extfieldsofstudy:
Computer Science
Medicine

text:
Automated nucleic acid chain tracing in real time

A method is presented for the automatic building of nucleotide chains into electron density which is fast enough to be used in interactive model-building software. Likely nucleotides lying in the vicinity of the current view are located and then grown into connected chains in a fraction of a second. When this development is combined with existing tools, assisted manual model building is as simple as or simpler than for proteins.

Background
The structural analysis of RNA and DNA molecules and complexes has become important in recent years, with the PDB now reporting nearly 3000 deposited structures containing nucleotides (Berman et al., 2007). The interpretation of nucleotide electron-density maps presents different challenges to the interpretation of protein maps: the resolution of the data is frequently lower (Keating & Pyle, 2010), the monomers are larger and therefore more flexible, and in some cases the structures are very large (see, for example, Wimberly et al., 2000). While graphical software such as Coot (Emsley et al., 2010) can be used for building nucleotides, the tools available are less mature than for proteins. A new approach is described which allows interactive backbone tracing of nucleotides in Coot, which may be used in combination with existing tools such as RCrane (Keating & Pyle, 2012) to produce a complete and accurate model.
There are a number of existing packages for automated or semi-automated nucleotide building. The RESOLVE software, now incorporated in phenix.autobuild, will build nucleotide chains in a fully automated manner when the data are of sufficient quality (Terwilliger et al., 2008). The ARP/ wARP package will also build nucleotide chains, but not assign the base types (Hattne & Lamzin, 2008). The LAFIRE package includes software for extending and rebuilding existing nucleotide chains (Yamashita et al., 2013). The RCrane software performs semi-automated building, in which the software aids the user in the location of the ribose and phosphate groups and then builds a chain through the resulting groups, and can be used interactively from within the Coot graphical model-building software.
The methods described here are complementary to the RCrane development as they provide a method for the instantaneous building of segments of nucleotide chain in the region of an electron-density map currently being viewed in the Coot program. These segments may then be corrected and rebuilt automatically using the RCrane tools. The primary design consideration for the method was speed, enabling the user to build tens or even hundreds of nucleotides in a few minutes. For interactive use the software must therefore be able to find a nucleotide chain in the general vicinity of the current view position and trace it in both directions as far as the density supports in a fraction of a second. This is achieved through a carefully designed search function which can rapidly score electron density according to the likely presence of a nucleotide feature at that position.

Methods
The nucleotide chain-tracing algorithm has two fundamental stages: (i) finding nucleotides as a starting point for building and (ii) growing extended nucleotide chains. Finding nucleotides is performed using an optimized search algorithm and a search 'target' describing the electron-density feature to be recognized; these will be described in x2.1. Growing the nucleotides into chains involves converting the search targets into single-nucleotide or binucleotide fragments and then extending these fragments by adding additional nucleotides using a database of chain conformations; these steps are described in x2.2.
2.1. Finding nucleotides as a starting point for building 2.1.1. Fragment-search algorithm. In order to achieve fully interactive performance, careful consideration must be given to the search algorithm. The location of three-dimensional model fragments usually requires a six-dimensional search over three positional and three orientation parameters. While it is possible to factor out the orientation search by searching for spherically averaged density (Vagin & Isupov, 2001), this involves discarding information about the shape of the fragment. The alternative is to search over all three orientation parameters and then use a highly optimized positional search.
One approach to optimizing the positional search is to make use of fast Fourier transforms (FFTs), as employed in molecular-replacement calculations (Navaza & Vernoslova, 1995) and related methods (Cowtan, 2001). However, a threedimensional FFT still requires of the order of 3log(n) computations per grid cell, where n is the number of grid cells along one edge of the unit cell. FFT-based methods also tend to be calculated over the entire unit cell or at least the asymmetric unit (Read & Schierbeek, 1988), although it is possible to relax this constraint. When working interactively the user often wants to build the electron-density feature that they are currently viewing (and continuations of this feature in the case of long polymers); this provides an additional optimization which can be exploited by non-FFT methods. Optimal performance is then achieved by minimizing the average number of computational operations required per grid cell to identify the presence of a model fragment in a given orientation at that position. Kleywegt & Jones (1997) developed a method for locating the presence of molecular fragments using a scoring metric based on the electron density at the atomic centres of the oriented fragment after translation to the current grid position. However, this approach has the limitation that a large 'blob' of high density which is larger than the fragment will score highly even if it does not resemble the fragment in shape. In the FFT-based method of Cowtan (2001) it was found that better fragment discrimination could be obtained by using a scoring function which requires both high density where there are expected to be atoms and low density where there are expected to be none.
The latter approach may be adapted for use in a fast non-FFT-based approach by defining a set of probe points describing the 'fingerprint' of the search fragment in terms of a set of positions where the electron density must be high if the oriented fragment is present and a second set of positions where the electron density must be low if the fragment is present: these will be referred to as high and low probe points, respectively. A scoring function is then used to evaluate the electron densities at the high and low probe points and return a single value to indicate the presence or absence of the fragment.
Let the positions of the high probe points relative to the centre of the oriented fragment be x h , the positions of the low probe points relative to the centre of the fragment be x l and n be the number of probe points of each type. (For simplicity of implementation the numbers of high and low probe points are constrained to be equal.) A simple score may be obtained from the difference between the means of the densities at the high and low probe points, However, this scoring function does not meet the criteria for very fast evaluation since it must be evaluated for every probe point. To reduce the computation to an average of a few operations per grid cell a different form is adopted, The advantage of this form is that in most cases only part of the expression needs to be evaluated. Since the minimum can only decrease and the maximum can only increase with the inclusion of further probe points, the score can only become worse as it is evaluated. Evaluation therefore takes place one pair of probe points at a time, and as soon as the partial score drops below a given threshold, evaluation is terminated and the existence of the oriented fragment at that position is rejected. The score threshold may start at zero (implying that for a fragment to be research letters accepted the lowest high probe must be greater than the highest low probe), although other values are possible.
Three further optimizations are employed.
(i) The positional offsets are rounded to the nearest grid vector, so that the electron density is evaluated at a grid point rather than by interpolation between grid points.
(ii) A target number of matches is chosen and the score threshold is periodically adjusted upwards to retain no more than the target number of matches and allow the earlier termination of further scoring steps.
(iii) Fragment translations where the density at the first high probe point is less than one standard deviation above the mean are eliminated immediately.
Rotation space is explored with an 18 search step, giving a total of 2792 orientations. The number of translations within 6 Å of the view centre depends on the grid resolution, but is typically around a thousand. Pruning based on the first high probe point typically reduces this number by a factor of six. Initially, a little under half of the remaining points are pruned as each new pair of probe points is included in s minmax ; however, as the calculation progresses and the score threshold is raised more translations are subject to early pruning.
The approximations mean that the calculation is crude but very efficient. In practice, this scoring method is used to provide a list of candidate fragment locations which may then be re-evaluated using the more sensitive s mean score using interpolated electron-density values. The calculation is repeated for each possible orientation of the fragment by a search over the three rotational parameters.
2.1.2. Search targets. Each nucleic acid monomer provides three rigid groups which may be used as search fragments: the pentose sugar, the phosphate group and the base. The sugar and phosphate groups are used for identifying the main chain: this is in contrast to the method of Hattne & Lamzin (2008), in which the base is located first. For each of these search fragments a set of high and low probe points must be identified.
The probe points were determined using the electron density of nucleotides from the structure with PDB code 1hr2 (Juneau et al., 2001): a total of 316 nucleotides, with the conserved atoms of the search fragment superimposed. Less common conformations of the atoms of the search target were removed by successively removing fragments in which the O2 0 atom (for sugars) or C5 0 and C3 0 atoms (for phosphates) were more than 1 Å from the ensemble mean for that fragment, leaving 280 sugar fragments and 226 phosphate fragments. An electron-density map for the structure was calculated from the observed data at 2.2 Å resolution. The maximum and minimum across all of the fragments in this map for each grid point around the oriented fragment were determined and used to calculate a minimum and maximum map.
For every grid point in the minimum map, every instance of the fragment has an electron-density value greater than or equal to the value at that position in the minimum map. As a result, a high value in the minimum map is a strong indication of a conserved electron-density feature which is consistent with the presence of the fragment. Similarly, a low value in the maximum map corresponds to a position where no instance of the fragment has high electron density, which indicates a conserved electron-density hole consistent with the presence of the fragment. The minimum and maximum maps may therefore be used to locate high and low probe points, respectively.
High probe points are placed on atoms of the search fragment (O3 0 , C1 0 , C2 0 , C3 0 , C4 0 , O4 0 and C5 0 for the sugar, and O3 0 , P, OP1, OP2 and O5 0 for the phosphate), omitting atoms which are not strongly conserved (e.g. O2 0 ). Additional probe points are allocated to represent density not consistently associated with a single atom; thus, for example, in the case of sugar fragments the base is represented by a probe point on the N1 or N9 atom and a second point at the far side of the associated ring, but not on an atomic centre.
Low probe points are dispersed around the perimeter of the fragment. The contour level of the maximum map was set to the lowest value in the map within a 6 Å radius of the fragment centre, and a low probe was placed at this point. The contour level was then gradually increased and probe points were placed in new density features as they appeared, subject to the constraint that a new probe point should not be too close to an existing probe point so as to provide independent information.
The minimum and maximum maps, and the corresponding high and low probe points, are shown for each of the two search fragments in Fig. 1. The coordinates of the probe points are provided as Supporting Information.

