Silk–PVA Hybrid Nanofibrous Scaffolds for Enhanced Primary Human Meniscal Cell Proliferation

In this study, silk fibroin nanofibrous scaffolds were developed to investigate the attachment and proliferation of primary human meniscal cells. Silk fibroin (SF)–polyvinyl alcohol (PVA) blended electrospun nanofibrous scaffolds with different blend ratios (2:1, 3:1, and 4:1) were prepared. Morphology of the scaffolds was characterized using atomic force microscopy (AFM). The hybrid nanofibrous mats were crosslinked using 25 % (v/v) glutaraldehyde vapor. In degradation study, the crosslinked nanofiber showed slow degradation of 20 % on weight after 35 days of incubation in simulated body fluid (SBF). The scaffolds were characterized with suitable techniques for its functional groups, porosity, and swelling ratio. Among the nanofibers, 3:1 SF:PVA blend showed uniform morphology and fiber diameter. The blended scaffolds had fluid uptake and swelling ratio of 80 % and 458 ± 21 %, respectively. Primary meniscal cells isolated from surgical debris after meniscectomy were subcultured and seeded onto these hybrid nanofibrous scaffolds. Meniscal cell attachment studies confirmed that 3:1 SF:PVA nanofibrous scaffolds supported better cell attachment and growth. The DNA and collagen content increased significantly with 3:1 SF:PVA. These results clearly indicate that a blend of SF:PVA at 3:1 ratio is suitable for meniscus cell proliferation when compared to pure SF-PVA nanofibers.     

Fabrication of conducting electrospun nanofibers scaffold for three-dimensional cells culture

Electrospinning is a versatile method to fabricate nanofibers of a range of polymeric and composite materials suitable as scaffolds for tissue engineering applications. In this study, we report the fabrication and characterization of polyaniline-carbon nanotube/poly(N-isopropyl acrylamide-co-methacrylic acid) (PANI-CNT/PNIPAm-co-MAA) composite nanofibers and PNIPAm-co-MAA nanofibers suitable as a three-dimensional (3D) conducting smart tissue scaffold using electrospinning. The chemical structure of the resulting nanofibers was characterized with FTIR and (1)H NMR spectroscopy. The surface morphology and average diameter of the nanofibers were observed by SEM. Cellular response of the nanofibers was studied with mice L929 fibroblasts. Cell viability was checked on 7th day of cell culture by double staining the cells with calcein-AM and PI dye. PANI-CNT/PNIPAm-co-MAA composite nanofibers were shown the highest cell growth and cell viability as compared to PNIPAm-co-MAA nanofibers. Cell viability in the composite nanofibers was obtained in order of 98% that indicates the composite nanofibers provide a better environment as a 3D scaffold for the cell proliferation and attachment suitable for tissue engineering applications.     

Specific adhesion of vesicles monitored by scanning force microscopy and quartz crystal microbalance

The specific adhesion of unilamellar vesicles with an average diameter of 100 nm on functionalized surfaces mediated by molecular recognition was investigated in detail. Two complementary techniques, scanning force microscopy (SFM) and quartz crystal microbalance (QCM) were used to study adhesion of liposomes consisting of 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine and varying concentrations of N-((6-biotinoyl)amino)hexanoyl)-1, 2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (biotin-X-DHPE). Monitoring the adhesion of the receptor-doped vesicles to avidin-coated gold surfaces by QCM (f(0) = 5 MHz) revealed an increased shift in resonance frequency with increasing biotin concentration up to 10 mol% biotin-X-DHPE. To address the question of how the morphology of the liposomes changes upon adhesion and how that contributes to the resonator's frequency response, we performed a detailed analysis of the liposome morphology by SFM. We found that, with increasing biotin-concentration, the height of the liposomes decreases considerably up to the point where vesicle rupture occurs. Thus, we conclude that the unexpected high frequency shifts of the quartz crystal (>500 Hz) can be attributed to a firm attachment of the spread bilayers, in which the number of contacts is responsible for the signal. These findings are compared with one of our recent studies on cell adhesion monitored by QCM.     

