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Novel ways to track and verify items of a high value or security is an ever-present need. Taggants made from deoxyribonucleic acid (DNA) have several advantageous properties, such as high information density and robust synthesis; however, existing methods require laboratory techniques to verify, limiting applications. Here, we leverage DNA nanotechnology to create DNA taggants that can be validated in the field in seconds to minutes with a simple equipment. The system is driven by toehold-mediated strand-displacement reactions where matching oligonucleotide sequences drive the generation of a fluorescent signal through the potential energy of base pairing. By pooling different "input" oligonucleotide sequences in a taggant and spatially separating "reporter" oligonucleotide sequences on a paper ticket, unique, sequence-driven patterns emerge for different taggant formulations. Algorithmically generated oligonucleotide sequences show no crosstalk and ink-embedded taggants maintain activity for at least 99 days at 60 °C (equivalent to nearly 2 years at room temperature). The resulting fluorescent signals can be analyzed by the eye or a smartphone when paired with a UV flashlight and filtered glasses.

Owing to its critical role in the development of female reproductive tissues and as a clinical biomarker, there is an urgent need to develop a rapid and cost-effective method to sensitively detect 17β-estradiol (E2). In this work, a folding aptasensor platform with microfluidic channels for the label-free electrochemical detection of E2 is described. The platform, designed with a delicate folding structure, integrating filter holes, microfluidic channels, reaction chambers, and a three-electrode system, is extremely easy to use. To increase the detection sensitivity and immobilize the aptamer, we synthesized a novel nanoassembly consisting of amine-functionalized single-walled carbon nanotube/new methylene blue/gold nanoparticles (AuNPs) and modified the working electrode with this nanoassembly. The calibration curve obtained from the experimental results exhibited a linear range between 10 pg mL-1 and 500 ng mL-1 (R2 = 0.993), and a detection limit of 5 pg mL-1 was achieved (S/N = 3). Furthermore, experiments to detect E2 in clinical serum were conducted, and the results were highly similar to those obtained using a large electrochemical luminescence apparatus. By integrating multiple functional components, adopting novel nanoassemblies, and using a folding structure, this paper-based platform not only has great potential as a simple and convenient integrated device for point-of-care testing of E2, but also as a portable, low-cost, and highly sensitive aptasensor platform capable of detecting many diagnostic biomarkers with the appropriate aptamers.

Several solution-based methods have recently been adapted for use in paper substrates for enzymatic amplification to increase the number of copies of DNA sequences. There is limited information available about the impact of a paper matrix on DNA amplification by enzymatic processes, and about how to optimize conditions to maximize yields. The work reported herein provides insights about the impact of physicochemical properties of a paper matrix, using nuclease-assisted amplification by exonuclease III and nicking endonuclease Nt. Bbv, and a quantum dot (QD) - based Forster Resonance Energy Transfer (FRET) assay to monitor the extent of amplification. The influence of several properties of paper on amplification efficiency and kinetics were investigated, such as surface adsorption of reactants, and pore size. Additional factors that impact amplification processes such as target length and the packing density of oligonucleotide probes on the nanoparticle surfaces were also studied. The work provides guidance for development of more efficient enzymatic target-recycling DNA amplification methods in paper substrates.

Hemoglobin A1c (HbA1c) is a standard biomarker to measure long-term average glucose concentration for diagnosis and monitoring of diabetes. Various methods have been reported for measuring HbA1c, however, portable and precise determination is still challenging. Herein, a new highly sensitive electrochemical nanobiosensor is developed for the specific determination of HbA1c. A nanocomposite of reduced graphene oxide (rGO) and gold with hierarchical architecture structure was electrochemically deposited on a cheap and flexible graphite sheet (GS) electrode. The nanocomposite increased the surface area, improved the electron transfer on the electrode surface and augmented the signal. It also provided a suitable substrate for linkage of thiolated DNA aptamer as a bioreceptor on the electrode surface by strong covalent bonding. The quantitative label free detection was carried out by differential pulse voltammetry (DPV) in a phosphate-buffered saline (PBS) solution containing redox probe Fe(CN)63−/4-. The detection is based on insulating the surface in presence of HbA1c and decreasing the current, which is directly related to the HbA1c concentration. The nanobiosensor demonstrated high sensitivity of 269.2 μA. cm−2, wide linear range of 1 nM–13.83 μM with a low detection limit of 1 nM. The biosensor was successfully used for measuring HbA1c in blood real sample. Furthermore, it is promising to use it as a part of a point of care device for low-invasive screening and management of diabetes.

Evasion of apoptosis is a hallmark of human cancer, and a desired endpoint of many anticancer agents is the induction of cell death. With the heterogeneity of cancer becoming increasingly apparent, to understand drug mechanisms of action and identify combination therapies in cell populations, the development of tools to assess drug effects at the single cell level is a necessity for future preclinical drug development. Herein we describe the development of pCasFSwitch, a genetically encoded reporter construct designed to identify cells undergoing caspase-3 mediated apoptosis, by a translocation of a GFP signal from the cell membrane into the nucleus. Anticipated cellular distribution was demonstrated by use of confocal microscopy and cleavage by caspase-3 was shown to be required for the translocation of the GFP signal seen in apoptotic cells. Quantification of apoptosis using the construct revealed similar levels to that obtained with a commercially available apoptosis imaging agent (22.6% versus 20.3%). Moreover, we demonstrated its capacity for use in a high-throughput setting making it a powerful tool for drug development pipelines.

Monodisperse aqueous upconverting nanoparticles (UCNPs) were covalently immobilized on aldehyde modified cellulose paper via reduction amination to develop a luminescence resonance energy transfer (LRET)-based nucleic acid hybridization assay. This first account of covalent immobilization of UCNPs on paper for a bioassay reports an optically responsive method that is sensitive, reproducible and robust. The immobilized UCNPs were decorated with oligonucleotide probes to capture HPRT1 housekeeping gene fragments, which in turn brought reporter conjugated quantum dots (QDs) in close proximity to the UCNPs for LRET. This sandwich assay could detect unlabeled oligonucleotide target, and had a limit of detection of 13 fmol and a dynamic range spanning nearly 3 orders of magnitude. The use of QDs, which are excellent LRET acceptors, demonstrated improved sensitivity, limit of detection, dynamic range and selectivity compared to similar assays that have used molecular fluorophores as acceptors. The selectivity of the assay was attributed to the decoration of the QDs with polyethylene glycol to eliminate non-specific adsorption. The kinetics of hybridization were determined to be diffusion limited and full signal development occurred within 3 min.

