extfieldsofstudy: Biology Medicine Chemistry

s2fieldsofstudy: Chemistry Biology

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Sialic Acid Mutarotation Is Catalyzed by the Escherichia coli β-Propeller Protein YjhT*

The acquisition of host-derived sialic acid is an important virulence factor for some bacterial pathogens, but in vivo this sugar acid is sequestered in sialoconjugates as the α-anomer. In solution, however, sialic acid is present mainly as the β-anomer, formed by a slow spontaneous mutarotation. We studied the Escherichia coli protein YjhT as a member of a family of uncharacterized proteins present in many sialic acid-utilizing pathogens. This protein is able to accelerate the equilibration of the α- and β-anomers of the sialic acid N-acetylneuraminic acid, thus describing a novel sialic acid mutarotase activity. The structure of this periplasmic protein, solved to 1.5Å resolution, reveals a dimeric 6-bladed unclosed β-propeller, the first of a bacterial Kelch domain protein. Mutagenesis of conserved residues in YjhT demonstrated an important role for Glu-209 and Arg-215 in mutarotase activity. We also present data suggesting that the ability to utilize α-N-acetylneuraminic acid released from complex sialoconjugates in vivo provides a physiological advantage to bacteria containing YjhT.

Sialic acids are an important family of related 9-carbon sugars acids, present on the surface of many different cells and functioning in a wide range of different biological processes (1)(2)(3). The most common sialic acid, N-acetylneuraminic acid (Neu5Ac), 3 is the predominant form present in humans and can be found as a terminal sugar on a wide range of surface glycoconjugates (2). A number of bacteria that can colonize humans make use of Neu5Ac as a nutrient source; for example, the intestinal bacterium Escherichia coli can grow on Neu5Ac as a sole carbon source (4). The intestinal mucous layer is rich in sialic acids, and E. coli genes required for sialic acid catabolism are induced in this environment and are important for colonization (5). Additionally, uropathogenic E. coli express genes for sialic acid catabolism during growth in human urine (6). Other bacteria, like the respiratory pathogen Haemophilus influenzae, have an additional use for sialic acid in an immune evasion mechanism by adding Neu5Ac to their lipopolysaccharide (7,8), which provides increased survival in human serum (9,10). H. influenzae is unable to synthesize Neu5Ac de novo and hence must acquire it from the host (9,11). This has recently been demonstrated to be dependent on a tripartite ATP-independent periplasmic (TRAP) transporter (12)(13)(14), which is encoded in a genomic region that also contains the genes for sialic acid catabolism (1,12). Directly downstream of the transporter genes (siaPQM) is an uncharacterized gene, HI0148 (Fig. 1), which encodes a predicted periplasmic protein that has been suggested to contain 7 Kelch motifs and be involved in sialic acid catabolism (15), but is not essential for Neu5Ac uptake (12). In E. coli the orthologue of this gene is yjhT, which is the second gene in a potential three gene operon, yjhATS, whose expression is induced by sialic acid (16). The first gene in this operon, yjhA, has already been demonstrated to encode a Neu5Ac-specific porin in the outer membrane of E. coli and has been renamed nanC (16). These data suggest a sialic acid-related function for the yjhT/HI0148 gene products.
Here we describe the function of YjhT as a representative of this family of uncharacterized periplasmic proteins from pathogenic bacteria and demonstrate that this protein functions, unexpectedly, as a sialic acid mutarotase. We report the structure of YjhT and identify amino acid residues important for enzyme activity in vitro. In addition, we provide in vivo data consistent with our suggested function for this protein.

EXPERIMENTAL PROCEDURES
Cloning and Expression of Native and Mutant YjhT-His 6 Proteins-The yjhT gene was amplified from genomic DNA of E. coli K12 MG1655 using KOD Hot Start polymerase (EMD Biosciences) and primers EcyjhT-NdeI and EcyjhT-XhoI (supplemental Table 1). The NdeI-and XhoI-cut PCR product was cloned into pET21b and confirmed by DNA sequencing. The resulting construct (pES5) encodes a C-terminally His 6 -tagged variant of YjhT, in which the tag is spaced from the protein by a Leu-Glu dipeptide. yjhT mutant alleles were generated using a modified QuikChange TM method (17) using pES5 as a template and KOD Hot Start polymerase. The mutagenic oligonucleotides used are listed in supplemental Table 1. Native and mutant YjhT proteins were expressed in BL21 (DE3) pLysS, which was grown at 25°C in 1.5 liters of LB with antibiotics to an OD 650 of 0.4 -0.5 before induction with 1 mM isopropyl 1-thio-␤-D-galactopyranoside overnight. Cells were harvested and incubated in SET buffer (0.5 M sucrose, 5 mM EDTA in 50 mM Tris-HCl, pH 8, 600 g/ml lysozyme) at 30°C for 1 h to release the periplasmic fraction, which was then clarified by centrifugation and dialyzed against 20 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 7.5. All YjhT variants were purified by nickel-affinity chromatography on a 1-ml HisTrap HP column (GE Healthcare) and dialyzed against double distilled water containing 100 mM NaCl; proteins were ϳ95% pure as judged based on SDS-PAGE. Protein concentrations were determined from the absorbance at 280 nm (A 280 ) using a theoretical molar absorption coefficient of 73,540 (Y309A mutant) or 74,820 cm Ϫ1 M Ϫ1 (all other variants). A SeMet derivative of YjhT was prepared as described previously (18) by growing BL21 (DE3) pLysS pES5 cells in double-strength M9 (19) supplemented with selenomethionine; the expression and purification procedures for this protein were those used for native YjhT. Throughout this paper we will refer to the concentration of YjhT as that of the monomer. The masses of all the recombinant proteins were confirmed using electrospray mass spectrometry (see supplemental Experimental Procedures), which demonstrated that all had been correctly processed by removal of their signal peptide.