2.2.
Growing an extended nucleotide chain 2.2.1. Converting the initial fragments to nucleotides. The initial candidate fragments are then grown into extended chain fragments. Candidate fragments which fail to grow to at least three nucleotides are rejected. This process comprises two steps. Firstly, the initial fragments must be extended to complete single nucleotides or binucleotides. Next, they are iteratively extended using an algorithm which successively adds additional nucleotides to either the 3 0 or the 5 0 end of the chain. High and low probe points (spheres and crosses, respectively) for (a) the sugar target and (b) the phosphate target. The average electron density over the reference set of nucleotides is shown.
Both of these processes involve a database of nucleotide chain linkages, which are determined from one (or potentially more than one) known high-quality nucleotide structure. The database consists of fragments of connected nucleotides, in which each nucleotide is represented by the main-chain atoms.
The key functionality of the database is to provide fragments matching one or more sugar rings (or phosphate groups). If a single sugar ring is provided, then a list of short chain fragments are returned with every sugar ring from the database superposed on the given sugar. If multiple sugar rings are provided then every possible chain fragment which is capable of linking those sugars is returned.
The default database is also constructed using the structure with PDB code 1hr2 (Juneau et al., 2001) and thus contains 316 nucleotides. Limiting the size of the database improves performance and has also been found to reduce the chance of the incorporation of incorrect rare conformations when data quality is poor. When the data are good a larger database can sometimes improve the results if interactive performance is not required.
Since the database relies upon oriented sugar rings for further building, the sugar rings located in the initial search may be used directly as a starting point for chain tracing. Phosphate groups, however, require an additional step. Every phosphate group from the database is superposed on the group identified from the electron density. The two neighbouring sugars are then scored against the density using s mean and the best-scoring binucleotide is stored as a starting point for chain tracing. The result of this step is a set of mono-nucleotides (from the location of sugar rings) and binucleotides (from the location of phosphate groups); however, in each case the terminal phosphate group is in an arbitrary conformation so that the fitted parts of the fragments actually correspond to nucleosides and 'suites', respectively (Murray et al., 2003).
The use of known structure fragments to describe the range of possible main-chain conformations is in contrast to the approach of Keating & Pyle (2010), who use the database of distinct sugar-sugar backbone conformers (or 'suites') determined by Murray et al. (2003). The latter approach is probably more efficient because redundant conformers are eliminated, and will be explored in future; however, the known structure database can also be used to bridge multi-nucleotide gaps using a method analogous to loop fitting in proteins (Cowtan, 2012).
2.2.2. Growing chains. Chains are then grown from the initial fragments by adding further nucleotides at either end. Each sugar ring from the database is superposed on the 3 0 or 5 0 sugar of a fragment, and the next (or previous) nucleotide from the database is extracted and scored against the map using the s mean score for both the new sugar and the intervening phosphate group. Building continues at each end of the chain until no sugar ring can be found whose score exceeds a threshold value. The threshold is determined by scoring 100 000 random translations and orientations of the search target in the current electron-density map to establish a probability distribution of score values. The threshold score is set such that the probability of a score exceeding this value is 0.1%.
The algorithm as described can often produce multiple overlapped chain traces starting from different initial fragments. Merging of these fragments and resolving branches is achieved using the method described for proteins in Cowtan (2006).
The sugar puckers are often ambiguous in the electron density, and the method as implemented does not attempt to resolve sugar puckers except to the extent that the sugar pucker is implicit in the sugar-phosphate-sugar backbone conformation. The effectiveness of this approach has not been investigated and therefore post-refinement using RCrane (Keating & Pyle, 2012) is advisable.

Search targets
The skill of the search target functions in locating sugar and phosphate groups was evaluated using a deposited structure, PDB entry 3cw5 (Barraud et al., 2008), consisting of 77 nucleotides. Experimental data were available to 3.1 Å resolution. Synthetic phases were created by starting from the refined structure and copying figures of merit (FOMs) from an experimentally phased structure, preserving the resolution and magnitude dependence of the FOMs. The mean FOM for the data to 3.1 Å resolution was 0.58. A random phase error was then selected from a distribution consistent with the FOM Comparison of the value of the search target score as a function of translational coordinate error for the correctly oriented sugar and phosphate targets, using either the s minmax or s mean scoring functions. The shading represents a density histogram of counts, with light shading for 1-4 or dark shading for 5+ counts per bin. Dashed lines show the regression of score on distance for distances less than 2.5 Å .
for that reflection and added to the phase. The performance of each search target was evaluated using the electron-density map calculated using the resulting phases and FOMs.
For each sugar or phosphate in the test structure, the search target was evaluated using the correct orientation and translations spanning a sphere of radius 4 Å around the true position. The values of s minmax and s mean were plotted against distance from the true position for each target, and are shown in Fig. 2.
The sugar search target shows significant discrimination using either the s minmax or s mean functions. The mean function is more effective in locating sugars; however, as discussed, it is less amenable to optimization.
The phosphate search target is less effective, with s minmax showing very little signal. The mean function provides a weak signal, but also starts producing false positives beyond 2.5 Å from the true position as the target function begins to pick up tetrahedral C atoms in the sugar ring.
The poor performance of the phosphate target is in contrast to that of Keating & Pyle (2012), in which the phosphates form a starting point for building. The difference arises from the form of the search functions. The phosphate is distinguishable by the higher density of the P atom; however, s minmax only reflects the lowest density at any of the high probe points and thus is blind to the phosphorus density. Similarly, in s mean the phosphorus density is diluted by averaging over the high probe points. As a result, the phosphate target is primarily of value when used in conjunction with the sugar target.