A strategy for aptamer-based biosensing termed linkage inversion assembled nano-aptasensors (LIANAs) is shown to be a generally applicable approach to the sensitive and specific detection of a target molecule in turn-on fluorescence solution-based and paper-based tests.

Controlled integration of features that enhance the analytical performance of a sensor chip is a challenging task in the development of paper sensors. A critical issue in the fabrication of low-cost biosensor chips is the activation of the device surface in a reliable and controllable manner compatible with large-scale production. Here, we report stable, well-adherent, and repeatable site-selective deposition of bioreactive amine functionalities and biorepellant polyethylene glycol-like (PEG) functionalities on paper sensors by aerosol-assisted, atmospheric-pressure, plasma-enhanced chemical vapor deposition. This approach requires only 20 s of deposition time, compared to previous reports on cellulose functionalization, which takes hours. A detailed analysis of the near-edge X-ray absorption fine structure (NEXAFS) and its sensitivity to the local electronic structure of the carbon and nitrogen functionalities. σ*, π*, and Rydberg transitions in C and N K-edges are presented. Application of the plasma-processed paper sensors in DNA detection is also demonstrated.

The development of simple fluorescent and colorimetric assays that enable point-of-care DNA and RNA detection has been a topic of significant research because of the utility of such assays in resource limited settings. The most common motifs utilize hybridization to a complementary detection strand coupled with a sensitive reporter molecule. Here, a paper-based colorimetric assay for DNA detection based on pyrrolidinyl peptide nucleic acid (acpcPNA)-induced nanoparticle aggregation is reported as an alternative to traditional colorimetric approaches. PNA probes are an attractive alternative to DNA and RNA probes because they are chemically and biologically stable, easily synthesized, and hybridize efficiently with the complementary DNA strands. The acpcPNA probe contains a single positive charge from the lysine at C-terminus and causes aggregation of citrate anion-stabilized silver nanoparticles (AgNPs) in the absence of complementary DNA. In the presence of target DNA, formation of the anionic DNA-acpcPNA duplex results in dispersion of the AgNPs as a result of electrostatic repulsion, giving rise to a detectable color change. Factors affecting the sensitivity and selectivity of this assay were investigated, including ionic strength, AgNP concentration, PNA concentration, and DNA strand mismatches. The method was used for screening of synthetic Middle East respiratory syndrome coronavirus (MERS-CoV), Mycobacterium tuberculosis (MTB), and human papillomavirus (HPV) DNA based on a colorimetric paper-based analytical device developed using the aforementioned principle. The oligonucleotide targets were detected by measuring the color change of AgNPs, giving detection limits of 1.53 (MERS-CoV), 1.27 (MTB), and 1.03 nM (HPV). The acpcPNA probe exhibited high selectivity for the complementary oligonucleotides over single-base-mismatch, two-base-mismatch, and noncomplementary DNA targets. The proposed paper-based colorimetric DNA sensor has potential to be an alternative approach for simple, rapid, sensitive, and selective DNA detection.

Microfluidic paper-based analytical devices (μPADs) fabricated by wax-printing are suitable platforms for the development of simple and affordable molecular diagnostic assays for infectious diseases, especially in resource-limited settings. Paper devices can be modified for biological assays by adding appropriate reagents to the test areas. For this purpose, the use of affinity immobilization strategies can be a good solution for bioactive paper fabrication. This paper describes a methodology to capture labeled-DNA strands and hybrids on paper via the anchoring of antibodies with a fusion protein that combines a family 3 carbohydrate binding module (CBM) from Clostridium thermocellum, with high affinity to cellulose, and the ZZ fragment of the staphyloccocal protein A, which recognizes IgG antibodies via their Fc portion. Antibodies immobilized via CBM-ZZ were able to capture appropriately labeled (biotin, fluorescein) DNA strands and DNA hybrids. The ability of an antibody specific to biotin to discriminate complementary from noncomplementary, biotin-labeled targets was demonstrated in both spot and microchannel assays. Hybridization was detected by fluorescence emission of the fluorescein-labeled DNA probe. The efficiency of the capture of labeled-DNA by antibodies immobilized on paper via the CBM-ZZ construct was significantly higher when compared with a physical adsorption method where antibodies were simply spotted on paper without the intermediation of other molecules. The experimental proof of concept of wax-printed μPADs functionalized with CBM-ZZ for DNA detection at room temperature presented in this study constitutes an important step toward the development of easy to use and affordable molecular diagnostic tests.

We demonstrate the hybridization-induced fluorescence detection of DNA on an origami-based paper analytical device (oPAD). The paper substrate was patterned by wax printing and controlled heating to construct hydrophilic channels and hydrophobic barriers in a three-dimensional fashion. A competitive assay was developed where the analyte, a single-stranded DNA (ssDNA), and a quencher-labeled ssDNA competed for hybridization with a fluorophore-labeled ssDNA probe. Upon hybridization of the analyte with the fluorophore-labeled ssDNA, a linear response of fluorescence vs. analyte concentration was observed with an extrapolated limit of detection < 5 nM and a sensitivity relative standard deviation as low as 3%. The oPAD setup was also tested against OR/AND logic gates, proving to be successful in both detection systems.

A multiplexed solid-phase nucleic acid hybridization assay on a paper-based platform is presented using multicolor immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET). The surface of paper was modified with imidazole groups to immobilize two types of QD-probe oligonucleotide conjugates that were assembled in solution. Green-emitting QDs (gQDs) and red-emitting QDs (rQDs) served as donors with Cy3 and Alexa Fluor 647 (A647) acceptors. The gQD/Cy3 FRET pair served as an internal standard, while the rQD/A647 FRET pair served as a detection channel, combining the control and analytical test zones in one physical location. Hybridization of dye-labeled oligonucleotide targets provided the proximity for FRET sensitized emission from the acceptor dyes, which served as an analytical signal. Hybridization assays in the multicolor format provided a limit of detection of 90 fmol and an upper limit of dynamic range of 3.5 pmol. The use of an array of detection zones was designed to provide improved analytical figures of merit compared to that which could be achieved on one type of array design in terms of relative concentration of multicolor QDs. The hybridization assays showed excellent resistance to nonspecific adsorption of oligonucleotides. Selectivity of the two-plex hybridization assay was demonstrated by single nucleotide polymorphism (SNP) detection at a contrast ratio of 50:1. Additionally, it is shown that the use of preformed QD-probe oligonucleotide conjugates and consideration of the relative number density of the two types of QD-probe conjugates in the two-color assay format is advantageous to maximize assay sensitivity and the upper limit of dynamic range.