Studies on the biotin flow at the duodenum of dairy cows fed diets with different concentrate levels and types of forages

Abstract

Biotin is involved in many vital metabolic pathways and must be provided for an efficient fermentation in the rumen, as well as for the intermediary metabolism of the host animal. Factors influencing ruminal biotin metabolism and output are widely unknown at present. Therefore, dairy cows fitted with permanent cannulas in the dorsal rumen and in the proximal duodenum were fed differently composed diets, and the biotin flow at the proximal duodenum was measured. The diets (on DM basis) consisted of 8.9 kg grass hay (Diet 1), 8.9 kg corn silage plus 2.0 kg concentrate (Diet 2), or 7.3 and 7.4 kg grass silage plus 10.0 kg concentrate (Diets 3 and 4). The concentrate in Diets 3 and 4 contained 87% wheat and corn grain, respectively. The cows were pre-fed the rations for 21 days. Thereafter duodenal digesta was sampled every two h for 5 days. Cr₂O₃ served as a flow marker and the microbial proportion of total nitrogen at the duodenum was estimated by near infrared spectroscopy (NIRS). The duodenal flow of biotin was not related to biotin intake, but to the amount of fermented organic matter (FOM) and the amount of microbial protein (Biotin [mg/d] = 0.518 · kg FOM?0.300; r = 0.85 and biotin [mg/d] = 0.012 · g microbial protein + 1.478; r = 0.84), irrespective of the composition of the diet fed. Mean daily biotin flow was 0.48 ± 0.11 mg/kg FOM without any systematic effect of diet composition. The ruminal biotin balance, calculated as the difference between biotin flow at the duodenum and biotin intake, was positive (1.4 – 2.0 mg/d) in cows fed the mixed roughage/concentrate diets and negative (?0.71 mg/d) when the pure hay diet was fed.     

A direct detection of Escherichia coli genomic DNA using gold nanoprobes

Background

Electrospun nanofibers have been widely used as substrata for mammalian cell culture owing to their structural similarity to natural extracellular matrices. Structurally consistent electrospun nanofibers can be produced with synthetic polymers but require chemical modification to graft cell-adhesive molecules to make the nanofibers functional. Development of a facile method of grafting functional molecules on the nanofibers will contribute to the production of diverse cell type-specific nanofiber substrata.

Results

Small molecules, peptides, and functionalized gold nanoparticles were successfully incorporated with polymethylglutarimide (PMGI) nanofibers through electrospinning. The PMGI nanofibers functionalized by the grafted AuNPs, which were labeled with cell-adhesive peptides, enhanced HeLa cell attachment and potentiated cardiomyocyte differentiation of human pluripotent stem cells.

Conclusions

PMGI nanofibers can be functionalized simply by co-electrospinning with the grafting materials. In addition, grafting functionalized AuNPs enable high-density localization of the cell-adhesive peptides on the nanofiber. The results of the present study suggest that more cell type-specific synthetic substrata can be fabricated with molecule-doped nanofibers, in which diverse functional molecules are grafted alone or in combination with other molecules at different concentrations.     

Noncovalent Immobilization of Streptavidin on In Vitro- and In Vivo-Biotinylated Bacterial Magnetic Particles

Biotinylated magnetic nanoparticles were constructed by displaying biotin acceptor peptide (BAP) or biotin carboxyl carrier protein (BCCP) on the surface of bacterial magnetic particles (BacMPs) synthesized by Magnetospirillum magneticum AMB-1. BAP-displaying BacMPs (BAP-BacMPs) were extracted from bacterial cells and incubated with biotin and Escherichia coli biotin ligase. Then the in vitro biotinylation of BAP-BacMPs was confirmed using alkaline phosphatase-labeled antibiotin antibody. In contrast, BacMPs displaying the intact 149 residues of AMB-1 BCCP (BCCP-BacMPs) and displaying the COOH-terminal 78 residues of BCCP (BCCP78-BacMPs) were biotinylated in AMB-1 cells. The in vivo biotinylation of BCCP-BacMPs and BCCP78-BacMPs was thought to be performed by endogenous AMB-1 biotin ligase. Streptavidin was introduced onto biotinylated BacMPs by simple mixing. In an analysis using tetramethyl rhodamine isocyanate-labeled streptavidin, approximately 15 streptavidin molecules were shown to be immobilized on a single BCCP-BacMP. Furthermore, gold nanoparticle-BacMP composites were constructed via the biotin-streptavidin interaction. The conjugation system developed in this work provides a simple, low-cost method for producing biotin- or streptavidin-labeled magnetic nanoparticles. Various functional materials can be site selectively immobilized on these specially designed BacMPs. By combining the site-selective biotinylation technology and the protein display technology, more innovative and attractive magnetic nanomaterials can be constructed.     