Model building
The method described here is optimized for interactive use, and so does not incorporate all of the model-completion steps or the time-consuming refinement step employed in automated model-building calculations such as ARP/wARP (Langer et al., 2008) or phenix.autobuild (Terwilliger et al., 2008). However, it is interesting to see how much of the model can be built in a single step with no refinement or improvement of the electron-density map. 35 nucleotide structures from the PDB for which experimental observations were available were used as test cases. For each structure, a simulated experimental data set was created using the same method as in x3.1. The resulting maps were used as a basis for finding search fragments and growing them into chains. The search algorithm was modified to search the whole asymmetric unit, rather than just a region around the current view centre.
The resulting partial models were then compared against the deposited structures and scored on the basis of the number of C1 0 atoms in the deposited structure for which a C1 0 atom was present in the partial model within 1.5 Å of the refined position. The results are shown in Table 1

Figure 3
Comparison of autobuilt nucleotides with the final structure for 3cw5 at 3.1 Å resolution. The backbone trace of the full structure is show using a worm representation, and backbone atoms for the autobuilt model are shown as bonds.
less common conformations. The deposited structure and the autobuilt model for the structure 3cw5 are shown in Fig. 3. In this case about half of the structure has been built. The test structures were also used to judge the utility of the phosphate target. Omitting the phosphate target from either the search or the growing steps decreases the total proportion of the structures built, although in the case of the growing step the difference is marginal.

Discussion
One notable result of this work is that fitting nucleotides appears to be a simpler problem than fitting proteins. This was initially surprising, given that nucleotides have greater torsional variability in the main chain (Murray et al., 2003) and typically higher thermal displacement parameters (Keating & Pyle, 2010), leading in turn to greater ambiguity in the features of the electron-density map. However, the rigid groups within each nucleotide are larger and the most common relationships between neighbouring nucleotides show less variation than might be expected from the torsional variability for individual bonds. Attempts to apply the same kind of techniques to proteins has not yielded comparable benefits except in the case of large secondary-structure features (Langer et al., 2006).
When applied to automated model building and refinement, the methods developed here are sufficiently fast that refinement becomes the rate-limiting step. The implementation of high-performance computing methods for crystallography (Sauter et al., 2013) might in future increase the speed of the refinement step, which would be of immediate value to comprehensive ligand searches and some molecular-replacement problems; however, with the developments described here the building of novel nucleotide structures may also benefit.
There is scope for further applications of fingerprint techniques. The determination of the high and low probe points may be automated either using ad hoc rules to emulate the manual approach adopted here, or through a more rigorous information theoretical approach. These are being explored in the context of carbohydrate model building, but should also be applicable to the location of rigid or semi-rigid ligands.

Software availability
The software is available as a part of the Coot modelbuilding package from version 0.7 using the scripting command find_nucleic_acids_local(6) or by a right mouse click on the top tool bar to add the 'NA build' button. From build 5199 it is also available through the 'Other Modelling Tools' menu. A slower implementation which includes model refinement is available in the CCP4 software suite from version 6.3.0 under the name Nautilus.


extfieldsofstudy: Biology Medicine Chemistry

text:
Molecular isothermal techniques for combating infectious diseases: towards low-cost point-of-care diagnostics

Nucleic acid amplification techniques such as PCR have facilitated rapid and accurate diagnosis in central laboratories over the past years. PCR-based amplifications require high-precision instruments to perform thermal cycling reactions. Such equipment is bulky, expensive and complex to operate. Progressive advances in isothermal amplification chemistries, microfluidics and detectors miniaturisation are paving the way for the introduction and use of compact ‘sample in-results out’ diagnostic devices. However, this paradigm shift towards decentralised testing poses diverse technological, economic and organizational challenges both in industrialized and developing countries. This review describes the landscape of molecular isothermal diagnostic techniques for infectious diseases, their characteristics, current state of development, and available products, with a focus on new directions towards point-of-care applications.

Diverse technologies are currently available for diseases diagnostics in laboratory environments. Culture remains the gold standard of bacteriological diagnosis despite being moderately sensitive, time-consuming and expensive. Culturing also requires specific laboratory infrastructure as well as trained operators. Rapid antigen tests have been described to provide modest diagnostic sensitivity, particularly for vaccinated populations, but they can deliver results in minutes with relatively simple equipment and are less expensive than culture. Tests that are based on nucleic acid amplification (NAAT), such as PCR, are available for many infectious diseases. PCR techniques provide high accuracy with a time to results of few hours and allow multianalyte testing. However, these techniques involve performance of thermal cycling amplifications in high-precision instruments. Such equipment is bulky, expensive and complex to operate, requiring specially trained operators and stringent conditions of laboratory compartmentalization. All these factors constrain performance of PCR out of centralized laboratories.
An ideal diagnostic test should meet the 'ASSURED' criteria: affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free and delivered to those who need it (accessible quality-assured diagnostics -2009 annual report) [1]. Less than US$10 per test and less than US$500 per device could be considered affordable prices for developing countries [2]. New directions are being explored to improve accessibility, rapidity and simplicity of diagnostic tests. Advances in microfluidics and miniaturization of signal detectors have allowed the integration of molecular diagnostics into microscale lab-on-achip devices that perform all necessary PCR steps automatically, from sample intake to cell lysis, DNA extraction, purification and amplification. However, there continues to be a gap between first-generation PCR-based point-ofcare (POC) tests and ideal 'ASSURED' tests in terms of portability, affordability and instrumentation requirements.
Isothermal DNA amplification technologies are emerging as a promising driver of molecular POC diagnostics aiming to meet 'ASSURED' attributes. Such techniques have been reported to have analytical sensitivities and specificities comparable to PCR and a higher tolerance to inhibitory compounds, while allowing shorter time to results easier use. Some of these chemistries have the capability of processing raw samples directly without previous DNA purification, thus contributing increased simplicity and additional reductions in cost and time [3].
Although currently isothermal reagents are not cheaper than those for PCR, isothermal techniques avoid the use of bulky, complex and expensive devices in contrast to PCR. Additionally, some of these techniques do not require DNA purification, allowing the use of raw samples. Therefore, isothermal assays pave the way for the development and introduction of inexpensive POC devices with prices that will progressively tend to be closer to lateral flow antigen tests than to PCR.
For example, a device for the detection of Salmonella by PCR (TaqMan Ò Salmonella enterica Detection Kit; Thermo Fisher Scientific, Life Technologies; CA, USA) costs about US $20,000-30,000, whereas a device for detection of the same pathogen by recombinase polymerase amplification (RPA) (TwistFlowÒ Salmonella kit, TwistDx; Cambridge, UK) is estimated to cost US$4500. It is also remarkable that those isothermal techniques processing raw samples skip purification steps, reducing by US$4-10 the total cost per sample in comparison with PCR. Overall, isothermal techniques have the potential to make performance easier, bringing additional savings in labor costs.
Diverse studies have described the evolution of isothermal molecular techniques [4,5]. This review describes the state-ofthe-art and new directions in the development of isothermal amplification technologies for diagnosis of infectious diseases with particular focus on those susceptible to be integrated in inexpensive molecular POC tests. Most promising techniques, their basic design, state of development and applications are classified and discussed. We also provide a glimpse at the evolution of these applications in the near future and the barriers to overcome for their successful deployment.