Crystallization Procedure-Purified SeMet-substituted YjhT was dialyzed against double distilled water and concentrated to 12 mg/ml. Crystallization experiments utilized the vapor diffusion method and a MOSQUITO nanoliter dispensing robot to set up sitting drops. Successful crystallization conditions were identified using the PACT screen (20). Crystals for data collection were grown from drops made up of 150 nl of SeMet-substituted YjhT and 150 nl of 200 mM sodium malonate, 0.1 M BisTris propane, pH 7.5, and 20% w/v polyethylene glycol 3350.
Data Collection and Structure Solution-A single wavelength data set extending to a resolution of 1.5 Å with a high redundancy was collected from a crystal of SeMet substituted YjhT at the ESRF, Grenoble, France, on beamline ID14-4 (Table 1). From the SAD signal with a resolution cutoff at 2.5 Å, the programs SHELXC and SHELXD (21) led to the location of all six selenium atoms in the asymmetric unit. Refinement of the selenium atom positions using the program MLPHARE provided experimental phase probability distributions in terms of the Hendrikson-Lattman coefficients. These served as restraints during the ARP/wARP-REFMAC model building process (22), which led to a model with 587 of 714 residues built and sequence coverage of 99%. The model was completed and refined in iterative cycles of REFMAC (23) and manual modeling in COOT (24). In subunit A there is clear electron density for residues 1-225 and 229 -349, with a disordered surface loop from 226 to 228. In subunit B, all residues from 1 to 349 are visible. The C terminus, residues 350 -357, is disordered in both subunits. Refinement statistics are given in Table 1. Coordinates and structure factors were deposited in the Pro-tein Data Bank (code 2uvk). The protein was compared with proteins of known structure using the DALI software (25). The size of the dimer interface was calculated using the Pro-Face software (26).
NMR Spectroscopy-All NMR experiments were performed in 50 mM deuterated acetate buffer, pH 5.5, 100 mM NaCl, 10% (v/v) 2 H 2 O. 1 H NMR spectra were acquired on a Bruker 700-MHz spectrometer at 25°C using 50 M 3-(trimethylsilyl)proprionic 2,2,3,3-d 4 acid sodium salt (Sigma) as a chemical shift (0 ppm) reference and using excitation sculpting (27) with gradients for suppression of the H 2 O signal. Saturation transfer difference (STD) (28) spectra of Neu5Ac (1 mM) with YjhT (10 M) were acquired with 8 scans and a 7-s saturation time with saturation at 0 ppm (saturation transfer) or at Ϫ20000 Hz (reference spectrum). A difference spectrum was obtained by subtracting the saturation transfer spectrum from the reference spectrum. One-dimensional spectra for the time courses were acquired with 128 scans and a 1-s delay between scans. Initially a "0" time point was acquired of 1 mM sialyllactose (␣(2-3) and ␣(2-6) mixture from human milk; Sigma). Then 0.6 units of Clostridium perfringens sialidase (type V, Sigma) and 0.5 M YjhT (or YjhT mutant) was added. Acquisition of the first spectrum took 5 min and was completed ϳ7 min after addition of the enzymes. Two-dimensional NOESY spectra were acquired with 1 mM Neu5Ac and 10 M YjhT, 64 scans, and a mixing time of 100 ms. The addition of protein (sialidase and/or YjhT) where I i is the intensity of the ith measurement of a reflection with indexes hkl, and ͗I͘ is the statistically weighted average reflection intensity. c R-factor ϭ ͚ʈF o ͉ Ϫ ͉F c ʈ/͚͉F o ͉, where F o and F c are the observed and calculated structure factor amplitudes, respectively. d R-free is the R-factor calculated with 5% of the reflections chosen at random and omitted from refinement. e Outer shell for refinement corresponds to 1.539 to 1.500 Å. f r.m.s.d. indicates root mean square deviation of bond lengths and bond angles from ideal geometry. g Percent of residues in favored/allowed/disallowed regions of the Ramachandran plot, according to MolProbity (61). and/or sugars did not alter the pH of the buffer. One-dimensional spectra were processed using Topspin (Bruker Bio-Spin) and SigmaPlot, whereas two-dimensional spectra were processed using NMRPipe (29) and viewed in NMRView (30).