A novel electrochemiluminescence (ECL) "sandwich" biosensor has been developed to detect cocaine. The sandwich biosensor was fabricated on the basis of the fact that a single aptamer could be split into two fragments and the two dissociated parts could form a folded, associated complex in the presence of targets. One of these (capture probe), which had hexane-thiol at its 5'-terminus, was immobilized on a gold electrode via thiol-gold binding. The other one (detection probe) was labeled with the ECL reagent tris(2,2'-bipyridyl)ruthenium(II)-doped silica nanoparticles (RuSiNPs) at its 3'-terminus. Owing to the weak interaction between the two fragments, the sensor exhibited a low ECL signal in the absence of cocaine. After the target cocaine had been added to the solution, it induced association of the two fragments and stabilized the associated complexes, leading to immobilization of RuSiNPs on the electrode surface, and the ECL detected on the electrode surface was enhanced. The enhanced ECL intensity was directly proportional to the logarithm of the cocaine concentration in the range from 1.0 × 10(-9) to 1.0 × 10(-11) mol/L, with a detection limit of 3.7 × 10(-12) mol/L. The biosensor was applied to detect trace amounts of cocaine on banknotes with satisfactory results.

The genetic and functional diversity of Bacillus and Bacillus-derived genera was analyzed in soil samples collected from three different fields near Century Paper Mill, Lal Kuan, Uttarakhand, India. Two had been subjected to concentrated and diluted effluent irrigation for the past 25 years and were designated as a concentrated effluent irrigated field (CEIF) and a dilute effluent irrigated field (DEIF), respectively. The field irrigated with fresh water was designated as a water irrigated field (WIF). Increase in pH, Na and Zn content and decrease in Fe content was observed due to effluent irrigation. The population count of Bacillus and Bacillus-derived was maximum in DEIF followed by WIF and CEIF. Variations in plant growth-promoting traits and extracellular enzymes were observed among the isolates from the three different field soils. Based on the amplified ribosomal DNA restriction analysis (ARDRA) with three restriction enzymes, all the selected 104 isolates were clustered into 14 groups. The sequencing of the representative isolates revealed that the majority belonged to the genus Bacillus, while three isolates belonged to Paenibacillus, Lysinibacillus and Orthinibacillus. There were a few species like Orthinibacillus contaminans, B. oleronius, B. safensis, B. methylotrophicus, B. stratosphericus, B. aryabhattai, B. asahii and B. bataviensis that were prevalent only in DEIF and CEIF but not in WIF field soil. The diversity index parameters like the Shannon Index, indices of species richness and species evenness based on biochemical profiling and ARDRA profiling revealed that Bacillaceae members in the fresh water irrigated field were diverse.

Foxing of paper is a deterioration phenomenon occurring in the form of brown-yellowish spots, the abiotic and/or biotic causes of which are not yet completely understood. Nevertheless, microbiological infection has been recognized that may contribute to paper damage and therefore it becomes important to know the taxonomic position and the degradative activity of the potential infectious biological agents which mostly are fungi, but also bacteria and yeasts. A cellulolytic bacterial strain isolated from a foxed paper sample exhibited morphological and physiological characteristics of the Bacillus genus. To study its taxonomic position, different identification methods were used: the Biolog system, the direct amplified polymorphic DNA-polymerase chain reaction analysis (DAPD-PCR) and the partial sequencing of the 16S rDNA gene. Biolog system and partial sequencing of 16S rDNA gene assigned the strain to the Paenibacillus polymyxa species. DAPD-PCR analysis indicated a high similarity with Bacillus circulans, by comparing the isolated strain with some closely related Bacillus species.

The purpose of this study was to assess the feasibility of establishing a library of ovarian cancer nucleic acids using paper matrix by: 1) confirming the stability of DNA stored on paper matrix over a prolonged period of time, 2) determining the amount of genetic material required for storage, and 3) establishing the stability of RNA. Tumor tissue from 66 patients with ovarian cancer was collected intraoperatively, frozen, and dissociated with collagenase and trypsin. A cell suspension was then prepared and spotted onto the paper. The numbers of cells that were stored on the paper were counted using a hemocytometer. The cell suspension was serially diluted and spotted on the paper matrix until the minimum cell number that can be stored and produce a PCR product was determined. PCR, STR genotyping and direct sequencing were performed on tissue stored on the paper matrix. FTA® paper was used as RNA template, and RT PCR converted the RNA to cDNA. Ten to 50 mg of ovarian cancer tissue was stored on FTA® paper. We stored 7 × 104cells on ISOcode® paper and 18 × 104cells on FTA® and obtained extractable DNA. PCR analysis on cards with DNA stored 18 months ago enabled us to establish the stability of DNA after storage. RNA was stable for 6 months when stored on FTA® cards. Since genetic material is extractable from the paper matrices after passage of time, it could be a suitable medium for the storage of genetic material in cancer tissue banks.

The aim of the present study was to subject the post-consumer waste, namely paper cups for vermicomposting along with cow dung in three different ratios for a period of 90–140 days employing Eisenia fetida. The post-consumer wastes are a menace in many developing countries including India. This waste was provided as feed for earthworms and was converted to vermicompost. Vermicompost prepared with paper cup waste was analyzed for their physicochemical properties. Based on the physicochemical properties, it was evident that the best manure is obtained from type A (paper cup/cow dung in the ratio 1:1) than type B (paper cup/cow dung in the ratio 1.5:0.5) and type C (paper cup/cow dung in the ratio 0.5:1.5). The results showed that earthworms accelerated the rate of mineralization and converted the wastes into compost with needed elements which could support the growth of crop plants. The predominant bacterial strains in the vermicompost were characterized biochemically as well as by 16S ribosomal RNA (rRNA) gene sequencing. The bacterial strains like Bacillus anthracis (KM289159), Bacillus endophyticus (KM289167), Bacillus funiculus (KM289165), Virigibacillius chiquenigi (KM289163), Bacillus thuringiensis (KM289164), Bacillus cereus (KM289160), Bacillus toyonensis (KM289161), Acinetobacter baumanni (KM289162), and Lactobacillus pantheries (KM289166) were isolated and identified from the final compost. The total protein content of E. fetida involved in vermicomposting was extracted, and the banding pattern was analyzed. During final stages of vermicomposting, it was observed that the earthworm did not act on the plastic material coated inside the paper cups and stagnated it around the rim of the tub. Further, the degradation of paper cup waste was confirmed by Fourier transform infrared spectroscopy analysis. Hence, vermicomposting was found to be an effective technology for the conversion of the paper cup waste material into a nutrient-rich manure, a value-added product.