Molecular isothermal techniques currently used for diagnosis
Nucleic acid sequence-based amplification Nucleic acid sequence-based amplification (NASBA) [6] and related techniques, such as transcription mediated amplification [7] and self-sustained sequence replication [8], were the first isothermal amplification reactions mimicking retroviral RNA replication. Emergence of HIV in the 1990s propelled the development of more rapid and reliable HIV detection by NASBA in the early dawn of isothermal NAATs [9]. NASBA is based on three different enzymes participating in the amplification process (SUPPLEMENTARY FIGURE 1; supplementary material can be found online at www.informahealthcare.com/suppl/ 10.1586/14737159.2014.940319).
The range of currently commercialized NASBA tests covers solutions integrated into a lateral flow format method (named 'oligochromatography') and fluorescent detection assays (TABLE 1). Among NASBA tests, PANTHER (Gene-Probe) system and TGRIS devices are already available for fully automated process from sample to result. However, because of their large volume and weight, as well as long time consumed to results, such solutions cannot be characterized as ASSURED tests.
Some attempts have been made for developing POC tests based on NASBA. An integrated system combining RNA purification (previously extracted from bacteria), NASBA and amplification detection [10] was described for detection of Escherichia coli RNA. Recently, advances toward a 'sample-in, answer-out' NASBA POC instrument have been done integrating preconcentration, nucleic acid extraction, amplification and real-time fluorescent detection into a single sample device [11].

Strand displacement amplification
Strand displacement amplification (SDA) is an isothermal amplification method that combines the action of a restriction endonuclease and the strand displacing DNA polymerase to amplify a DNA target sequence [12] (SUPPLEMENTARY FIGURE 2). The amplification reaction takes place at low astringency conditions (37˚C), which produce nonspecific primer binding and nonspecific amplification, as occurs in the case of NASBA. Such mechanism is especially problematic for diagnostic devices utilized in clinical environments since samples contain more abundant human DNA than the desired target. Additionally, initial amplification time of 2 h makes SDA inadequate for POC applications.
At present, a laboratory diagnostic device named BD ProbeTec TM ET system for Chlamydia trachomatis and Neisseria gonorrhoeae detection is in commercialization by BD Diagnostics. Nonetheless, this device requires intensive hand labor (TABLE 1). Approaches to SDA integration into a POC device have been done through development of an automated instrument combining sample preparation from whole cells, SDA and detection [13]. Long time for detection (2.5 h) is an obstacle for using SDA in ASSURED tests.

Loop-mediated isothermal amplification
Loop-mediated isothermal amplification (LAMP) [14] is one of the most widely described one-step amplification techniques. This technology is based on the strand-displacement activity of the Bst DNA polymerase, which avoids a continuous DNA amplification reaction. Four primers are necessary for the reaction: F3/B3, FIP/BIP (FIGURE 1A); additional LF/LB primers accelerate the reaction [15] (FIGURE 1B). This pool of primers combined with the restrictive annealing and a reaction temperature of 60-65˚C makes the reaction highly specific, allowing an 'amplification is detection' scheme in 15-60 min [16]. The common template for LAMP is DNA, but RNA template could also be detected by adding the reverse transcriptase together with DNA polymerase. This specific variant has been termed reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) [17]. White precipitates of magnesium pyrophosphate produced during LAMP reaction allow turbidimetric detection of DNA amplification by both qualitative visual methods and real-time quantitative turbidimetry [18]. Several detection methods such as intercalating fluorescence dyes [ LAMP does not need initial denaturation at 95˚C to allow strand separation, which is an interesting characteristic for POC applications. Nevertheless, higher levels of sensitivity (both analytical and diagnostic) have been obtained when including heating denaturation [26] with detection limits as low as one copy per reaction [27]. Importantly, the Bst DNA polymerase used in LAMP reaction shows high tolerance to    [35].
Eiken Chemical Company (Tokyo, Japan) has developed and commercialized the LAMP method for both DNA and RNA amplification kits targeting laboratories that develop home-made techniques. In addition, this company offers a variety of kits for the detection of human pathogens in food or environment such as Salmonella, E. coli O157, Listeria monocytogenes, Campylobacter, Legionella, Giardia and Cryptosporidium. In addition, Eiken Chemical in collaboration with Foundation for Innovative New Diagnostics and the Hospital for Tropical Diseases has recently developed a field-stable LAMP kit for malaria for the detection of all species of human malaria parasites [36,37]. The validation of a commercial loop-mediated isothermal amplification assay (TB-LAMP) for the detection of tuberculosis has been recently reported by Eiken and is expected to be launched into the market soon together with a kit for human African trypanosomiasis. The LAMP technology has also been implemented in the Illumigene (Meridian Bioscience, Cincinnati, OH, USA) assay for rapid diagnosis of Clostridium difficile, Mycoplasma pneumonia, Group A Streptococcus and Group B Streptococcus. In addition, Amplex Biosystems (Gießen, Germany) have commercialized the eazyplex Ò Super-Bug that detects carbapenemases antibiotic resistances (covering KPC, NDM, OXA-48, VIM and the Acinetobacter baumannii belonging Oxa-groups OXA-23 like, OXA-40 like and OXA-58 like) and the eazyplex SuperBug CRE, which also includes ESBL genes of CTX-M-1 and CTX-M-9 families. All these tests take between 15 and 30 min for results. It is also of interest to note that real-time PCR devices can be used for isothermal amplification and detection although less complex instruments are specifically aimed for this purpose. Among others, both Instrument Genie Ò II (Optigene, Horsham, UK) and Instrument Genie III allow isothermal amplification and detection by fluorescence, whereas Ilumipro-10 (Meridian Bioscience, Cincinnati, OH) detects amplification products by turbidity.
Due to its rapidness, specificity, robustness, simple equipment and the possibility to process almost raw samples, LAMP is a suitable method for low-cost POC utilization, in combination with other technologies such as microfluidics. Since a first microfluidic disc for detection of hepatitis B virus reached an analytical sensitivity of about 50 copies per reaction within 60 min measured by turbidity [38], a significant number of POC devices based on LAMP have been developed: that is, a multichannel microfluidic device based on LAMP turbidity with a need of only 0.4 ml for detection of the swine virus in less than 1 h [39] or a multiplex microfluidic RT-LAMP device able to distinguish three types of influenza A substrains with a detection limit of 20 copies per reaction in 30 min [40]. In a similar way, a colorimetric device based on a RT-LAMP assay has been reported to detect hepatitis E virus by visual detection with a limit of detection of 10 copies and low handling and equipment requirement [41]. Last year a completely automated POC device termed Microfluidic Biomolecular Amplification Reader was reported to process raw samples through combination of molecular, microfluidic, optical and electronic technologies. This battery-powered instrument, which uses light emitting diodes and phototransistors, is able to detect the HIV-1 integrase gen in 70 min [42]. Recently, a novel microfluidic biochip has been manufactured that integrates LAMP, line probes assay and giant magnetoresistive sensors, having high analytical sensitivity for hepatitis B virus detection (10 copies per ml -1 ) in 1 h [41].