Construction of Deletion
Mutants-For this study we used three strains from the Keio collection (31) with Kan R cassettes in the nanC (JW4274), yjhT (JW4273), and yjhS (JW4272) genes as well as the parental strain BW25113. We removed the Kan R cassette by transformation with pCP20 followed by plasmid curing as described previously (32). Removal of the Kan R cassette was confirmed by PCR, and the ⌬yjhS and ⌬yjhT regions were sequenced. Of note, the ⌬yjhS strain contained an additional base in the Scar peptide-coding sequence, resulting in an extended Scar peptide about 60 amino acids long.
Neu5Ac Uptake Assay-For [ 14 C]Neu5Ac uptake assays, single colonies from freshly streaked LB plates were inoculated in LB medium and grown for 8 h. Cells were harvested, washed once in M9 minimal medium, and inoculated into M9 with 0.4% v/v glycerol to an initial OD 650 of 0.05 and grown overnight. Cells were harvested, washed three times in M9, and then inoculated into M9 Neu5Ac (1 mg/ml) to an initial OD 650 of 0.1 and allowed to grow until mid-log phase (OD 650 ϳ0.4), when they were harvested, washed four times in M9, and finally resuspended to an OD 650 of 2 in 1 ml of M9 salts (no carbon source added), and stored on ice. To assay sialic acid uptake, such suspensions were diluted to an OD 650 of 0.3 in M9 salts prewarmed at 37°C and incubated with stirring at 37°C for 2 min, when uptake was initiated by adding [ 14 C]Neu5Ac (Sigma) to a final concentration of 0.25 M. At the indicated times, 400-l aliquots were taken, filtered through 0.45-m pore-size nitrocellulose filters (Millipore), and washed with 3 ml of M9 salts, and the radioactivity within them was measured as described (33). The protein concentration within each sample was determined by a modified Lowry assay (34).
Sequence Analysis and Bioinformatics-Sequence and genome contexts of yjhT orthologues were obtained from the SEED (35) with additional manual curation. Sequences were aligned using ClustalX (36).

YjhT Is a Periplasmic Protein That Interacts Weakly with
Sialic Acid-To investigate the function of this family of uncharacterized proteins, we studied the protein from the model bacterium E. coli K12 MG1655 (37,38), which, like other members of this family, is encoded within a sialic acid-induced operon known to contain other genes involved in sialic acid utilization (Fig. 1). The YjhT protein was overexpressed and extracted from E. coli periplasm as a C-terminally hexahistidine-tagged protein and then purified to apparent homogeneity in a single step using nickel affinity chromatography (supplemental Fig. 1, inset). The molecular weight of recombinant YjhT was examined using electrospray mass spectrometry (supplemental Fig. 1), which revealed two distinct sets of m/z peaks that were identified as having masses of 38,685 Ϯ 4 and 77,371 Ϯ 8. These are consistent with the monomeric (38,686) and dimeric (77,372) forms of the C-terminally His-tagged version of YjhT that had been processed to remove the 19-amino acid signal peptide upon export to the periplasm.
To examine whether YjhT was able to interact with Neu5Ac, we used 1 H NMR spectroscopy, a method used successfully to study other aspects of sialic acid biology (39). A 1 H NMR spectrum of Neu5Ac is shown in Fig. 2 (lower spectrum) where peaks have been assigned based on published spectra of Neu5Ac (40). The STD (28) spectrum of YjhT in the presence of Neu5Ac (Fig. 2, upper spectrum) demonstrates an interaction ; NanR, sialic acid-responsive repressor of the FadR/GntR family of transcriptional regulators; NanA, sialic acid aldolase; NanT, Neu5Ac transporter; NanE, N-acetylmannosamine-6-P epimerase; NanK,N-acetylmannosamine kinase; YhcH, unknown function; NanC, Neu5Ac-specific porin; YjhS, unknown function; YjhB, NanT-like transporter of unknown function; YjhC, unknown function; NagA, N-acetylglucosamine-6-P deacetylase; NagB, glucosamine-6-P deaminase; SiaPQM, Neu5Ac TRAP transporter; NanH, sialidase. In E. coli the yjhBC genes belong to the NanR regulon as do the nanATEKyhcH operon and the putative nanCyjhTS operon (1). *, Shigella spp. and pathogenic E. coli strains display the same genetic arrangements as E. coli K12, except that Shigella spp. lack yjhBC. The genetic structure of the Pasteurella multocida sialometabolic locus is very similar to that of H. influenzae (1,12). FEBRUARY 22, 2008 • VOLUME 283 • NUMBER 8 between YjhT and Neu5Ac. This STD signal was not observed with Neu5Ac alone, and STD signals were not observed in the presence of other sugars, specifically galactose, N-acetylglucosamine, and N-acetylmannosamine (data not shown).