A chemical approach for the localization of membrane sites involved in lymphocyte activation.

The aldehyde groups formed on periodate oxidation of cell surface sialyl residues were used to insert a mitogenic site onto the lymphocyte membrane by attachment of biotin hydrazide or 2,4-dinitrophenyl hydrazine. The biotin- or 2,4-dinitrophenyl-conjugated cells were both agglutinated and stimulated when cultured with avidin or anti-2,4-dinitrophenyl antibody respectively. On the other hand, biotin or DNP-conjugated cells, modified via functional groups on the membrane proteins, were agglutinated but not stimulated when cultured with avidin or anti-DNP antibody respectively. Our results show that the specific interaction of a protein at the periodate oxidation site leads to blastogenesis.     

A novel fed-batch based strategy for enhancing cell-density and recombinant cyprosin B production in bioreactors

Nowadays, the dairy industry is continuously looking for new and more efficient clotting enzymes to create innovative products. Cyprosin B is a plant aspartic protease characterized by clotting activity that was previously cloned in Saccharomyces cerevisiae BJ1991 strain. The production of recombinant cyprosin B by a batch and fed-batch culture was compared using glucose and galactose as carbon sources. The strategy for fed-batch cultivation involved two steps: in the first batch phase, the culture medium presented glucose 1 % (w/v) and galactose 0.5 % (w/v), while in the feed step the culture medium was constituted by 5 % (w/v) galactose with the aim to minimize the GAL7 promoter repression. Based on fed-batch, in comparison to batch growth, an increase in biomass (6.6-fold), protein concentration (59 %) and cyprosin B activity (91 %) was achieved. The recombinant cyprosin B was purified by a single hydrophobic chromatography, presenting a specific activity of 6 × 10⁴ U·mg^?1, corresponding to a purification degree of 12.5-fold and a recovery yield of 16.4 %. The SDS-PAGE analysis showed that recovery procedure is suitable for achieving the purified recombinant cyprosin B. The results show that the recombinant cyprosin B production can be improved based on two distinct steps during the fed-batch, presenting that this strategy, associated with a simplified purification procedure, could be applied to large-scale production, constituting a new and efficient alternative for animal and fungal enzymes widely used in cheese making.     

Enzymatic production of 3,6-anhydro-l-galactose from agarose and its purification and in vitro skin whitening and anti-inflammatory activities

3,6-Anhydro-l-galactose (L-AHG) constitutes 50 % of agarose, which is the main component of red macroalgae. No information is currently available on the mass production, metabolic fate, or physiological effects of L-AHG. Here, agarose was converted to L-AHG in the following three steps: pre-hydrolysis of agarose into agaro-oligosaccharides by using acetic acid, hydrolysis of the agaro-oligosaccharides into neoagarobiose by an exo-agarase, and hydrolysis of neoagarobiose into L-AHG and galactose by a neoagarobiose hydrolase. After these three steps, L-AHG was purified by adsorption and gel permeation chromatographies. The final product obtained was 95.6 % pure L-AHG at a final yield of 4.0 % based on the initial agarose. In a cell proliferation assay, L-AHG at a concentration of 100 or 200 μg/?mL did not exhibit any significant cytotoxicity. In a skin whitening assay, 100 μg/?mL of L-AHG showed significantly lower melanin production compared to arbutin. L-AHG at 100 and 200 μg/?mL showed strong anti-inflammatory activity, indicating the significant suppression of nitrite production. This is the first report on the production of high-purity L-AHG and its physiological activities.     

Coupling of biotin-(poly(ethylene glycol))amine to poly(D,L-lactide-co-glycolide) nanoparticles for versatile surface modification