Helicase-dependent amplification
Helicase-dependent amplification (HDA) [43] is an elegant method based on a natural DNA replication mechanism, without the need of a denaturation step like PCR. Originally HDA process was performed through three different proteins (plus Polymerase): the DNA helicase, which unwinds the dsDNA, and two additional proteins (FIGURE 2). Nevertheless, the new thermostable helicase Tte-UvrD from Thermoanaerobacter tengcongenesis is able to work at 65˚C, allowing more specific reactions without both MutL and SSB [44]. HDA amplification usually takes 60-120 min, at least for low-copy numbers, but possible modifications for optimization can be done [45]. Although only a single set of primers is needed for HDA, few multiplex HDA applications have been developed so far such as a duplex-detection of pathogens N. gonorrhoeae and Staphylococcus aureus by combining HDA with microarray-based detection [46], and a 4-plex detection assay for four different genes from the same organisms [47]. HDA is compatible with diverse detection mechanisms like fluorescent detection [48] or electrochemical DNA detection with gold nanoparticles [49]. This technology has been successfully used for different types of samples including urine [45], stool [50], blood [3] and plasma [51].
BioHelix (Beverly, MA, USA), the developers of HDA technology, commercialize diverse kits for end point or real-time HDA detection such as IsoAmp Ò II Universal tHDA and IsoAmp III Universal tHDA Kit. These kits are aimed for further development of home-made techniques. In addition, a BESt Cassette Type II for lateral flow detection in 5-10 min has reached the stage of early commercialization. Several assays have being developed for the detection of, for example, S. aureus [52], or HIV-1 [51] by lateral flow with a time of detection of 20-40 min. However, only the kit for Herpes simplex virus detection (IsoAmp HSV) by Biohelix is currently available in the market (TABLE 1).
Andresen et al. [46] developed a HDA/microarray for the detection of N. gonorrhoeae and S. aureus in 2 h, in single or a duplex way and therefore suitable for the simultaneous detection of various pathogens. This microarray could be regarded as the first fully integrated microfluidic device that combines sample preparation and real-time HDA with capability to detect 100 genomic copies. Nonetheless, this method does not improve time to detection of PCR techniques and neither does the device developed by Mahalanabis et al. for E. coli detection [50]. In contrast, Ramalingam et al. [53] designed a microfluidic device where all flow is by capillarity and absorbent pad, without pumps or valves. Such device allows for HDA real-time detection of SARS-coronavirus in 30 min. In addition, Zhang et al. [54] presented a droplet microfluidic integrative technique where isolation, HDA amplification and detection occur in the same chip. Finally, steps in the direction of free-electricity pathogen detection have been done combining a nearly power-free nucleic acid extraction with an electricity-powered isothermal amplification assay for diarrhea diagnosis [55].
HDA seems to be an appropriate method for low-cost POC utilization. However, the relatively modest detection limit <100 copies achieved demands further improvement in sensitivity.

Recombinase polymerase amplification
Recombinase polymerase amplification is a low-temperature (37-42˚C) isothermal amplification method that uses three proteins in the reaction [56]: recombinase protein, DNA polymerase and DNA-binding protein (gp32) to prevent reannealing. RPA facilitates amplification in 20-40 min (FIGURE 3). Detection methods such as intercalating dyes, molecular beacons and TaqMan technologies are incompatible with RPA. For this reason, other detection strategies such as Twist-Ampexo and TwistAmp fpg have been developed by using sequence-specific probes.
Successful detection of less than 10 copies of HIV-1 DNA within 20 min has been documented for a RPA assay [57]. Likewise, a real-time reverse transcription recombinase polymerase amplification assay has been developed for fast detection of foot-and-mouth disease virus within 10 min [58] or bovine coronavirus in 10-20 min [59] as well as for targeting different viruses and bacteria biothreat agents [60].
TwistDX (Cambridge, UK) commercializes a variety of RPA products for developing both end point and real-time assays such as TwisAMP and TwistAmp Ò exo (probe included), but primers and template have to be supplied by the user. This company also has commercial kits for detection of Salmonella, L. monocytogenes, Campylobacter and Red snapper (fish) determination (TABLE 1), together with some devices to perform amplification and detection like the T-16 Isothermal Device, Twirla TM and Twista Ò portable realtime fluorometer. Being a novel technology, there is little background about RPA. However, it appears to be one of the fastest amplification techniques, which is an appealing feature for adaptation to POC devices. In this regard, a fully automated chip with a centrifugal disc format has been developed by Lutz et al. This device, with pre-stored freeze-dried RPA reagents, has been described to allow detection of gene mecA of S. aureus in 20 min [61]. Novel development of digital RPA on a SlipChip indicates that RPA may potentially be fast and sensitive with low volumes [62].

Nicking & extension amplification reaction
Nicking and extension amplification reaction (NEAR) is based on an isothermal method termed exponential amplification reaction [63]. Exponential amplification reaction uses nickingenzymes that by nicking the DNA target expose sites for primer annealing and allow DNA amplification in a few minutes. This method seems to be strongly limited by the requirement of native restriction flanking the target sequence to be amplified [64]. By contrast, NEAR allows amplification of any target by adding restriction sites in flank regions. Only one strand is nicked in NEAR while, comparatively, SDA avoids the requirement of dATPaS (5´-O-l-thiotriphosphate) to prevent double-stranded cleavage [65]. NEAR meets two basic criteria that are desirable for integration into POC tests: no DNA denaturation is needed and higher tolerance to inhibition than PCR is achieved [65].
Little is known about the NEAR technology, although first reports have described high analytical sensitivity for detection of N. gonorrhoeae and C. trachomatis (10 DNA copy number) and Clavibactermichiganesis and Ralstoniasolnacearum (50 copies of detection level), both of them with time to results within 10 min [66,67].

Rolling circle amplification
Rolling circle amplification (RCA) [68] is an isothermal method based on strand displacing DNA polymerase f29DNAP that is able to amplify continuously circular ssDNA (SUPPLEMENTARY FIGURE 3). A peculiar feature of RCA is that the unique concatenated DNA molecule that results as a product of the reaction allows in situ amplification [69]. This characteristic could be interesting for applications in which maintenance of cellular morphology is important. Another interesting application of RCA is the ultrasensitive protein detection by sandwich immunoassay [70]. Intriguingly, in spite of having been described almost 20 years ago, no RCA diagnostic test targeting pathogens has reached the market yet.
Different approaches to integrate RCA into a chip have been performed up to now. Sato et al. created a microchip that integrates all operating processes, including RCA [71]. Mahmoudian et al. combined RCA amplification followed by electrophoresis in a lab-on-a-chip assay for Vibrio cholerae detection in less than 65 min [72]. Reiss et al. performed a RCA in a flow-through system that allows stretching the amplified product and visualizing it under fluorescence microscopy [73].

