that the yellow colour's intensity is inversely proportional to the concentration of the analytes in the sample, which means, the more intense the yellow, the lower the concentration of pesticides in the sample. The concentrations applied in this study meet the parameters proposed by the Ontario Drinking Water Quality Standards (ODWQS) and may be compared to other paper-based devices described in the literature.Both propoxur and carbofuran presented consistent results that were observed by a steady decrease of the B values related to their concentration. In contrast, the chlorpyrifos presented a non-linear curve, which can be attributed to the higher amount of methanol used as a co-solvent to dissolve it in water due to its extremely poor solubility.The dip-and-fold device presented an exclusive, eco-friendly, and economical testing platform for pesticide detection in water, which offers an attractive solution for water assessment in vulnerable areas exposed to these toxic chemicals. Figure 1

Hybrid paper sheets with improved barrier properties.

Hybrid paper sheets were prepared by applying a thin coating layer of cross-linked polydimethylsiloxane (PDMS) and inorganic particles onto Whatman Grade 1 filter paper substrates. Several coatings with different inorganic particle contents and types were applied onto the paper substrates to investigate the effect of the variation in the coating formulation on the (i) wetting, (ii) water barrier properties, (iii) air barrier properties, (iv) surface roughness, and (v) mechanical properties of the samples. It was revealed that the superhydrophobic hybrid paper sheets with significantly low air permeability and high water barrier properties could be prepared which is an indication that the method proposed can be used for the preparation of packaging materials.

Hemp-Based Microfluidics.

Hemp is a sustainable, recyclable, and high-yield annual crop that can be used to produce textiles, plastics, composites, concrete, fibers, biofuels, bionutrients, and paper. The integration of microfluidic paper-based analytical devices (µPADs) with hemp paper can improve the environmental friendliness and high-throughputness of µPADs. However, there is a lack of sufficient scientific studies exploring the functionality, pros, and cons of hemp as a substrate for µPADs. Herein, we used a desktop pen plotter and commercial markers to pattern hydrophobic barriers on hemp paper, in a single step, in order to characterize the ability of markers to form water-resistant patterns on hemp. In addition, since a higher resolution results in densely packed, cost-effective devices with a minimized need for costly reagents, we examined the smallest and thinnest water-resistant patterns plottable on hemp-based papers. Furthermore, the wicking speed and distance of fluids with different viscosities on Whatman No. 1 and hemp papers were compared. Additionally, the wettability of hemp and Whatman grade 1 paper was compared by measuring their contact angles. Besides, the effects of various channel sizes, as well as the number of branches, on the wicking distance of the channeled hemp paper was studied. The governing equations for the wicking distance on channels with laser-cut and hydrophobic side boundaries are presented and were evaluated with our experimental data, elucidating the applicability of the modified Washburn equation for modeling the wicking distance of fluids on hemp paper-based microfluidic devices. Finally, we validated hemp paper as a substrate for the detection and analysis of the potassium concentration in artificial urine.

A practical method for storage, preservation and transportation of anuran urine samples using filter paper for hormone analysis

Anurans (frogs and toads) expelled urine when handled and it could provide insights into their physiological status. However, storage, preservation and transportation are often challenging. The study aimed to standardize and validate a field method for short-term storage and preserve of anuran urine samples using Whatman filter papers. To examine the efficacy of storage conditions and type of papers, urinary based enzyme immunoassays were used to measure progesterone and testosterone hormone metabolites. High-Performance Liquid Chromatography was performed and revealed immunoreactive progesterone and testosterone metabolites in the urine samples. Urinary hormone metabolites concentration stored in filter paper at room temperature and control samples stored in -20°C for the same period were similar. Whatman grade 50 was found to be more suitable for storage of hormones than grade 3 paper for the experiments performed.•A cheap and simple storage method for storage of anuran urine in field conditions using filter papers.•Anuran urine could be preserved and transported under ambient conditions without significant changes and loss of hormones.•This method would facilitate the endocrine monitoring of anurans in remote areas where limited logistics are available.