Generally, polymeric nanoparticles (NP) for drug targeting are designed to entrap the drug moiety in the core and to present the targeting moiety on the surface. However, in most cases, common preparation techniques of polymeric NP need to be specifically arranged for each compound to be entrapped or attached. In the present work, we introduce a method for versatile conjugation of targeting moieties to the surface of preformed, polymeric NP. Moreover, due to taking advantage of biotin-avidin interactions, our regime opens the additional possibility of a rapid fluorescence labeling of NP. Poly(D,L-lactide-co-glycolide) (PLGA) NP in the size of 210 nm were prepared by the classic oil-in-water method. Such NP were functionalized with biotin-(poly(ethylene glycol))amine (BPEG) by means of cyanuric chloride chemistry. The amount of surface-associated biotin was 850 pmol per milligram of polymer, corresponding to roughly 2650 molecules of biotin per NP. When drawn to scale, such surface coating appeared to be well-suited for subsequent binding of avidin or avidin-linked ligands. By resonant mirror measurements, we could prove specific binding of biotinylated NP to a NeutrAvidin (NAv)-coated surface. Furthermore, after coupling of NAv-linked fluorescence dyes to BPEG-functionalized NP, differences in binding and uptake could be demonstrated using two epithelial cell lines (Caco-2, A549).     

Bacterial cellulose nanofibers for albumin depletion from human serum

Cibacron Blue F3GA (CB) was covalently attached onto the bacterial cellulose (BC) nanofibers for human serum albumin (HSA) depletion from human serum. The BC nanofibers were produced by Acetobacter xylinum in the Hestrin–Schramm medium in a static condition for 14 days. The CB content of the BC nanofibers was 178 μmol/g. The specific surface area of the BC nanofibers was determined to be 914 m²/g. HSA adsorption experiments were performed by stirred-batch adsorption. The non-specific adsorption of HSA on the BC nanofibers was very low (1.4 mg/g polymer). CB attachment onto the BC nanofibers significantly increased the HSA adsorption (1800 mg/g). The maximum HSA adsorption was observed at pH 5.0. The HSA adsorption capacity decreased drastically with an increase of the aqueous phase concentration of sodium chloride. The elution studies were performed by adding 1 M NaCl to the HSA solutions in which adsorption equilibria had been reached. The elution results demonstrated that the binding of HSA to the adsorbent was reversible. The depletion efficiencies for HSA were above 96.5% for all studied concentrations. Proteins in the serum and eluted portion were analyzed by SDS-PAGE for testing the efficiency of HSA depletion from human serum. Eluted proteins include mainly HSA.     

Safety, Formulation and In Vitro Antiviral Activity of the Antimicrobial Peptide Subtilosin Against Herpes Simplex Virus Type 1

In the present study, the antiviral properties of the bacteriocin subtilosin against Herpes simplex virus type 1 (HSV-1) and the safety and efficacy of a subtilosin-based nanofiber formulation were determined. High concentrations of subtilosin, the cyclical antimicrobial peptide produced by Bacillus amyloliquefaciens, were virucidal against HSV-1. Interestingly, at non-virucidal concentrations, subtilosin inhibited wild type HSV-1 and aciclovir-resistant mutants in a dose-dependent manner. Although the exact antiviral mechanism is not fully understood, time of addition experiments and western blot analysis suggest that subtilosin does not affect viral multiplication steps prior to protein synthesis. Poly(vinyl alcohol)-based subtilosin nanofibers with a width of 278 nm were produced by the electrospinning process. The retained antimicrobial activity of the subtilosin-based fibers was determined via an agar well diffusion assay. The loading capacity of the fibers was 2.4 mg subtilosin/g fiber, and loading efficiency was 31.6 %. Furthermore, the nanofibers with and without incorporated subtilosin were shown to be non-toxic to human epidermal tissues using an in vitro human tissue model. Taking together these results, subtilosin-based nanofibers should be further studied as a novel alternative method for treatment and/or control of HSV-1 infection.     

Human palatal saliva: Adsorption behaviour and the role of low-molecular weight proteins

In situ ellipsometry was employed to study adsorption from human palatal saliva (HPalS) in terms of dependence on surface wettability and saliva concentration ( ? 1%). Adsorbed amounts, kinetics, and elutability with buffer and sodium dodecyl sulphate (SDS) were determined. The low-molecular weight protein content of bulk HPalS was also investigated using two-dimensional gel electrophoresis, and this revealed the presence of a large group of proteins < 100 kDa in size. Adsorption to pure (hydrophilic) and methylated (hydrophobized) silica surfaces revealed that the total adsorbed amounts were greater on hydrophobized silica. Below concentrations of 0.5 and 0.25% saliva, adsorption was concentration dependent on hydrophobized and hydrophilic surfaces, respectively. The initial adsorption ( ? 30 min) was faster on hydrophobized surfaces. Addition of SDS removed more material than buffer rinsing on both surfaces. Analysis of the adsorption kinetics indicated that the presence of low-molecular weight proteins plays a role in adsorption from HPalS.     