Resin acids are the major toxicants in pulp and paper mill effluents (PPMEs), and they form pitch interfering with papermaking. Efficient and reliable resin acid removal is critically important to prevent toxicity discharge and ensure proper functioning of paper machines. Two resin-acid-degrading bacteria, Pseudomonas abietaniphila BKME-9 and Zoogloea resiniphila DhA-35, were tested in laboratory sequencing batch reactors (SBRs) for their ability to enhance resin acid removal by biomass from a full-scale biotreatment system treating PPMEs. Both bacteria enhanced resin acid removal but not removal of total organic carbon (TOC) by either pH-shocked or starved activated sludge. These two bacteria also increased resin acid removal when the sludge was given high concentration (200 μM) of resin acid. A most-probable-number polymerase chain reaction (MPN-PCR) assay showed that these two bacteria were initially not detectable (detection limit: 102 bacterial cells/ml) in the sludge community and were persistent after inoculation. Both bacteria did not substantially change the indigenous microbial community composition, as assayed by ribosomal intergenic spacer analysis (RISA). Our results suggest that it is feasible and potentially useful to enhance resin acid removal by bioaugmentation using resin-acid-degrading bacteria such as BKME-9 and DhA-35.

The majority of pulp and paper mills now biotreat their combined effluents using activated sludge. On the assumption that their wood-based effluents have negligible fixed N, and that activated-sludge microorganisms will not fix significant N, these mills routinely spend large amounts adding ammonia or urea to their aeration tanks (bioreactors) to permit normal biomass growth. N 2 fixation in seven Eastern Canadian pulp and paper mill effluent treatment systems was analyzed using acetylene reduction assays, quantitative nitrogenase ( nifH ) gene probing, and bacterial isolations. In situ N 2 fixation was undetectable in all seven bioreactors but was present in six associated primary clarifiers. One primary clarifier was studied in greater detail. Approximately 50% of all culturable cells in the clarifier contained nifH , of which >90% were Klebsiella strains. All primary-clarifier coliform bacteria growing on MacConkey agar were identified as klebsiellas, and all those probed contained nifH . In contrast, analysis of 48 random coliform isolates from other mill water system locations showed that only 24 (50%) possessed the nifH gene, and only 13 (27%) showed inducible N 2 -fixing activity. Thus, all the pulp and paper mill primary clarifiers tested appeared to be sites of active N 2 fixation (0.87 to 4.90 mg of N liter −1 day −1 ) and a microbial community strongly biased toward this activity. This may also explain why coliform bacteria, especially klebsiellas, are indigenous in pulp and paper mill water systems.

Aims—To study the distribution of Epstein-Barr virus (EBV) variants containing mutations in the latent membrane protein 1 (LMP-1) oncogene and promoter in EBV associated Hodgkin's disease and infectious mononucleosis compared with previous findings in asymptomatic EBV carriers. Methods—Sequence analysis of the EBV LMP-1 promoter and gene in isolates from Danish patients with Hodgkin's disease (n = 61) and infectious mononucleosis (n = 10). Results—Viruses (previously designated group D) that contain two mutations in the activating transcription factor/cAMP response element (ATF/CRE) in the LMP-1 promoter, which are known to decrease promoter activity greatly, were significantly less frequent in Hodgkin's disease than in both infectious mononucleosis (p = 0.0081) and asymptomatic EBV carriers (p = 0.0084). In some cases, the LMP-1 gene contained mutations in a recently identified cytotoxic T cell (CTL) epitope. Most viral isolates contained mutations shown to increase nuclear factor κB (NF-κB) activation and had one of two newly identified C-terminal activation regions 3 (CTAR-3) deleted. The exon 1 Xho-I restriction site in the LMP-1 gene could be lost through a range of different mutations. Conclusions—These findings indicate selection pressure against EBV strains with weak LMP-1 promoter activity in Hodgkin's disease and thus provide further strong circumstantial evidence for the pathogenic role of EBV (and LMP-1) in this disease. Mutation of the CTL epitope suggests immune selection of EBV strains. Many EBV isolates contain functionally important mutations in the LMP-1 gene. Loss of the Xho-I restriction site should not be used as a marker of specific LMP-1 variants.

Whatman 3MM paper was chemically modified to generate nickel-charged iminodiacetic acid paper (Ni2+-IDA paper). Bacteria were transformed with Escherichia coli expression plasmids coding for either unmodified proliferating cell nuclear antigen (PCNA) or PCNA containing a genetically engineered polyhistidine tract (his-tag) located at its NH2 terminus. They were then grown, induced, and lysed, and macromolecules were transferred to Ni2+-IDA paper. After exhaustive washing, his-tagged PCNA but not unmodified PCNA remained bound to the paper. Moreover, bound his-tagged PCNA was biochemically active in an in situ DNA synthesis assay with exogenous template-primer and purified calf thymus DNA polymerase delta (pol delta). Ni2+-IDA paper was used to identify a PCNA- point mutant that, relative to wild-type PCNA, promotes increased DNA synthesis by pol delta beyond a model abasic template site. In addition, metal-charged IDA paper promises to be generally useful for functional screening of cells expressing cloned proteins.