YjhT Is a Sialic Acid Mutarotase
The ability to interact with Neu5Ac, the periplasmic localization and the predicted ␤-propeller structure (15), suggested two potential functions for YjhT. The first, as a lectin-like multivalent sialic-binding protein, such as the Psathyrella velutina lectin (PVL) (41), was not supported by a ligand binding assay that detects stable complexes between protein and Neu5Ac (supplemental Fig. 2). The second, as a sialidase (42,43), was tested using 1 H NMR spectroscopy and sialyllactose as a substrate. Only in the presence of a sialidase was there release of Neu5Ac from sialyllactose. In the presence of YjhT or with no added protein, sialyllactose was stable after overnight incubation (supplemental Fig. 3). These data suggest a novel function for this periplasmic protein related to its interaction with Neu5Ac.
YjhT Has a Neu5Ac Mutarotase Activity-In vertebrates, sialic acid is mostly present as part of larger sialoglycoconju-gates, where it is linked to the rest of the macromolecule exclusively by ␣-linkages (2). Free sialic acid is released by sialidase action as the ␣-anomer, which mutarotates spontaneously into ␤-Neu5Ac, which is the most abundant anomer at equilibrium (over 90%) (39,44). The spontaneous mutarotation rate of Neu5Ac is relatively slow, for example at pH 5.4 the t 0.5 is 80 min (45). Given that the doubling time of an E. coli cell (20 min) is faster than the spontaneous mutarotation rate, we reasoned that bacteria competing for a limited supply of sialic acid might gain a selective advantage by being able to mutarotate the ␣-anomer more quickly.
We investigated the possible Neu5Ac mutarotase activity of YjhT using 1 H NMR, a technique used previously to characterize other sugar mutarotases (46 -48). Unlike other techniques traditionally used to characterize these enzymes (for example, polarimetry), 1 H NMR also allows the detection of chemical exchange between anomers in solutions at equilibrium (46 -48), which is advantageous given the lack of commercially available ␣-Neu5Ac. Thus, we compared two-dimensional 1 H NOESY spectra of Neu5Ac acquired without (Fig. 3A) and with added YjhT (Fig. 3B); in these experiments anomeric exchange is detected only when this is quite fast, that is when it occurs within the mixing time of individual scans (100 ms). The spectrum in the presence of YjhT contains clear cross-peaks connecting the ␣ and ␤ frequencies that are not observed in its absence, indicating that in the presence of the protein the exchange between the two anomers has been accelerated so much that it is now within the detection limit of the 1 H NOESY experiment.
Next we wished to investigate YjhT-catalyzed Neu5Ac mutarotation in more in vivo like conditions where an excess of newly formed ␣-Neu5Ac mutarotates over time into the ␤-anomer. To overcome the limitation of the lack of commercial preparations of pure ␣-Neu5Ac, we used an NMR-based assay in which a sialidase is used to release ␣-Neu5Ac in situ from sialyllactose, the mutarotation of which is then followed over time in the absence (Fig. 4A) or in the presence of YjhT (Fig.  4B). The use of sialidases to release ␣-Neu5Ac in situ from a sialoglycoconjugate is an established method to produce nonequilibrium populations of the two anomers that mimics the in vivo situation (39). In the absence of YjhT, ␣-Neu5Ac accumulates as a consequence of sialidase action (Fig.  4A). This is particularly evident in the signal for the H3ax of ␣-Neu5Ac (H peak); the signal for the H3eq of ␣-Neu5Ac overlaps with those for the same hydrogen of the sialyllactose mixture (peak B), but it is still the most abundant peak. The intensities of the peaks for ␤-Neu5Ac (H3ax is peak E and H3eq is peak D) slowly increase during the time course of the assay because of spontaneous mutarotation, as observed in other studies (39), and only after 1 h is ␤-Neu5Ac the predominant species. When the experiment is  repeated in the presence of 0.5 M YjhT (Fig. 4B), strikingly ␣-Neu5Ac does not accumulate, and ␤-Neu5Ac is already the most abundant species after 7 min. These spectra are identical to the 1 H NMR spectrum observed for a solution of Neu5Ac at chemical equilibrium (for example in Fig. 2, lower spectrum).