Two Nonnegligible Factors Influencing Lignocellulosic Biomass Valorization: Filtration Method after Pretreatment and Solid Loading during Enzymatic Hydrolysis

Valorization of lignocellulosic biomass into biofuels has received considerable attention for decades with tremendous thermochemical pretreatments proposed. However, inconsistencies among studies in lignin removal can often be misleading and even violate the chemical mechanism, which might be attributed to the variation in the filtration method used after pretreatment. A phenomenon, promoting pretreatment strategies based on sugar conversion efficiency or yield only from single and random low-solid enzymatic hydrolysis, was also observed. Herein, the effects of filtration methods using a slow drip metal filter (SDMF) with an aperture of 0.2 mm and Whatman grade 4 filter paper (WGFP) with an aperture of 20–25 μm under vacuum filtration after liquid hot water (LHW), acid, and alkali pretreatments on the chemical composition of the solid and liquid, sugar recoveries, and lignin removal and solid loading (2–15%) on enzymatic hydrolysis of corn stover and poplar wood were investigated. Results showed that SDMF increased the glucan content (1.1–3.0%) in pretreated biomass and induced solid loss (3.8–12.6%) into the filtrates, resulting in lower sugar recoveries and overestimated lignin removal compared to WGFP. 5-Hydroxymethylfurfural (HMF) and furfural derived from sugar degradation triggered pseudolignin formation, inducing a negative lignin removal for both LHW and acid pretreated biomass. In comparison, alkali pretreatment neutralized the weak acids released from sugar decomposition and achieved a robust delignification (67.8–90.8%). Solid loading used for enzymatic hydrolysis of pretreated biomass determined sugar concentration, conversion efficiency, and yield. This result indicates that promoting pretreatment technologies based on sugar conversion efficiency or yield of enzymatic hydrolysis at low solid loading may be misleading. This work demonstrates that the filtration method used for solid and liquid separations after pretreatment and solid loading for enzymatic hydrolysis should be cautiously selected and implemented when valorizing biomass into biofuels.

XPS and Raman Characterized Lime Stabilized Lead Contaminated Soil

Artificially lead (Pb) contaminated soil was stabilised with hydrated lime and the leaching behaviour of the element was investigated. A 5 grams’ sample of contaminated soil was thoroughly mixed with 0.1g of lime, and The mixture containing 50 ml of leaching solution was shaken for 2hours, and then the solution was filtered using a Whatman Grade 540. filter paper. The extent of lead leaching, with and without the addition of lime, was determined after adjusting the solution pH between (1-12). More than 90% of the lead was retained when the pH was 12.0. Results from the analysis of X-ray photoelectron spectroscopy and Raman, on the soil samples retained on the filter paper, showed the formation of lead sulphate, lead oxide, and lead carbonate. which is in agreement with the analysis of X-ray diffraction and FTIR measurements.

Feasibility test of cellulose filter for collection of sulfuric acid mists

Abstract Exploring affordable and reliable filters to sample sulfuric acid mists is of interest because the polytetrafluoroethylene filters recommended by NIOSH Method 7908 are relatively costly. In this study, the feasibility of hardened cellulose filters such as Whatman grade 540, 541, and 542 filters were investigated for sampling sulfuric acid mists. Thirty-minute collection efficiency tests showed low collection efficiency for Whatman grade 540 and 541 filters while Whatman grade 542 filters exhibited high collection efficiency because of its smaller pore size. Eight-hour collection efficiency tests for the concentration of ca. 5000 µg/m3 showed Whatman grade 542 filters retained 95% collection efficiency. The collection efficiency over eight hours neither increased nor decreased because the mists might have been absorbed or have formed a thin layer on the fiber and the filter structural integrity did not change. The higher collection efficiency was at the expense of a higher pressure drop; however, the pressure drop was still under the back pressure limit for most personal pumps. The integrity test showed that under the typical concentration in the occupational environment the hardened cellulose filters can maintain integrity for more than seven days. In summary, Whatman grade 542 filter is a suitable alternative for measuring sulfuric acid mists.