Four strains of filamentous bacteria were isolated from slimes collected in different paper mill factories. Morphological and physiological characterization of the isolates indicated an affiliation with the genus Sphaerotilus . However, while the physiological properties of the isolates were almost identical, pronounced physiological differences between the isolates and Sphaerotilus natans DSM 6575 T , DSM 565, and DSM 566 with respect to their ability to metabolize complex polysaccharides, sugars, polyalcohols, or organic acids as carbon sources were detected. In contrast to the analyzed culture collection strains of S. natans , all paper mill isolates were able to grow at elevated temperatures of up to 40°C. Comparative sequence analysis of nearly complete 16S ribosomal DNA (rDNA) sequences from the four new isolates demonstrated that the retrieved sequences were highly similar to each other (99.6 to 99.8% similarity) and to previously published partial 16S rDNA sequences of S. natans DSM 6575 T and ATCC 15291. Polyphasic characterization of the isolated Sphaerotilus strains revealed interesting adaptations of the strains to the environmental paper mill conditions with regard to temperature tolerance and utilization of cellulose and starch.

The collection of dried blood spots (DBS) on filter paper provides a powerful approach for the development of large-scale, population-based screening programs. DBS methods are particularly valuable in developing countries and isolated rural regions where resources are limited. Large numbers of field specimens can be economically collected and shipped to centralized reference laboratories for genetic and (or) serological analysis. Alternatively, the dried blood can be stored and used as an archival resource to rapidly establish the frequency and distribution of newly recognized mutations, confirm patient identity or track the origins and emergence of newly identified pathogens. In this report, we describe how PCR-based technologies are beginning to interface with international screening programmes for the diagnosis and genetic characterization of human immunodeficiency virus type 1 (HIV-1). In particular, we review recent progress using DBS specimens to resolve the HIV-1 infection status of neonates, monitor the genetic evolution of HIV-1 during early infancy and establish a sentinel surveillance system for the systematic monitoring of HIV-1 genetic variation in Asia.

A gene expressing xylanase activity was isolated from a genomic library of Thermotoga sp. strain FjSS3-B.1. The sequence of the gene shows that it encodes a single domain, family 10 xylanase. The recombinant enzyme has extremely high thermal stability, activity over a relatively broad pH range, and activity on Pinus radiata kraft pulp.

Two novel cellulase genes, cbh6A and egGH45, were cloned from Phialophora sp. G5 and successfully expressed in Pichia pastoris. The putative polypeptide of CBH6A consists of a family 1 CBM and a catalytic domain of glycosyl hydrolase family 6 cellobiohydrolases, while deduced EgGH45 only contains a catalytic domain of family 45 endoglucanases. CBH6A and EgGH45 were optimally active at pH 7.0 and 65°C, and pH 6.0 and 60°C, respectively. Both enzymes exhibited high activities and stabilities over a wide pH range and had good thermostability at 70°C. CBH6A and EgGH45 had significant resistance to SDS (10mM), remaining 35% and 54% activities, respectively. These enzymes had synergic effect on the hydrolysis of filter paper, showing the highest efficiency in the ratio of CBH6A to EgGH45 at 80:20. The properties make this enzyme combination potential for application in textile and detergents industries.

Human immunodeficiency virus type 1 (HIV-1) in cultured cells, peripheral blood samples and sera were adsorbed on filter paper disks and inactivated by heat or ethanol. Two procedures, the polymerase chain reaction (PCR) and microtiter plate assay (HMPA) were used to detect the nucleic acid. The sensitivity after different heat treatments with nested PCR for HIV-1 DNA (or nested reverse transcription-PCR for HIV-1 RNA) was identical regardless of whether the samples were examined immediately or one month later. Inactivation by ethanol treatment resulted in a slight loss of sensitivity. The HMPA proved to be as reliable and specific as the conventional PCR technique. We conclude that the heat-treated filter paper disk assay is suitable for identifying HIV nucleic acid in clinical samples sent to the laboratory from a distance, e.g. in an envelope.

A morphologically complex cellulosic substance (e.g., commercial filter paper) was employed as a substrate for DNA immobilization and successive recognition. A uniform ultrathin zirconia gel film was first deposited on each cellulose nanofiber in bulk filter paper by a facile sol-gel process. Relying on the large surface area of filter paper and the strong affinity of zirconia for the phosphate group, terminal-phosphate probe DNA was abundantly immobilized on the zirconia-modified filter paper so as to convert the composite to a biofunctional material for the sensitive and repetitive recognition of the corresponding complementary target DNA on the nanomolar level. By contrast, in spite of the viability of the immobilization of the probe DNA and the recognition of target DNA on the quartz plate, the amount of captured probe DNA or recognized target DNA on such a flat substrate was much less than that captured or recognized on filter paper, resulting in a relatively insensitive recognition event. Moreover, control experiments on bare filter paper (without a zirconia nanocoating) suggested that the zirconia gel film was essential to probe DNA immobilization and subsequent target DNA recognition.

White spot syndrome virus (WSSV) is an important shrimp pathogen responsible for large economic losses for the shrimp culture industry worldwide. The nucleic acids of the virus must be adequately preserved and transported from the field to the laboratory before molecular diagnostic analysis is performed. Here, we developed a new method to isolate WSSV-DNA using Flinders Technology Associates filter paper (FTA matrix card; Whatman) without centrifugation or hazardous steps involved. FTA technology is a new method allowing the simple collection, shipment and archiving of nucleic acids from haemolymph samples providing DNA protection against nucleases, oxidation, UV damage, microbial and fungal attack. DNA samples prepared from 10-fold dilutions of moribund shrimp haemolymph using FTA matrix cards were analysed using semi-quantitative and quantitative polymerase chain reaction (PCR) and were compared with two commercially available DNA isolation methods, the blood GenomicPrep Mini Spin Kit (GE Healthcare) and the DNAzol (Invitrogen). Sequence analysis was performed for the DNA samples prepared using the various isolation procedures and no differences in the sequence among these methods were identified. Results based on the initial copy number of DNA prepared from the GenomicPrep Mini Spin Kit are a little more sensitive than the DNA prepared from FTA matrix cards, whereas the DNAzol method is not suitable for blood samples. Our data shows the efficiency of retention capacity of WSSV-DNA samples from impregnated FTA matrix cards. Matrix cards were easy to store and ship for long periods of time. They provide ease of handling and are a reliable alternative for sample collection and for molecular detection and characterization of WSSV isolates.