Smart amplification process & signal mediated amplification of RNA technology
The novel LAMP-based smart amplification process (SMAP) v.2 technology [74] should not be confounded with the Signal mediated amplification of RNA technology (SmartAmp2). SMAP2 primes and enzymes are similar to LAMP, but it adopts an asymmetric primer design. SMAP2 is used to distinguish single nucleotide polymorphism clones differing by only one nucleotide, in contrast to LAMP reaction where targets have lower identity and primer design is more flexible. Therefore, in SMAP2 reactions, primers could anneal with wild-type and single nucleotide polymorphism variant through different amplification pathways by two priming event: primer priming (specific) and self-priming (nonspecific). An asymmetrical design of primers allows reducing the selfprimer event that contributes to the 'background/wild-type' amplification product.
This specific design takes into account that wild-type and target sequences could just differ in one nucleotide. Five primers are required: turn-back primers (TP), folding primers (FP), boost primer (BP) and two outer primers (OP1 and OP2). A second special feature of SMAP2 is the use of Thermusaquaticus MutS, an enzyme that scans DNA and identifies

Isothermal techniques for infection
Review informahealthcare.com mismatches with single-nucleotide sensitivity (FIGURE 4). When MutS finds a mismatch, it binds to it in an irreversible way, blocking the action of the DNA polymerase and, consequently, DNA amplification. Both asymmetric primer design and MutS allow single-nucleotide discrimination, features that make this technique very useful for single nucleotide polymorphism identification [75].
Little information can be found in the literature about SMAP2, but this technology has been documented to have not only high specificity but also high analytical sensitivity (three copies) [74]. Precise design of primers is needed to achieve desirable specificity and sensitivity, especially in relation to TP primers [76] and FP primers [77]. The use of SMAP2 for infectious disease diagnosis is still limited. A first progress has been made in the detection of pandemic flu within 40 min based on this technique coupled to a reverse transcriptase reaction (RT-SMAP2) [78]. However, as far as we know, kits for research or diagnostic utilization have not been commercialized yet.

Isothermal & chimeric primer-initiated amplification of nucleic acids
Isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN) [79] is an isothermal method (around 55C ) based on 5´-DNA-RNA-3´chimeric primers, thermo-stable RNaseH and BcaBEST DNA polymerase with stranddisplacing activity. Chimeric primers bind to ssDNA template, after initial dsDNA denaturation by heat, starting the synthesis of the new DNA strands. These strands are straightaway nicked by the RNase action at the penultimate 3´RNA residue allowing DNA amplification in a repetitive cycle until the primer is too short (FIGURE 5).
The amplification product generated by ICAN technology can be detected in real time in a more sensitive way (25 times) than the conventional PCR method [80]. At least 2-plex detection systems have been described for simultaneous detection of C. trachomatis and N. gonorrhoeae, by luminescent probe [81]. In this case, analytical sensitivity between 10 and 100 copies was achieved after 3.5 h for detection.
A few approaches have been done in order to incorporate this technology into POC assays. A semiautomated POC test able to detect spores of Bacillus subtilis by fluorescence has been described [82] as an example of biological warfare agents detection. Nonetheless, low analytical sensitivity (10 4 spores) and long time for detection (2 h) require further improvement for effective POC application. Another POC approach is the ICAN-chromatostrip for detecting Salmonella invA, which was documented to have higher diagnostic sensitivity than a standard PCR assay [83]. An N. gonorrhoeae test kit (ICAN NG-QR) and a Salmonella Detection Kit for DNA chips (ICAN TM DNA) were ICAN-based products commercialized by Takara Bio (Otsu, Japan). However, no ICAN products for research or diagnostic use are available in the market at present. (1)

Expert commentary
The current landscape of emerging isothermal diagnostic solutions for infectious diseases covers a variety of technologies (TABLE 2) that allow amplification of both DNA (ssDNA or dsDNA) or RNA (with a previous reverse transcriptase step). Interestingly, some methods such as LAMP, RCA and SMAP2 produce a continuous concatemeric DNA strand that could be useful for in situ detection. It is noteworthy that the majority of isothermal techniques have been attributed high analytical sensitivity within a range of 1-10 copies in previous studies, whereas SDA, ICAN and NEAR seem to be less sensitive. Nevertheless, since most evaluations have been performed at the development stage in academic laboratories, further studies on precommercial or available products should be undertaken to confirm these data. Certain techniques (LAMP, SMAP2, SDA, HDA, RPA and NEAR) appear to show mid-to-high tolerance to biological compounds and allow a low-or nonpretreated sample process. This could be an interesting characteristic for POC use and procedural simplicity. Denaturation is a previously needed step for some technologies as it obviously is for PCR. Since heating requirements at 95˚C for DNA extraction and denaturation are moderate, this factor may facilitate their integration into power-free POC instruments that would be particularly suitable for use in remote settings [55]. The integration of isothermal techniques into POC devices offers the advantages of simple instrumentation and the possibility of certain technologies to process raw samples, thus reducing costs of products and processes involved. However, most emerging isothermal near-POC or POC applications have only reached a proof-of-concept stage. Further evaluation studies are needed to determine their feasibility for commercialization.

Five-year view
Interest and expectation around POC disease diagnostics are increasing. Specifically, there has been an exponential growth of near-POC or POC developments based on PCR and isothermal amplification technologies for diagnosis of infectious diseases in the last years. Because of their competitive advantages, isothermal technologies are likely to become reference chemistries for future POC applications and could gradually displace PCR-based solutions. Diverse isothermalbased technologies are tacking technical challenges at the micro-scale and finding their way to market. Low sample volume handling, low concentration of target, complex signal amplification due to Table 2. low concentration, target instability and compatibility and integration of the various device subsystems have been identified as major hurdles for the development of POC assays. It appears that LAMP has reached a higher degree of maturity than other isothermal techniques to overcome such hurdles, according to the number of related developments and products. However, technical potentialities of LAMP or any other competing chemistries do not automatically guarantee their future adoption. In addition to further validation in clinical labs to obtain solid results, a range of cultural, organizational, economic and regulatory barriers may undermine successful deployment of diagnostics at the point of care, either isothermal or based on other technologies. One of the main challenges for effective implementation in the upcoming years will be to gain a positive perception from end users and healthcare providers. Since rapid diagnosis by POC assays alleviates patient anxiety and eliminates return visits for results, it appears that delivery of diagnosis on site will be well accepted by end users. However, acceptance of decentralized diagnostics by healthcare providers will imply important changes in clinical practices, workloads and logistics. New frameworks will need to be designed at the point of contact between the patient and the healthcare system to ensure adequate on-site sample collection, test performance and quality control of results [84].
Similarly, protocols of communication between healthcare centers and public health agencies will have to be put in place so that results of POC assays are properly captured and registered for adequate centralized surveillance and control of infectious diseases. Another challenge to be met in the near future will be to establish the final price for a POC test that is acceptable to payers, not only in terms of low cost but also of willingness to pay. It will be important to optimize costs of reagents, materials and systems of mass production and assembly of device components. Nonetheless, the final decision to purchase a POC test or a centralized laboratory test will not strictly be which test is cheapest or faster but rather the cost of the test needed to provide the desired clinical benefit [85]. On the other hand, future widespread use of POC devices will be constrained by nonharmonized regulatory conditions for commercialization of POC devices in diverse geographical markets, especially in Western countries.
Being aware that unexpected events may encourage or limit evolution toward near-patient diagnostics, we envision two scenarios where initial adoption of isothermal POC tests is more plausible within a timeframe of 5 years. Core laboratories, which are usually located in referral hospitals of industrialized countries, will presumably be the first market segment to be penetrated. Such environments are already familiarized with performance of PCR and other NAATs and can easily evaluate characteristics of isothermal POC solutions against current methods. Minor organizational changes may be needed for implementation of isothermal assays in specialized laboratories, but it will be fundamental that they prove to have sensitivity and specificity comparable to gold standard techniques for pathologies of complex etiology, most prevalent in referral hospitals. The demonstration of cost-effectiveness will also be required prior to adoption to ensure sustainability of diagnostic services. POC applications that would be best valued referral hospitals could be assays targeting diagnosis of time-critical, chronic or highly transmissible infections, as well as differential diagnosis of infections with common symptoms and different treatments and those requiring complex, toxic or expensive therapies. In a mid-term time, POC applications may be able to migrate from core laboratories to other areas within the referral hospital (emergency wards, intensive care units) and beyond (second-level hospitals, primary care), provided that they maintain diagnostic accuracy, rapidity and cost-effectiveness and allow use by nonspecifically trained personnel.
A second scenario for the implementation of POC testing in the next years will be the global health market. The WHO estimated that in 2008 resource-limited countries experienced 241 million cases of malaria, 2.8 million new infections with HIV, 7.8 million cases of reactivated TB and 217 million cases of bacterial pneumonia [86]. In fact, simple inexpensive POC tests based on antibody detection that are close to the 'ASSURED' ideal are already available in most developing countries in a lateral-flow or dipstick format (i.e., for the diagnosis of human immunodeficiency virus infection). It also needs to be highlighted that the majority of population in the developing world resides in remote rural areas, so patients often travel long distances to primary healthcare settings and thus may be unable to return for test results. Moreover, primary care infrastructures may have poor availability of trained technicians and limited electricity supply for equipment or refrigerators for the storage of reagents. Portable, extremely low-cost, very easy-to-use isothermal POC tests that provide rapid visual readouts with minimal instrumentation and reagents stable at room temperature would have wide acceptance in this scenario. In particular, it is expectable that mobile phones, which are prevalent in the developing world, will become fundamental complements of POC applications since they can contribute capabilities of battery-powered supply, readout and connectivity for transmission of results. The big size of the global health market and its urgent need for more accessible diagnostics offer attractive opportunities for manufacturers that have the ability to scale up POC applications and reduce prices. While margins may be low in the poorest countries, POC products may allow profitable commercialization in emerging middle-income countries. Those applications that target infections with a heavy burden on global health or that match with those occurring in developed countries may have higher potential for success in the short term.