Because ␣-Neu5Ac is the primary product of sialidase activity (and we have shown that YjhT is not a sialidase), this indicates that the ␣-anomer is being rapidly mutarotated into the ␤-anomer (see scheme in Fig. 5). Addition of YjhT also resulted in faster hydrolysis of sialyllactose, which is consistent with YjhT scavenging the sialidase primary product and thus pulling the reaction forward. These data demonstrate that YjhT is able to catalyze the exchange of the ␣and ␤-anomers at equilibrium and also can accelerate mutarotation in the direction which is likely to occur in vivo in nonequilibrium solutions. Investigation of the in Vivo Function of YjhT-Given the biochemical data demonstrating the mutarotase activity of YjhT, we wished to identify an in vivo phenotype for E. coli lacking a functional yjhT gene. Using a [ 14 C]Neu5Ac uptake assay, we were able to measure a 20% drop in the rate of Neu5Ac uptake using subsaturating amounts of substrate (Fig. 6). Significantly, neither a ⌬yjhS mutant (Fig. 6) nor a ⌬nanC mutant (data not shown) showed any uptake defect in these conditions demonstrating the ⌬yjhT specificity of this phenotype. The small phenotype is best explained by assuming that the E. coli Neu5Ac transporter NanT recognizes the abundant ␤-anomer so that only in the presence of the mutarotase is the ␣-Neu5Ac pool available to NanT during the time course of the assay. This assumption is consistent with the observation of a similar phenotype when these assays are repeated at pH 5.5 in MES minimal medium (data now shown), where the spontaneous mutarotation rate is decreased by at least 3-fold (45). In these conditions, the amount of total Neu5Ac taken up during the assay (20%) is twice that of the available ␣-Neu5Ac pool (Ͻ10%), and given that spontaneous mutarotation is slow (t 0.5 of 80 min at pH 5.5), this is not compatible with the linear uptake we observe.
We then investigated whether the ⌬yjhT mutant displayed any noticeable growth phenotype on Neu5Ac. We were able to grow E. coli cells on minimal medium supplemented with colominic acid, an ␣(2-8)-homopolymer of Neu5Ac that per se does not serve as a carbon source for E. coli but can be utilized if sialidase is supplied to release monomeric ␣-Neu5Ac (supplemental Fig. 4). Under these conditions a very small but highly reproducible growth delay was observed during growth of the ⌬yjhT mutant over the first few hours of incubation (supplemental Fig. 4A). This result is consistent with a transient period in which ␣-Neu5Ac is present in excess over its concentration at equilibrium allowing cells expressing yjhT to have a growth advantage. This defect was not observed for the ⌬yjhS mutant thus ruling out any polarity effects of the yjhT mutant. When free Neu5Ac (over 90% ␤) was used as sole carbon source, the ⌬yjhT mutant showed no growth defect even when the sugar was provided at concentrations sustaining growth rates comparable with those observed in colominic acid medium (supplemental Fig. 4B). No growth defect was observed also when cells were pregrown with glycerol rather than Neu5Ac (data not shown), indicating that expression of yjhT is required for this small growth advantage. Taken together these results are supportive of a function for YjhT in the efficient use of exogenous ␣-Neu5Ac.
YjhT Is a Kelch Domain Containing Protein That Forms a 6-Bladed ␤-Propeller-To further study the YjhT protein, we crystallized full-length protein containing a C-terminal hexahistidine tag and solved the structure to 1.5 Å (Table 1 and Fig.  7A). The structure is a dimer in the crystal, with each subunit consisting of a 6-bladed ␤-propeller that contains Kelch motifs (49), defined by residues 14 -47, 60 -100, 112-185, 195-233, 247-302, and 311-348 ( Fig. 7 and supplemental Fig. 5). The dimer interface is formed by a large continuous patch with a buried surface area of ϳ2500 Å 2 and contains multiple hydrogen bonds between the two subunits. The dimeric species of YjhT was visible in electrospray mass spectrometry analysis of the protein (supplemental Fig. 1), so we used analytical ultra-  FEBRUARY 22, 2008 • VOLUME 283 • NUMBER 8 centrifugation to confirm that YjhT is a dimer in solution. Data from sedimentation equilibrium experiments fit well to a single species of the dimeric molecular mass of 73.9 kDa (supplemental Fig. 6A), which does not change with protein concentration (supplemental Fig. 6B) implying that the dimer is highly stable.

YjhT Is a Sialic Acid Mutarotase
Extensions from blades II and III extend upwards from the top of the ␤-propeller and form a surface against which the second subunit packs at about a 90°angle to the first subunit, providing a larger interaction face than simply packing the two ␤-propellers against one another (43). The ␤-propeller is not closed using the typical "Velcro" mechanism seen in most other ␤-propellers (50) but is rather "unclosed" as seen first in the prolyl oligopeptidase (51). A DALI search revealed the best hit to be human Keap1 (52) with the second hit to the fungal galactose oxidase (53). Although these eukaryotic proteins have quite different functions, they both contain a Kelch motif ␤-propeller structure. This motif was first observed in the Drosophila Kelch ORF1 protein as a repeating element of around 50 amino acids, which is present in a range of other organisms performing a wide range of functions (49,54,55). The motif is characterized by a run of four hydrophobic residues followed by conserved twin glycines, which are followed by two conserved aromatic residues at a particular spacing (49). The Keap1 protein has an almost perfect set of 6 Kelch domains with all elements present in all 6 blades, but in YjhT there are some slight variations in a number of the blades (supplemental Fig. 5). Of note is a conserved pair of small/polar amino acids (N/D or G) found between strands a and b in all 6 blades, which are exposed to solvent in the structure.