Glue for Manufacturing Heel Prick Filter Cards Does Not Interfere with the Measurement of Analytes for Newborn Screening

Population-based newborn screening (NBS) using blood collected and dried on filter paper was developed in the 1960s and remains the international standard for NBS programs. Glue, used in the manufacture of dried blood collection cards, may present a source of contamination and is often considered as a possible cause of anomalous results in routine screening. Our study evaluates this potential contamination on NBS analyses. EBF#1003 glue was blotted onto dried blood collection cards made of Whatman grade 903 filter paper (Whatman#903) and adult whole blood was pipetted onto the dried glue blots. In addition, blank glue blots (i.e., no blood) and dried blood spots (DBSs) in the absence of glue were prepared. The DBSs and blank samples were run in duplicate as routine samples for NBS. DBS absorption time and diameter, the effect of glue on measurements (concentrations and variation) were assessed. DBS absorption time and shape were equivalent for DBSs prepared in the absence and presence of undiluted glue. DBS diameter increased when prepared in the presence of glue. When EBF#1003 was diluted prior to use, DBS absorption time increased, and DBSs were non-uniform. Glue, diluted or not, did not produce measurements above the established Limit of Detection (LOD) for all assays used in the current Dutch screening programme. For all analytes with concentrations in the quantifiable range, contamination with glue had no effect on measurement variation, as it appeared equivalent to variation in untreated DBSs. Our data show that, in the unlikely event of contamination of Whatman#903 with EBF#1003, there is no effect on the measured concentration of analytes.

Rapid, Secure Drug Testing Using Fingerprint Development and Paper Spray Mass Spectrometry.

Paper spray mass spectrometry (PS-MS) is a technique that has recently emerged and has shown excellent analytical sensitivity to a number of drugs in blood. As an alternative to blood, fingerprints have been shown to provide a noninvasive and traceable sampling matrix. Our goal was to validate the use of fingerprint samples to detect cocaine use. Samples were collected on triangular pieces (168 mm2) of washed Whatman Grade I chromatography paper. Following application of internal standard, spray solvent and a voltage were applied to the paper before mass spectrometry detection. A fingerprint visualization step was incorporated into the analysis procedure by addition of silver nitrate solution and exposing the sample to ultraviolet light. Limits of detection for cocaine, benzoylecgonine, and methylecgonine were 1, 2, and 31 ng/mL respectively, with relative standard deviations < 33%. No matrix effects were observed. Analysis of 239 fingerprint samples yielded a 99% true-positive rate and a 2.5% false-positive rate, based on the detection of cocaine, benzoylecgonine, or methylecgonine with use of a single fingerprint. The method offers a qualitative and noninvasive screening test for cocaine use. The analysis method developed is rapid (4 min/sample) and requires no sample preparation.

Assessment of the RNASound RNA Sampling Card for the Preservation of Influenza Virus RNA

Shipping influenza virus specimens, isolates or purified RNA is normally conducted at ultra-low temperatures using dry ice to ensure minimal degradation of the samples but this is expensive and requires special packaging and shipping conditions. Therefore, alternative methods for shipping influenza viruses or RNA at ambient temperatures would be desirable. The RNASound RNA Sampling Card (FortiusBio LLC, San Diego, CA, USA) is a device that enables specimens or isolates to be applied to a card, whereby viruses are inactivated, while RNA is preserved and purified RNA can also easily be eluted. To evaluate this card, we applied influenza virus cell culture isolate supernatants to either the RNASound card or Whatman Grade No. 1 filter paper (GE Healthcare, Rydalmere, NSW, Australia) and compared the preservation to that of material stored in liquid form. Preservation was tested using influenza A and B viruses at two different storage temperatures [cool (2–8°C) or room temperature (18–22°C)] and these were compared with control material stored at -80°C, for 7, 14, or 28 days. The quality of the RNA recovered was assessed using real time RT-PCR and Sanger sequencing. The RNASound card was effective in preserving influenza RNA at room temperature for up to 28 days, with only a minor change in real-time RT-PCR cycle threshold values for selected gene targets when comparing between viruses applied to the card or stored at -80°C. Similar results were obtained with filter paper, whilst virus in liquid form performed the worst. Nevertheless, as the RNASound card also has the capability to inactivate viruses in addition to preserving RNA at room temperature for many weeks, this makes it feasible to send samples to laboratories using regular mail, and thus avoid the need for expensive shipping conditions requiring biohazard containers and dry ice. Moreover, the quick and simple RNA recovery from the RNASound card allows recipient labs to obtain RNA without the need for special reagents or equipment.