The majority of bioassays utilize thermosensitive reagents (e.g., biomolecules) and laboratory conditions for analysis. The developing world, however, requires inexpensive, simple-to-perform tests that do not require refrigeration or access to highly trained technicians. To address this need, paper-based bioassays using gold nanoparticle (AuNP) colorimetric probes have been developed. In the two prototype DNase I and adenosine-sensing assays, blue (or black)-colored DNA-cross-linked AuNP aggregates were spotted on paper substrates. The addition of target DNase I (or adenosine) solution dissociated the gold aggregates into dispersed AuNPs, which generated an intense red color on paper within one minute. Both hydrophobic and (poly(vinyl alcohol)-coated) hydrophilic paper substrates were suitable for this biosensing platform; by contrast, uncoated hydrophilic paper caused "bleeding" and premature cessation of the assay due to surface drying. The assays are surprisingly thermally stable. During preparation, AuNP aggregate-coated papers can be dried at elevated temperatures (e.g., 90 degrees C) without significant loss of biosensing performance, which suggests the paper substrate protects AuNP aggregate probes from external nonspecific stimuli (e.g., heat). Moreover, the dried AuNP aggregate-coated papers can be stored for at least several weeks without loss of the biosensing function. The combination of paper substrates and AuNP colorimetric probes makes the final products inexpensive, low-volume, portable, disposable, and easy-to-use. We believe this simple, practical bioassay platform will be of interest for use in areas such as disease diagnostics, pathogen detection, and quality monitoring of food and water.

Denitrification is applied in the tertiary treatment of wastewater to reduce nitrogen pollution. Fluorescence in situ hybridization (FISH), catalyzed reporter deposition (CARD)-FISH, cloning, and scanning electron microscopy (SEM) were applied to follow the evolution of the microbial composition and structure of granular sludge in chemolithotrophic denitrifying bioreactors fed with nitrate and thiosulfate. FISH oligonucleotide probes for the chemolitoautotrophic denitrifiers Thiobacillus denitrificans and Thiomicrospira denitrificans were designed and their utility tested. CARD-FISH and cloning data showed that bacterial diversity in the biofilms changed during the reactor operation. Chemoorganotrophic fermentative Gram-positive strains in the phyla, Actinobacteria and Firmicutes, were dominant in the methanogenic inoculum, both in terms of biodiversity and in number. Other significant phyla were Bacteroidetes and Chloroflexi. After 6 months of operation, Proteobacteria became dominant (83% of the clones). The diversity of Gram-positive bacteria was partially maintained although their abundance decreased notably. After 110 d of operation, the abundance of Tb. denitrificans cells increased considerably, from 1% to 35% of total DAPI-stained cells and from no isolated clones to 15% of the total clones. Tm. denitrificans only represented a minor fraction of the microorganisms in the sludge (1-4% of the DAPI-stained cells). These findings confirm that Tb. denitrificans was the dominant chemolitoautotrophic denitrifying microorganism in the bioreactors. The Archaeal diversity remained almost unchanged and it was represented mostly by Methanosaeta soehngenii. SEM results indicated a considerable loss in the integrity of the sludge granules during the operation, with risk of sludge buoyancy.

The combination of Flinders Technology Associates filter papers (FTA® cards) and real-time PCR was examined to establish a simple and rapid technique for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) from whole pig blood. A modified live PRRS vaccine was diluted with either sterilised saline or pig whole blood, and the suspensions were applied onto the FTA® cards. The real-time RT-PCR detection of PRRSV was performed directly with the samples applied to the FTA® card without the RNA extraction step. Six whole blood samples from at random selected piglets in the PRRSV infected farm were also assayed in this study. The expected PCR product was successfully amplified from either saline diluted or pig whole blood diluted vaccine. The same PCR ampliocon was detected from all blood samples assayed in this study. This study suggested that the combination of an FTA® card and real-time PCR is a rapid and easy technique for the detection of PRRSV. This technique can remarkably shorten the time required for PRRSV detection from whole blood and makes the procedure much easier.

This study evaluates the feasibility of the use of the FTA® Gene Guard System (a commercial product consisting of filter paper impregnated with patented chemicals supplied by the Whatman company) for the shipment, storage and detection of RNA rabies viruses by a simplified hemi-nested reverse transcriptase polymerase chain reaction. HnRT-PCR of the rabies virus nucleoprotein gene with specific primers showed that viral RNA extracted from crude infected tissues remained stable after fixation on the filter paper under diverse environmental conditions for at least 35 days. The sequence analysis of the products amplified from five out of the seven known genotypes of Lyssaviruses showed the stability of viral RNA viruses after fixation on the filter paper. Furthermore, the sensitivity of the hnRT-PCR following RNA fixation on the filter paper was equivalent to that of standard hnRT-PCR. In conclusion, the stability of viral RNA and the inactivation of infectivity make the FTA® technology useful for the storage, transport, collection and subsequent molecular analysis of viral rabies RNA, facilitating epidemiological investigations in the field.

We recently described a method for digitally labelling objects with DNA. Here we show that, using DNA methyltransferases to create polymorphic DNA templates, it is possible to significantly increase the number of labels that can be generated by this method. Nine double-stranded DNA templates of different length were methylated with either M.HaeIII or M.AluI methyltransferase, or both. Different mixtures of methylated and unmethylated versions of this template set were used to 'invisibly' label paper. The mixtures were eluted from the paper and the methylated status of the templates in each mixture successfully determined, and the labels read, by digestion with the complementary restriction endonuclease, followed by a polymerase chain reaction and agarose gel electrophoresis. One methylated DNA label was read after it had been left on paper for two months.

The family 9 cellulase gene celI of Clostridium thermocellum , was previously cloned, expressed, and characterized (G. P. Hazlewood, K. Davidson, J. I. Laurie, N. S. Huskisson, and H. J. Gilbert, J. Gen. Microbiol. 139:307-316, 1993). We have recloned and sequenced the entire celI gene and found that the published sequence contained a 53-bp deletion that generated a frameshift mutation, resulting in a truncated and modified C-terminal segment of the protein. The enzymatic properties of the wild-type protein were characterized and found to conform to those of other family 9 glycoside hydrolases with a so-called theme B architecture, where the catalytic module is fused to a family 3c carbohydrate-binding module (CBM3c); CelI also contains a C-terminal CBM3b. The intact recombinant CelI exhibited high levels of activity on all cellulosic substrates tested, with pH and temperature optima of 5.5 and 70°C, respectively, using carboxymethylcellulose as a substrate. Native CelI was capable of solubilizing filter paper, and the distribution of reducing sugar between the soluble and insoluble fractions suggests that the enzyme acts as a processive cellulase. A truncated form of the enzyme, lacking the C terminal CBM3b, failed to bind to crystalline cellulose and displayed reduced activity toward insoluble substrates. A truncated form of the enzyme, in which both the cellulose-binding CBM3b and the fused CBM3c were removed, failed to exhibit significant levels of activity on any of the substrates examined. This study underscores the general nature of this type of enzymatic theme, whereby the fused CBM3c plays a critical accessory role for the family 9 catalytic domain and changes its character to facilitate processive cleavage of recalcitrant cellulose substrates.