Growth of the yeast Kluyveromyces marxianus CBS 6556 on different sugar combinations as sole carbon and energy source

The yeast Kluyveromyces marxianus has been pointed out as a promising microorganism for a variety of industrial bioprocesses. Although genetic tools have been developed for this yeast and different potential applications have been investigated, quantitative physiological studies have rarely been reported. Here, we report and discuss the growth, substrate consumption, metabolite formation, and respiratory parameters of K. marxianus CBS 6556 during aerobic batch bioreactor cultivations, using a defined medium with different sugars as sole carbon and energy source, at 30 and 37 °C. Cultivations were carried out both on single sugars and on binary sugar mixtures. Carbon balances closed within 95 to 101 % in all experiments. Biomass and CO₂ were the main products of cell metabolism, whereas by-products were always present in very low proportion (<3 % of the carbon consumed), as long as full aerobiosis was guaranteed. On all sugars tested as sole carbon and energy source (glucose, fructose, sucrose, lactose, and galactose), the maximum specific growth rate remained between 0.39 and 0.49 h^?1, except for galactose at 37 °C, which only supported growth at 0.31 h^?1. Different growth behaviors were observed on the binary sugar mixtures investigated (glucose and lactose, glucose and galactose, lactose and galactose, glucose and fructose, galactose and fructose, fructose and lactose), and the observations were in agreement with previously published data on the sugar transport systems in K. marxianus. We conclude that K. marxianus CBS 6556 does not present any special nutritional requirements; grows well in the range of 30 to 37 °C on different sugars; is capable of growing on sugar mixtures in a shorter period of time than Saccharomyces cerevisiae, which is interesting from an industrial point of view; and deviates tiny amounts of carbon towards metabolite formation, as long as full aerobiosis is maintained.     

Development of novel robust nanobiocatalyst for detergents formulations and the other applications of alkaline protease

Alkaline protease from alkaliphilic Bacillus sp. NPST-AK15 was immobilized onto functionalized and non-functionalized rattle-type magnetic core@mesoporous shell silica (RT-MCMSS) nanoparticles by physical adsorption and covalent attachment. However, the covalent attachment approach was superior for NPST-AK15 protease immobilization onto the activated RT-MCMSS-NH₂ nanoparticles and was used for further studies. In comparison to free protease, the immobilized enzyme exhibited a shift in the optimal temperature and pH from 60 to 65 °C and pH 10.5–11.0, respectively. While free protease was completely inactivated after treatment for 1 h at 60 °C, the immobilized enzyme maintained 66.5 % of its initial activity at similar conditions. The immobilized protease showed higher k _cat and K _m , than the soluble enzyme by about 1.3-, and 1.2-fold, respectively. In addition, the results revealed significant improvement of NPST-AK15 protease stability in variety of organic solvents, surfactants, and commercial laundry detergents, upon immobilization onto activated RT-MCMSS-NH₂ nanoparticles. Importantly, the immobilized protease maintained significant catalytic efficiency for ten consecutive reaction cycles, and was separated easily from the reaction mixture using an external magnetic field. To the best of our knowledge this is the first report about protease immobilization onto rattle-type magnetic core@mesoporous shell silica nanoparticles that also defied activity-stability tradeoff. The results clearly suggest that the developed immobilized enzyme system is a promising nanobiocatalyst for various bioprocess applications requiring a protease.     

Functional polymer brushes via surface-initiated atom transfer radical graft polymerization for combating marine biofouling

Dense and uniform polymer brush coatings were developed to combat marine biofouling. Nonionic hydrophilic, nonionic hydrophobic, cationic, anionic and zwitterionic polymer brush coatings were synthesized via surface-initiated atom transfer radical polymerization (SI-ATRP) of 2-hydroxyethyl methacrylate, 2,3,4,5,6-pentafluorostyrene, 2-(methacryloyloxy)ethyl trimethylammonium chloride, 4-styrenesulfonic acid sodium and N,N′-dimethyl-(methylmethacryloyl ethyl) ammonium propanesulfonate, respectively. The functionalized surfaces had different efficacies in preventing adsorption of bovine serum albumin (BSA), adhesion of the Gram-negative bacterium Pseudomonas sp. NCIMB 2021 and the Gram-positive Staphylococcus aureus, and settlement of cyprids of the barnacle Amphibalanus amphitrite (=Balanus amphitrite). The nonionic hydrophilic, anionic and zwitterionic polymer brushes resisted BSA adsorption during a 2 h exposure period. The nonionic hydrophilic, cationic and zwitterionic brushes exhibited resistance to bacterial fouling (24 h exposure) and cyprid settlement (24 and 48 h incubation). The hydrophobic brushes moderately reduced protein adsorption, and bacteria and cyprid settlement. The anionic brushes were least effective in preventing attachment of bacteria and barnacle cyprids. Thus, the best approach to combat biofouling involves a combination of nonionic hydrophilic and zwitterionic polymer brush coatings on material surfaces.     