Financial & competing interests disclosure
The authors acknowledge support from The Seventh framework programme of the European Union (FP7-SME-2013, grant 606488). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. This includes employment, consultancies, honoraria,

Isothermal techniques for infection
Review informahealthcare.com stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.

Key issues
• An ideal diagnostic test should meet the 'ASSURED' criteria: affordable, sensitive, specific, user-friendly, rapid and robust, equipmentfree and delivered to those who need it.
• Current PCR-based devices, even those intended for application at the point of care, need to be instrumented for performing thermal cycling amplification steps and have limited portability and affordability.
• Molecular isothermal amplification technologies may facilitate development of electricity-free, non-or minimally instrumented point-ofcare devices that could be particularly suitable for use in developing countries.
• Proof-of-concept studies in academic laboratory environments have reported that most isothermal techniques have similar or better sensitivity, specificity and rapidness than PCR assays.
• Further studies in real field conditions are needed to validate the promising diagnostic characteristics of isothermal techniques.
• Loop-mediated isothermal amplification, smart amplification process & signal mediated amplification of RNA technology, helicasedependent amplification, strand displacement amplification, recombinase polymerase amplification and nicking and extension amplification reaction are isothermal techniques with mid/high tolerance to inhibitory compounds that allow the use of raw samples without any pretreatment step, which may be an interesting feature for PCR-based point-of-care (POC) testing.
• Isothermal techniques such as loop-mediated isothermal amplification, nicking and extension amplification reaction and recombinase polymerase amplification could be adequate for integration into POC tests due to their rapidness.
• The introduction and use of isothermal POC applications will need to meet the diverse organizational, economic and regulatory challenges before successful adoption.
• Central laboratories in industrialized countries and primary health centers in resource-limited countries are the most plausible scenarios for the deployment of isothermal POC tests in a 5-year view.


Synthesis of Eu2+-activated Rb–Ba–Sc–Si–O glass phosphors using melt synthesis technique

Eu2+-activated RbBaScSiO glass phosphor was synthesized by a melt synthesis technique using arc-imaging furnace and their photoluminescence properties were characterized. In addition, the obtained glass phosphors annealed in a flow of 5%H2 95%Ar gas to enhance the luminescent efficiency. The emission spectra of the Eu2+-activated glass phosphors presents yellow emission band centered at 537 nm due to the 4f65d1 1⁄4 4f7 transition of Eu2+, and the emission intensity was effectively enhanced by the annealing treatment.

Introduction
Phosphor-converted white light-emitting diodes (pc-white LEDs) have been widely applied in displays and lamps as an alternative to conventional incandescent and fluorescent lamps. White LEDs have a number of advantages, such as a high luminescent efficiency, a long lifetime, compactness, environmental friendliness, and designable features. 1) However, the pc-white LEDs have some problems because they are generally embedded in epoxy or silicone resins. 2)4) The large difference of refractive index between phosphors and organic resins will lead to high amount of scattering light. Furthermore, these organic resins have the poor heat-or light-resistance, which will also attribute to the degradation of luminescence emission intensity and the change of emission color. Recent years, therefore, the glass phosphors have been extensively investigated as a promising materials for white LEDs, because they have efficient luminescence, excellent heator light-resistance and ease of formability. 5) 11) Among several phosphor materials, Eu 2+ -activated phosphors are suitable for the tri-color phosphor converting white LEDs. These phosphors have excellent optical absorption at near UV and blue light region due to the 4f 7 ¼ 4f 6 5d 1 transitions of Eu 2+ coupled to the host lattice. 12) In particular, Eu 2+ -activated silicate phosphors have been widely investigated because they can show good luminescence properties due to the strong crystal fields with the covalent SiO bond. 13)15) The strong crystal field strength around Eu 2+ in the host lattice was contribute to the increase of the energy splitting of 4f 6 5d excited level, therefore, the silicate phosphors are expected to be obtained the excitation and emission band in the longer wavelength range.
In this study, we are synthesized the Eu 2+ -activated Rb BaScSiO glass phosphors by the melt synthesis technique using arc-imaging furnace. We have previously demonstrated that the RbBaScSi 3 O 9 :Eu 2+ powder phosphor exhibit the blue-green emission with good luminescence properties under near-UV excitation. 16) In addition, the melt synthesis technique using arcimaging furnace is suitable for synthesizing powder phosphors with high luminescence efficiency. 17)19) This method has an interesting feature because it is a high-temperature approach for synthesis, which is rather opposite from the solid-state syntheses. The melt reactions are extremely rapid and homogeneous because of liquid mixing and rapid diffusion in the liquid phase, in contrast with the conventional solid-state reactions. Melting using arc-imaging furnace requires no container at various atmospheres up to high temperatures even greater than 2000°C. Furthermore, this method has rapid cooling process, which indicates that this method is not only suitable for synthesizing powder phosphors but also suitable for synthesizing glass phosphors.