Site-directed Mutagenesis of Conserved Amino Acids within YjhT Reveals a Potential Active Site-To investigate the enzymatic function of YjhT further, we used a sequence-based sitedirected mutagenesis strategy to identify residues potentially important for mutarotase activity. Selected YjhT homologues from other bacteria (see Fig. 1) were collected and aligned with   the secondary structural elements of YjhT (supplemental Fig.  7). There are a small number of conserved residues besides those that are part of the Kelch motifs, which all sit in the same region on the surface of the ␤-propeller (Fig. 8). We mutated these seven conserved residues (indicated on the alignment in supplemental Fig. 7) to alanine and expressed and purified the resulting recombinant proteins. We investigated their ability to enhance the mutarotation rate using the in vitro time course assay of sialic acid release and mutarotation. Significantly a E209A mutant of YjhT is severely impaired in its ability to enhance the mutarotation rate above the spontaneous rate (Fig.  4C), whereas an R215A mutant gives a decreased but observable activity (Fig. 4D). Replacement of Lys-11, His-278, Lys-283, Tyr-309, or Glu-325 with alanine residues has no effect on the activity of the protein in this assay (data not shown). The circular dichroism spectra of the E209A and R215A mutant are indistinguishable from that of the wild-type protein, suggesting that the phenotype was not because of misfolding of YjhT (data not shown). To further characterize the E209A mutant we acquired a 1 H NOESY spectrum of this protein in the presence of Neu5Ac and were unable to detect cross-peaks indicative of fast mutarotation at equilibrium as observed in the presence of the wild-type protein (data not shown). Interestingly the Glu-209 residue sits at the bottom of a deep cleft on the upper surface of YjhT and is very close to Arg-215 (supplemental Fig. 8), suggesting that this region of the protein is important for the activity of the enzyme. The mechanism of this enzyme is currently under further investigation.

DISCUSSION
We here report the in vitro and preliminary in vivo characterization of the product of the E. coli yjhT gene. Our in vitro analyses demonstrated that this ␤-propeller protein has a function as a mutarotase that accelerates the equilibration between the ␣and ␤-anomers of Neu5Ac. This is both a novel enzymatic activity related to bacterial sialic acid utilization and represents a new fold for a mutarotase. In accordance with the nomenclature of sialic acid metabolic genes in E. coli, we propose to rename the yjhT gene nanM (for N-acetylneuraminate mutarotase).
The existence of a sialic acid mutarotase has not been anticipated despite many years of work on the biology of this important cell surface sugar, presumably as this reaction does occur spontaneously. After release of the ␣-anomer from sialoglycoconjugates by sialidase action, Neu5Ac spontaneously mutarotates to reach an equilibrium position with over 90% in the ␤-anomer (see Fig. 4A) (39,44). In vitro NanM accelerates the conversion of newly released ␣-Neu5Ac into ␤-Neu5Ac so quickly that the ␣-anomer never accumulates after its release; thus it seems reasonable that the physiological role of NanM might be to facilitate a sialidase-negative bacterium such as E. coli to compete successfully for limited amounts of extracellular Neu5Ac, which is likely to be taken up as ␤-Neu5Ac. Also, given that endogenously released sialic acid is an inflammatory indicator in the host (56), its rapid removal from solution might be advantageous to the bacterium to damp down host responses. Sialic acid is an important molecule in the life-style of both pathogenic and commensal strains of E. coli, and although its significance in vivo has been normally associated with the synthesis of a polysialic acid capsule by E. coli K1 and K92 strains, there is now evidence demonstrating that unencapsulated strains of E. coli can also use sialic acid to their advantage as a carbon and nitrogen source in vivo. Mucosal surfaces that are colonized by E. coli contain high levels of sialylated mucins, and free sialic acid can be made available to E. coli by the action of sialidases expressed by other bacteria in the same niche or by host sialidases in the course of inflammation (56). A number of genes for sialic acid utilization are induced specifically during growth in mucus, including genes in both the catabolic nanATEK operon and the nanCMyjhS operon containing nanM (5). Remarkably, a nanAT mutant was impaired in the colonization of the mouse intestine (5), providing direct evidence for an important role of sialic acid metabolism in this environment. In E. coli sialic acid also inhibits the expression of type 1 fimbriae, which are well known virulence factors in uropathogenic strains of E. coli (56), thus reducing the number of antigenic structures exposed on the bacterial cell surface. In this context E. coli cells might use sialic acid as an indicator of inflammation (56), and expressing a mutarotase might allow the cell to perceive the immune response of the host at earlier stages.