Aim—To test the hypothesis that, because the human papillomavirus (HPV) E2 protein represses viral early gene transcription, E2 gene sequence variation or disruption could play a part in the induction of the numerical chromosome abnormalities that have been described in squamous cervical lesions. Methods—The integrity and sequence of the E2 gene from 11 cervical intraepithelial neoplasia (CIN) grade 3 lesions and 14 invasive squamous cell carcinomas, all of which contained HPV-16, were analysed by the polymerase chain reaction (PCR). The E2 gene was amplified in three overlapping fragments and PCR products sequenced directly. Chromosome abnormalities were identified by interphase cytogenetics using chromosome specific probes for chromosomes 1, 3, 11, 17, 18, and X. Results—E2 gene disruption was present in significantly more invasive carcinomas (eight of 14) than CIN 3 lesions (one of 11) (p = 0.03). No association was found between E2 disruption and the presence of a numerical chromosome abnormality. The E2 gene from the non-disrupted isolates was sequenced and wild-type (n = 5) and variant (n = 11) sequences identified. Variant sequences belonged to European and African classes and contained from one to 15 amino acid substitutions. Although numerical chromosome abnormalities were significantly more frequent in invasive squamous cell carcinoma than CIN 3 (p = 0.04), there was no significant relation between the presence of sequence variation and either histological diagnosis or chromosome abnormality. Conclusions—These data do not support the hypothesis that E2 gene disruption or variation is important in the induction of chromosome imbalance in these lesions. However, there is a relation between E2 gene disruption and the presence of invasive disease.

Background—Two mouse monoclonal antibodies (mAbs) have been described recently; namely, mAb 2C7 (IgG2bκ), which is directed against the major house dust mite allergen Der p 1, and mAb 2G10 (IgG1κ), which is an anti-idiotypic antibody raised against mAb 2C7. The anti-idiotype mAb 2G10 does not block the binding of mAb 2C7 to Der p 1, which means that mAb 2C7 can simultaneously bind to Der p 1 and to mAb 2G10, thereby generating a trimolecular complex consisting of antigen–idiotype–anti-idiotype. Aims—To sequence and model the V region of the anti-idiotypic antibody mAb 2G10 to enable the prediction of the interacting surfaces in the trimolecular complex consisting of Der p 1–mAb 2C7–mAb 2G10. Methods—DNA sequencing of mAb 2G10 was carried out and the Swiss Model and Swiss PDB-Viewer programs were used to build a three dimensional model of the trimolecular complex. Results—Complementarity of shape and charge was revealed when comparing the protrusion of the previously determined Der p 1 epitope (Leu147–Gln160) with the cavity formed by the complementarity determining regions (CDRs) of mAb 2C7. Such complementarity was also observed between the mAb 2C7 epitope predicted to be recognised by mAb 2G10 (residues Lys19 from framework region 1 (FRW1) and Ser74–Gln81 from FRW3) and residues from the CDRs of mAb 2G10 (a negatively charged patch flanked by the residues Asp55H/Glu58H and Glu27L/Glu27cL). As expected, the location of the mAb 2C7 epitope recognised by mAb 2G10 does not appear to interfere with the binding of Der p 1 to mAb 2C7. Conclusion—Although the results obtained represent only an approximation, they nevertheless provide a rare insight into how an antigen (Der p 1) might bind to its antibody (mAb 2C7) while in complex with an anti-idiotype (mAb 2G10).

The gene ce1A, encoding an endoglucanase from the strain Bacillus sp. BP-23, was cloned and expressed in Escherichia coli. The nucleotide sequence of a 1867-bp DNA fragment containing the ce1A gene was determined, revealing an open reading frame of 1200 nucleotides that encodes a protein of 44,803 Da. The deduced amino acid sequence of the encoded enzyme shows high homology to those of enzymes belonging to subtype 4 of the family-A cellulases. The ce1A gene product synthesized in E. coli showed activity on carboxymethylcellulose and lichenan but no activity was found on Avicel. Activity was enhanced in the presence of 10 mM Mg2+ and Ca2+ and showed its maximum at 40 degrees C and pH 4.0. Study of the performance of Ce1A on paper manufacture from agricultural fibres showed that treatment with the enzyme improved the properties of the pulp and the quality of paper. Ce1A treatment enhanced the physical properties (stretch and tensile index) of paper from wheat straw, while dewatering properties were slightly diminished. Electron-microscope analysis showed that the surface of straw fibres was modified by Ce1A.

Escherichia coli KO11 was previously constructed to produce ethanol from acid hydrolysates of hemicellulose (pentoses and hexoses) by the chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB). Klebsiella oxytoca P2 was constructed in an analogous fashion for the simultaneous saccharification and fermentation of cellulose and contains PTS enzymes for cellobiose. In this study, KO11 was further engineered for the fermentation of cellulose by adding the K. oxytoca casAB genes encoding Enzyme IIcellobiose and phospho-beta-glucosidase. Although the two K. oxytoca genes were well expressed in cloning hosts such as DH5 alpha, both were expressed poorly in E. coli KO11, a derivative of E. coli B. Spontaneous mutants which exhibited more than 15-fold-higher specific activities for cellobiose metabolism were isolated. The mutations of these mutants resided in the plasmid rather than the host. Three mutants were characterized by sequence analysis. All contained similar internal deletions which eliminated the casAB promoter and operator regions and placed the lacZ Shine-Dalgarno region immediately upstream from the casA Shine-Dalgarno region. KO11 harboring mutant plasmids (pLOI1908, pLOI1909, or pLOI1910) rapidly fermented cellobiose to ethanol, and the yield was more than 90% of the theoretical yield. Two of these strains were used with commercial cellulase to ferment mixed-waste office paper to ethanol.