Experimental
Rb 2 CO 3 , BaCO 3 , SiO 2 , Sc 2 O 3 and Eu 2 O 3 were mixed in a stoichiometric ratio using a mortar with acetone, in which the Eu 2+ content was adjusted between 0.5 and 5 mol %. The homogeneous mixture placed on a copper hearth, and then melted in a flow of 5%H 2 95%Ar gas using arc-imaging furnace. Finally, the glasses were annealed at 900 and 950°C for 12 h in a flow of 5%H 2 95%Ar gas. Figure 1 illustrates the schematic diagram of arc-imaging furnace. Arc-imaging furnace use the xenon lamp (6 kW) as a light source. The emitted strong light from xenon lamp was reflected by ellipsoidal aluminum mirror and the high energy light corrected on the cupper sample holder. The sample can be heated rapidly up to the high temperature over 2000°C in 1 min and also cooled by removing the sample stage from the mirror focus. The cooling rate was estimated as more than 100°C/s. Powder XRD data were obtained using a diffractometer (MX-Labo; Mac Science Ltd.) with Cu K¡ radiation. The photoluminescence emission and excitation spectra were measured at room temperature with a spectrofluorometer (Jasco Corp. FP-6500/6600). The transmittance curves were measured with a ultravioletvisible spectrometer (Jasco Corp. V-550). The Commission International de I'Eclairage (CIE) chromaticity coordinate date for the phosphor were measured at room temperature with a luminescence colorimeter (Hamamatsu C7473-36 PMA-11).

Results and discussion
The XRD patterns of the x mol % Eu 2+ -activated RbBa ScSiO (0.5¯x¯5) glass phosphors synthesized by the melt synthesis technique using are-imaging furnace were obtained completely amorphous patterns without peaks as shown in Fig. 2. The maximum luminescent intensity was observed for 1 mol % Eu 2+ -activated RbBaScSiO glass phosphor by the optimization of the Eu 2+ concentration. Therefore, the Eu 2+ concentration in the RbBaScSiO glass phosphor was fixed at 1 mol %. The photographs of the 1 mol % Eu 2+ -activated Rb BaScSiO glass phosphor before annealing treatment are shown in Fig. 3. The body color of the obtained glass phosphor was yellow, and presented the yellow emission under excitation at 365 nm light irradiation. In contrast, under excitation at 254 nm light, the obtained glass phosphor show the red emission, which is considered to be due to the Eu 3+ existing in the glass phosphor. The emission intensity of the obtained 1 mol % Eu 2+ -activated RbBaScSiO glass phosphor compared with that of a commercial Y 3 Al 5 O 12 :Ce 3+ (P-46) powder phosphor was attained up to only 9%, which is too low for use in white LEDs. Therefore, the Eu 2+ -activated RbBaScSiO glass phosphor annealed in a flow of 5%H 2 95%Ar gas to enhance the emission intensity.
The XRD patterns of the 1 mol % Eu 2+ -activated RbBaSc SiO glass phosphors before and after annealing treatment at 900 and 950°C are shown in Fig. 4. The samples prepared before and after annealing treatment at 900°C were obtained completely amorphous patterns. In contrast, the sample annealed at 950°C observed the surface crystallization and the XRD pattern of this sample is in good agreement with that of the single phase RbBaScSi 3 O 9 structure. These results indicate that the crystallization of this glass phosphor has occurred beyond 900°C. Figure 5 shows the transmittance curves of the 1 mol % Eu 2+activated RbBaScSiO glass phosphors before and after annealing treatment at 900°C. Since these glass phosphors has    yellow body color, the optical absorption behavior was observed in all samples in the region of wavelength shorter than 500 nm (i.e., in the blue region), which is the complementary color to yellow. In addition, the 1 mol % Eu 2+ -activated RbBaScSiO glass phosphors before and after annealing treatment at 900°C shows high transmittance properties, although the transmittance of the obtained glass phosphor was slightly decreased by the annealing treatment. The transmittances of the 1 mol % Eu 2+activated RbBaScSiO glass phosphors before and after annealing treatment at 900°C were 60 and 55% at 700 nm, respectively. Figure 6 illustrates the excitation and emission spectra of the 1 mol % Eu 2+ -activated RbBaScSiO glass phosphors before and after annealing treatment at 900 and 950°C. The excitation spectra of the samples prepared before and after annealing treatment at 900°C are consisted of a broad band covering the region from the UV to blue light part. The emission spectrum under excitation at 330 nm exhibits a broad yellow emission band centering at 537 nm, corresponding to the allowed 4f 6 5d 1 ¼ 4f 7 transition of Eu 2+ . In the sample before annealing treatment, a sharp peak observed at 610 nm are assigned to the 5 D 0 ¼ 7 F 2 transition of Eu 3+ , while no red line emissions from Eu 3+ are observed in the sample after annealing treatment at 900°C. This indicates that the dopant trivalent Eu 3+ ions were completely reduced to the divalent Eu 2+ ions by the annealing treatment, and result in the excitation and emission peaks intensities were effectively enhanced about two times. The relative emission intensity of the grounded powder sample after annealing treatment at 900°C was 20% compared with that of the commercial Y 3 Al 5 O 12 :Ce 3+ (P-46) powder phosphor. Although the emission intensity of the obtained glass phosphor is lower than that of the conventional powder phosphor, since the amount of absorption due to glass phosphor is generally smaller than that of the powder phosphor, it is expected to be a suitable for material of the remote phosphor or high powder large materials. On the order hand, the sample annealed at 950°C presents the broad blue-green emission band peaking at 500 nm, which is similar to that of RbBaSc-Si 3 O 9 :Eu 3+ powder phosphor reported in our previous study, 16) and the emission intensity became smaller than that of the samples annealed at 900°C. Figure 7 presents the CIE chromaticity diagram for the 1 mol % Eu 2+ -activated RbBaScSiO glass phosphor after annealing treatment at 900°C. Results for a commercial Y 3 Al 5 O 12 :Ce 3+ (P-46) phosphor is also included for comparison. The CIE chromaticity coordinate values for the 1 mol % Eu 2+ -activated RbBa ScSiO glass phosphor were x = 0.45 and y = 0.43 under near-UV irradiation. The x and y values of this glass phosphor are more close to white light compared with a commercial Y 3 Al 5 O 12 :Ce 3+ (P-46) phosphor (x = 0.47, y = 0.49). Figure 8 shows the normalized emission spectra of the 1 mol % Eu 2+ -activated Rb    BaScSiO glass phosphor after annealing treatment at 900°C and a commercial Y 3 Al 5 O 12 :Ce 3+ (P-46) phosphor. The emission band of the 1 mol % Eu 2+ -activated RbBaScSiO glass phosphor has the larger full width at half maximum (FWHM) of emission band than that of a commercial Y 3 Al 5 O 12 :Ce 3+ (P-46) phosphor and it was widely curved in the high spectral luminous efficacy region. In addition, the glass phosphor obtained in the present study show the high transparency (Fig. 5). These results suggest that that Eu 2+ -activated RbBaScSiO glass phosphors are a suitable candidate for the yellow-emitting phosphor of near UV based white LEDs lamps using remote phosphor. 20)23) In addition, this glass phosphor was expected to be also used in the laser excited high powder optical devices.

Conclusions
The Eu 2+ -activated RbBaScSiO glass phosphor was synthesized by the melt synthesis technique using arc-imaging furnace. The obtained glass phosphor was annealed in a flow of 5%H 2 95%Ar gas to increase the reduction effect, as a result, the emission intensity was effectively enhanced by the annealing treatment. The excitation spectra of the Eu 2+ -activated glass phosphors are consisted of broad band covering the region from 220 to 450 nm, and presents yellow emission band centered at 537 nm due to the 4f 6 5d 1 ¼ 4f 7 transition of Eu 2+ . These results indicate that Eu 2+ -activated RbBaScSiO glass phosphors are a suitable candidate for the yellow-emitting phosphor of near UV based white LEDs lamps using remote phosphors.