Evaluation of dried blood spot collection paper blotters for avian sexing by direct PCR

1. Avian sexing by polymerase chain reaction (PCR) plays an important role in sexual identification of avian species with similar phenotypes. Dried blood spots (DBSs) on paper blotters can help reduce the cost and problem of sample transportation and processing.2. In the first experiment, several kinds of papers were evaluated for collecting DBS for chicken sexing by direct PCR with different processing methods. The most practical method with cost optimality was the utilisation of Whatman grade 1 filter paper with the combination of methanol fixation and boiling.3. A second experiment was performed to determine whether cross-contamination could occur among samples cut with the same scissors. No-clean scissors were compared with ones cleaned with bleach-ethanol combination or 0.3N HCl. The PCR results showed that all three methods provided correct amplicon sizes without any false-positives regardless of the utilisation of cleaning intervention.4. In conclusion, a technique that is suitable for DBS collection for avian sexing by direct PCR with cost efficacy was developed, and it was also shown that the utilisation of the same pair of scissors for several DBS samples did not affect the PCR results.

预处理方式对香蒲和芦苇种子萌发的影响

Global changes and human activities have negative effects on the wetland habitats,which decrease the area of wetland,reduce species diversity,and hinder the ecological functions. Wetland restoration,especially dominant plants restoration is critical and urgent to improve wetland conditions and areas worldwide. As the largest tributary of the Heilong River,Songhua River drains nearly 70% of the Northeast China's land area. The riparian wetland along the downstream of Songhua River is suffering from ecological degradation of native vegetation. Typha domingensis(T. domingensis) and Phragmites australis(P. australis) are two dominant species in this basin,and play an important role in sustaining regional environmental functions. Seeds are produced annually,which would allow ample opportunities for colonization by sexual propagation. Seed propagation is considered to be an effective and feasible technique for vegetation restoration. In order to explore the optimal seed-soaking reagents to accelerate seed germination of T. domingensis and P. australis,the pure live seeds of both species were collected from a riparian wetland along the downstream of Songhua River(47°16'39.4.″ N,132°02'44. 2″ E),and the seeds germination experiment was conducted in incubator(LRH-250-GS II,China) with an alternating diurnal regime of 12 h of daylight at 25 ℃ and 12 h of darkness at 15 ℃ in laboratory. Effects of 8 different pretreatments(untreated,soaking with distilled water,soaking with 0.1% H2O2,soaking with 0.1% H2O2and then rinsing with distilled water,soaking with 0.1% KNO3,soaking with 0.1% KNO3and then rinsing with distilled water,soaking with0.1% KMnO4,soaking with 0.1% KMnO4and then rinsing with distilled water,respectively) on germination percentage(GP) and germination speed(GS) of T. domingensis and P. australis seeds are observed for 10 days. For each treatment,100 seeds(four replicates) were placed on a layer of Whatman grade No.1 filter paper(pH 7) in 90 mm Petri dishes with distilled water to keep moist during the whole experiment. Results showed as follows:(1) Differences between untreated seeds and pretreated ones with reagent soaking on GP and GS of T. domingensis and P. australis were significant;(2) GP and GS of T. domingensis under the pretreatment of soaking with 0.1% KMnO4and then rinsing with distilled water were significantly greater than that of the rest pretreatments,and the average GP of T. domingensis was about 3.1 times greater than the untreated and the GS was up to 16. 17 ± 0. 80;(3) Seed germination of P. australis performed best under the treatment of soaking with 0.1% concentration KNO3and rinsing with distilled water,and its GP and GS were up to 96%—99% and 28.43±0.71,respectively. The results indicate that seed pretreatment(seed-soaking reagent) is one of the most indispensable and efficient methods of accelerating the seed germination and seedling growth of T. domingensis and P.australis. Rapid and effective restoration of the two dominant plants in the riverine wetlands along the downstream of Songhua River,therefore,could be expected through collecting their pure live seeds and pretreating the seeds with 0.1%KMnO4and 0.1% KNO3and then rinsing with distilled water respectively before dispersal.