Our characterization of the role(s) of NanM in vivo has been complicated by the very small percentage (below 10%) of ␣-Neu5Ac in solution. We did observe a highly reproducible growth defect when a nanM mutant was grown on ␣-Neu5Ac generated in situ from colominic acid (poly-␣(2-8)-sialic acid); however this defect is transient, which can probably be ascribed to the gradual accumulation of ␤-Neu5Ac in the experiment through sialidase activity and spontaneous mutarotation. The nanM mutant grew normally on Neu5Ac provided as monomeric substrate, consistent with the preferential utilization of the ␤-anomer by the cells. Our attempts to increase this growth defect by changing the composition of the minimal medium (i.e. pH and ionic strength) were unsuccessful (data not shown). A similar lack of phenotype for growth on Neu5Ac was YjhT Is a Sialic Acid Mutarotase FEBRUARY 22, 2008 • VOLUME 283 • NUMBER 8 observed in an E. coli nanC mutant (16), and together these observations suggest that the function of the nanCMyjhS operon is unlikely to be for utilization of normal equilibrium solutions of Neu5Ac, but rather that they might allow utilization of host-derived sialic acids being released slowly from a mucosal surface. Also, in vivo phenotypes for other mutarotases have not been observed in wild-type cells and have required the use of artificial substrates (57,58). In contrast, the nanM mutant displayed a clear sialic acid uptake phenotype with a rate about 80% that of the WT. As sialic acid uptake rates were determined using concentrations of [ 14 C]Neu5Ac well below the reported K s of 30 M for sialic acid uptake in E. coli (1), these results are consistent with the nanM mutant being unable to use efficiently all the available Neu5Ac. By extrapolation, it is conceivable that in the presence of an excess of ␣-Neu5Ac, immediately after sialic acid cleavage from sialoglycoconjugates, the contribution of NanM to sialic acid uptake may be more significant.
Orthologues of nanM from various pathogens cluster with other sialic acid utilization/acquisition genes (Fig. 1) in a variety of different arrangements. Proteomic data from H. influenzae revealed that HI0148 is a relatively abundant periplasmic protein (59), although it is not essential for uptake of free Neu5Ac (12). Interestingly, Vibrio cholerae and Vibrio vulnificus encode two NanM orthologues (Fig. 1), only one of which has a signal peptide, indicating that the second is located in the cytoplasm. This suggests that these organisms have a requirement for a cytoplasmic as well as periplasmic mutarotase. There is also a clear nanM orthologue in the free-living marine bacterium Psychromonas sp. CNPT3, suggesting that sialic acid mutarotation might be required in other environments.
The structure of the ␤-propeller in NanM is interesting as this is the first prokaryotic example of a Kelch domain protein to be solved. Kelch domains are rare in prokaryotes (currently about 200 from all sequenced bacteria in InterPro (IPR006652)), and they are scattered in a range of organisms and contained within a wide range of different protein types, none of which have been characterized functionally. Kelch domains are much more abundant in eukaryotic organisms, with humans possessing 71 Kelch domain proteins with many cellular functions (55), and it is possible that prokaryotic Kelch domain proteins may have been acquired by horizontal gene transfer from eukaryotic systems.
We identified Glu-209 and Arg-215 as being important for full activity of NanM in our assay conditions. Although the mechanism of this enzyme is currently under investigation, it is of note that a Glu residue is essential for the activity of the galactose mutarotase (60). In this enzyme, the Glu and a His function in a general acid/base mechanism initiated by abstraction of a proton by the conserved Glu-304 from the C1 hydroxyl group of galactose followed by protonation of the ring oxygen by the conserved His-170 residue, which leads to ring opening. Mutarotation can then occur, and the ring closes again by reversal of the ring opening steps (60). Therefore it is possible that Glu-209 might function to abstract a proton from the C2 hydroxyl in Neu5Ac; however, the complementary functional group for this mechanism does not appear to be the conserved His-278, as mutation of this amino acid results in wild-type activity in our assay. The role of the Arg-215 might relate to modulation of the function of Glu-209 to which it is hydrogen bonded, or it might have a direct function in coordinating the Neu5Ac via a salt bridge to the C1 carboxylate group.
In conclusion, we have identified a novel and unexpected mutarotase enzyme that acts on sialic acid, an important cell surface molecule, which may have a function in vivo to aid in the acquisition of host-derived sialic acid.


Release of gas-phase halogens from sodium halide substrates: heterogeneous oxidation of frozen solutions and desiccated salts by hydroxyl radicals

Motivated by the need to determine the mechanism of the initial release of halogens from sea ice and marine aerosol substrates, a study of the interactions of OH radicals with a variety of halide-containing surfaces has been performed in a coated-wall flow tube using chemical ionization mass spectrometry for gas-phase analysis. The salts studied were NaCl with 0.01% and 0.002% impurities of Br− and I− respectively, and NaCl/NaBr mixtures with Cl−/Br− seawater ratios. The surfaces were desiccated salts, desiccated salts exposed to elevated relative humidity, and frozen solutions. In all cases, gas-phase Br2 and BrCl were formed, with the Br2 yield (defined as a molar ratio of halogen produced to OH lost) larger than 0.2 and the BrCl yield as roughly 0.01. For the first time, an observation of heterogeneous release of iodine-containing halogens (in particular IBr) was made with yields comparable to those of BrCl. We note that pH neutral frozen solutions demonstrated halogen release, although the yields were higher for acidic frozen solutions.