A thermophilic xylanase from Bacillus strain D3 suitable for use as a bleach booster in the paper pulping industry has been identified and characterized. The enzyme is suited to the high temperature and alkaline conditions needed for using xylanases in the pulp industry. The xylanase is stable at 60 degrees C and relatively stable at high temperatures, with a temperature optimum of 75 degrees C. The pH optimum is 6, but the enzyme is active over a broad pH range. The xylanase has been cloned and sequenced, and the crystal structure has been determined. The structure of Bacillus D3 xylanase reveals an unusual feature of surface aromatic residues, which form clusters or "sticky patches" between pairs of molecules. These "sticky patches" on the surface of the enzyme are responsible for the tendency of the protein to aggregate at high concentrations in the absence of reagents such as ethylene glycol. The formation of dimers and higher order polymers via these hydrophobic contacts may also contribute to the thermostability of this xylanase.

The sequence-specific detection of DNA hybridization has attracted considerable interest in numerous fields. Although traditional DNA biosensors have been widely explored due to their high sensitivity, it is still challenging to develop a low-cost, portable, disposable, fast, and easy-to-use DNA detection method for public use at home or in the field. To address these challenges, herein, we report a novel microfluidic photoelectrochemical (PEC) paper-based analytical platform, integrated with an internal chemiluminescent light source, a novel paper supercapacitor (PS) amplifier, and a terminal digital multi-meter (DMM) detector, for sensitive DNA detection using a graphene-modified porous Au-paper electrode as the working electrode to obtain enhanced PEC responses. The quantification mechanism of this strategy is based on the charging of this PS, which was constructed on a paper-based analytical platform through a simple "drawing and soaking" method, by the generated photocurrent. After a fixed period, the PS was automatically shorted under the control of a novel built-in fluidic delay-switch to output an instantaneously amplified current, which could be sensitively detected by the DMM. At optimal conditions, this paper-based analytical platform can detect DNA at concentrations at femtomolar level. This approach also shows excellent specificity toward single nucleotide mismatches.

This study evaluated an enzyme immunoassay (EIA) as a screening tool for detection of the most common mutation (delta F508) in cystic fibrosis (CF). Guthrie card bloodspots were extracted to remove whole blood polymerase chain reaction (PCR) inhibitors. The washed filter paper was directly amplified under standard (98 bp amplicons) or modified PCR conditions (491 bp amplicons) for the delta F508 mutation. Monoclonal anti-double stranded deoxyribonucleic acid antibody was used to detect competent hybrid formation between PCR products and normal (N) and mutant (M) cDNA (deoxyribonucleic acid) probes coated to microtiter plate wells. Under standard conditions, mean relative color production (N/M) via an enzyme-linked horseradish peroxidase secondary antibody was 8.8, 0.6 and 0.04 for individuals normal, heterozygous and homozygous for the CF delta F508 mutation, respectively (n = 27). Comparison of EIA results to those obtained by tris-borate-EDTA/8 percent polyacrylamide gel electrophoresis (TBE-PAGE) yielded excellent correlation (100 percent) for all three genotypes (n = 27). In comparison to TBE-PAGE, the EIA was 5 to 10 fold more sensitive when serially diluted PCR samples were evaluated. Larger PCR products (491 bp amplicons) for the CF delta F508 mutation obtained under modified conditions were not resolved by TBE-PAGE. The EIA, however, demonstrated equal sensitivity to the 98 bp and 491 bp amplicons. Performance time for TBE-PAGE analysis was substantially shorter (25 percent) than the EIA (3.5 to 4 h and 4.5 to 5 h, respectively) when small batches of samples (n = 5) were analyzed. The TBE-PAGE was not, however, convenient for screening large numbers of PCR-amplified samples (n15).

A method for detection of proviral human immunodeficiency virus DNA in dried blood spots on filter paper by direct polymerase chain reaction (PCR) has been developed. To develop the method, a standard system was used which was prepared from cells each containing a single integrated provirus and titrated with normal donor blood. This rapid procedure provides virtually quantitative yields of nuclear DNA and exploits most of the standard methodology described for blood specimens. A nested PCR using SK38-SK39 gag as the internal primer pair was also designed; this PCR detected a single copy of provirus per filter at near theoretical frequency with SK19 probe. The utility of the procedure was demonstrated with clinical specimens. Blood spot filters from human immunodeficiency virus-infected and uninfected individuals were readily and unequivocally discriminated. The method is designed for ultimate use with large (1.5-ml) sample preparation tubes that are compatible as PCR tubes with thermal cyclers. This will permit convenient, direct single-tube PCR of dried blood specimens on filters. It should be adaptable to analysis of dried blood spots for a variety of infectious or genetic diseases.

Paper strips containing DNA-conjugated microgels (MG) are used to achieve sensitive DNA detection in three steps: target DNA promoted ligation of a DNA primer to the MG-bound DNA, rolling circle amplification (RCA) between the primer and a circle DNA, and hybridization of the RCA products and a fluorescent DNA probe.

Kinetoplast DNA (kDNA) has been isolated from the human cutaneous Leishmania isolates, L. tropica major, L. aethiopica and an unknown Kenyan isolate, Leishmania SP48. DNA sequence relationships among these isolates have been studied by restriction enzyme digestion and two phase hybridisation to Southern blots of kDNA covalently coupled to diazobenzyloxymethyl (DBM) paper. The results of this analysis confirm that rapid kDNA sequence evolution is occurring in the Old World leishmanias although some sequence conservation in defined regions of the mini-circle sequence is present. These results emphasise the danger of constructing a rigid Leishmania classification on buoyant density data alone. The covalent binding of kDNA electrophoretic separations to DBM paper permits the construction of a DNA sequence "library' which can be used in the classification and diagnosis of unknown Leishmania isolates.

Yeast ENO1 promoter was prepared by a chemical synthetic method. Two variant promoters containing a pyrimidine-rich region (CT block), located between the TATA box and the transcription start site, either 32 or 34 base pairs (bp) longer than the native ENO1 promoter were isolated during the chemical synthesis. Gene expression of variant promoters was compared with that of the native promoter by measuring the amount of mRNA and the activity of beta-galactosidase by constructing ENO1-lacZ gene fusions. No significant differences were observed between the native and variant promoters in transcription levels. The start site of transcription was mapped on CAAG, a consensus sequence of transcription start site of yeast glycolytic genes. The results suggest a longer CT block in ENO1 promoter may not affect the expression of the yeast ENO1 gene. In addition, the level of ENO1 gene expression was found to be higher in stationary phase cells than in log phase cells.