Ethanol production from galactose by a newly isolated Saccharomyces cerevisiae KL17

A wild-type yeast strain with a good galactose-utilization efficiency was newly isolated from the soil, and identified and named Saccharomyces cerevisiae KL17 by 18s RNA sequencing. Its performance of producing ethanol from galactose was investigated in flask cultures with media containing various combination and concentrations of galactose and glucose. When the initial galactose concentration was 20 g/L, it showed 2.2 g/L/h of substrate consumption rate and 0.63 g/L/h of ethanol productivity. Although they were about 70 % of those with glucose, such performance of S. cerevisiae KL17 with galactose was considered to be quite high compared with other strains reported to date. Its additional merit was that its galactose metabolism was not repressed by the existence of glucose. Its capability of ethanol production under a high ethanol concentration was demonstrated by fed-batch fermentation in a bioreactor. A high ethanol productivity of 3.03 g/L/h was obtained with an ethanol concentration and yield of 95 and 0.39 g/L, respectively, when the cells were pre-cultured on glucose. When the cells were pre-cultured on galactose instead of glucose, fermentation time could be reduced significantly, resulting in an improved ethanol productivity of 3.46 g/L/h. The inhibitory effects of two major impurities in a crude galactose solution obtained from acid hydrolysis of galactan were assessed. Only 5-Hydroxymethylfurfural (5-HMF) significantly inhibited ethanol fermentation, while levulinic acid (LA) was benign in the range up to 10 g/L.     

Enhancing the catalytic properties of porcine pancreatic lipase by immobilization on SBA-15 modified by functionalized ionic liquid

The mesoporous silica SBA-15 was modified by carboxyl-functionalized ionic liquid (COOH-IL-SBA). The prepared support was used to immobilize porcine pancreatic lipase (PPL) by physical adsorption (PPL-COOH-IL-SBA) and covalent attachment (PPL-CON-IL-SBA). Enzymatic properties of the immobilized PPL were investigated in the triacetin hydrolysis reaction. It was found that carboxyl functionalized ionic liquid modification of the support surface was an effective method to improve the properties of immobilized PPL. Incorporating into the functionalized SBA-15 made PPL more resistant to temperature and pH changes, compared with PPL immobilized on parent SBA-15 (PPL-SBA). Especially, after the covalent attachment to a functionalized support, the stability of PPL was improved obviously, which retained 81.25% and 52.50% of the original activity after incubation for 20 days and four times recycling, respectively, whereas PPL-SBA exhibited only 58.80% and 27.78% of the original activity under the same conditions. In addition, physical and chemical properties of the supports and immobilized PPL were characterized by small-angle X-ray powder diffraction (SAXRD), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscope (SEM), nitrogen adsorption, nuclear magnetic resonance (NMR) and thermogravimetry (TG). The images and data confirmed chemical modification in SBA-15 and PPL immobilization on the tested support.     

Nonradioactive labeling of synthetic oligonucleotide probes with terminal deoxynucleotidyl transferase

Synthetic oligonucleotides were tailed at the 3' end using terminal deoxynucleotidyl transferase. Nucleotide triphosphates with free primary amines at the end of side chains were compared for their tailing efficiency and/or detection sensitivity, using biotin-11-dUTP as a reference. Free primary amines were tagged with activated biotin or fluorescein isothiocyanate. The probes were then detected with either streptavidin-alkaline phosphatase complex or anti-fluorescein antibodies and alkaline phosphatase-conjugated secondary antibodies. Tailing conditions were optimized and the probes were tested for detection of Escherichia coli ST1a enterotoxin DNA and rotavirus RNA.