Storage and stability of IgG and IgM monoclonal antibodies dried on filter paper and utility in Neisseria meningitidis serotyping by Dot-blot ELISA

BackgroundA simple filter paper method was developed for, the transport and storage of monoclonal antibodies (Mabs) at room temperature or -20°C after spotting on filter paper, for subsequent serotyping of outer membrane antigens of N.meningitidis by dot-blot ELISA.MethodsMonoclonal antibodies (Mabs) were spotted within a 0.5–1 cm diameter area of Whatman grade 903 paper, which were stored individually at room temperature or at -20°C. These MAbs were stored and analyzed after periods of one week, 4 weeks, 12 months, or 13 years in the case of frozen Mab aliquots, or after 4 weeks at -20°C or at room temperature (RT) in the case of Mabs dried on filter paper strips. Assays were performed in parallel using dot-blot ELISA. In addition to the MAbs specific for serotyping class 1, 2 or 3, we used a larger number of Mabs for polysaccharides, lipooligosaccharides (LOS), class 5 and cross-reactive antigens for native outer membrane of N.meningitidis. The Mabs dried on filter paper were eluted with phosphate-buffered saline (PBS) containing 0.2% gelatin.ResultsMabs of the isotypes IgG and IgM dried on filter papers were not affected by duration of storage. The detection by serotyping Mabs was generally consistent for dried filter paper MAb samples stored frozen for over 1 year at -20°C, and although decreased reactive antibody titers were found after storage, this did not interfere with the specificity of the Mabs used after 13 years as dry spots on filter paper.ConclusionThe use of filter paper is an inexpensive and convenient method for collecting, storing, and transporting Mab samples for serotyping studies. In addition, the samples occupy little space and can be readily transported without freezing. The efficiency of using immunoglobulin G (IgG) or M (IgM) eluted was found to be consistent with measurement of IgG or IgM titers in most corresponding, ascites Mabs stored frozen for over 1 year. The application of meningococcal typing methods and designations depend on the question being asked.

A Viable Amino Acid Editing Activity in the Leucyl-tRNA Synthetase CP1-splicing Domain Is Not Required in the Yeast Mitochondria

Aminoacyl-tRNA synthetases are a family of enzymes that are responsible for translating the genetic code in the first step of protein synthesis. Some aminoacyl-tRNA synthetases have editing activities to clear their mistakes and enhance fidelity. Leucyl-tRNA synthetases have a hydrolytic active site that resides in a discrete amino acid editing domain called CP1. Mutational analysis within yeast mitochondrial leucyl-tRNA synthetase showed that the enzyme has maintained an editing active site that is competent for post-transfer editing of mischarged tRNA similar to other leucyl-tRNA synthetases. These mutations that altered or abolished leucyl-tRNA synthetase editing were introduced into complementation assays. Cell viability and mitochondrial function were largely unaffected in the presence of high levels of non-leucine amino acids. In contrast, these editing-defective mutations limited cell viability in Escherichia coli. It is possible that the yeast mitochondria have evolved to tolerate lower levels of fidelity in protein synthesis or have developed alternate mechanisms to enhance discrimination of leucine from non-cognate amino acids that can be misactivated by leucyl-tRNA synthetase.