Introduction
It is well established that halogen chemistry plays a significant role in selected tropospheric environments (e.g. Honninger 2003, von Glasow et al 2004). Arctic ozone depletion events with concomitant increases of BrO (Barrie et al 1988) have been observed consistently over the past two decades. Subsequent field campaigns have further investigated the role of halogens at high latitudes (Hausmann and Platt 1994, Foster et al 2001, Avallone et al 2003 and other environments such as dry lake beds (Hebestreit et al 1999 and the marine boundary layer (Saiz-Lopez et al 2006, Bloss et al 2005.
Gas-phase chemistry has proven incapable of explaining the observed extended ozone depletion events. Therefore, 1 Author to whom any correspondence should be addressed. heterogeneous reaction mechanisms, such as the autocatalytic bromine explosion (Vogt et al 1996), have been invoked to provide an additional halogen source. In the marine boundary layer aged aerosols have displayed depleted bromine to sodium ratios with respect to seawater (Sander et al 2003). This suggests that bromine has been preferentially removed from the aerosol over its lifetime. The release of halogens from dry salts (Finlayson-Pitts 2003, Rossi 2003 has also been investigated in laboratory studies. Despite these studies, the mechanisms that release halogens to initiate these cycles remain ill-defined, which limits our predictive capabilities. There are a number of possibilities, including a dark reaction involving O 3 (Oum et al 1998b), interactions with NO x (Finlayson-Pitts et al 1990), and free radical processes (Mozurkewich 1995, Matthew et al 2003. In this regard, Frinak and Abbatt (2006) investigated halogen release from NaCl/NaBr solutions by heterogeneous oxidation involving hydroxyl radicals (OH). Both Br 2 and Cl 2 were released from acidic solutions. In this letter, we extend this work by examining the release of halogens by OH interactions with desiccated salts and frozen sodium halide solutions. Of interest is whether the relationship between acidity and halogen release is maintained on ice surfaces and whether halogen release occurs from neutral substrates, as described by Finlayson-Pitts (2003). To our knowledge, this is the first investigation of the interactions of OH with halide surfaces of this type.

Experimental details
Observations of gas-phase halogens were obtained with a chemical ionization mass spectrometer (CIMS) coupled to a coated-wall flow tube. This apparatus allows for controlled exposure of hydroxyl radicals to prepared surfaces, as described in detail in Frinak and Abbatt (2006). Briefly, the operating pressure of the flow tube was 90 Torr with a total flow rate of 1.5 lpm of N 2 . Water vapor mixing ratios were controlled by passing a fraction of the carrier gas through a bubbler before entering the flow tube. Within the CIMS ionization region, a trace flow of SF 6 in 10.5 lpm of dry N 2 passed over a 210 Po ion source. SF − 6 was chosen as the reagent ion (Huey et al 1995, Bertram et al 2001 because it allowed for simultaneous detection of NO 2 as well as multiple halogencontaining species (Br 2 , BrCl, IBr, HCl, and Cl 2 ).
A 3 ml aliquot of sodium halide solution was added to a 1.5 cm i.d., 30 cm-long rotatable glass insert that had been washed first with a 5% HF solution and then with lots of 18 M water. For preparation of desiccated salts, the solution was gradually dried for a period of roughly two hours while rotating the tube under vacuum. This will have prepared surfaces that will not have liquid water present but will have significant amounts of strongly adsorbed water. In particular, the pressure within the flow tube was gradually lowered from ambient to 90 Torr with a low flow (<1 lpm) of dry N 2 passing over the substrate. For frozen solutions, the aliquot was lowered to a temperature of 248 K, with a humidified flow (<1 lpm) of N 2 . Once frozen, the pressure of the flow tube was lowered to 90 Torr. Solutions used were 3.4 M NaCl (ACP 0.01% Br − , 0.002% I − impurities by mass) i.e. Solutions that we refer to as pH neutral had a pH of 6.5-7.0.
When the desired substrate phase had been obtained, a 100 sccm flow of Ar-containing trace H 2 was flowed through a microwave generated plasma. The resulting hydrogen atoms were then introduced into a controlled flow of a known quantity of NO 2 gas to generate OH, within a glass injector positioned with its tip at the upstream end of the coated glass insert.
H + NO 2 → OH + NO. (1) The initial OH concentrations used in the experiments are high with a median number density of 5×10 11 molecules cm −3 . As described in Frinak and Abbatt (2006), these concentrations were determined by assuming that all of the NO 2 loss was due to formation of OH and we consider them to have an absolute uncertainty of roughly a factor of two.
As determined by calibrations using known flows from gas bulb standards, the limits of detection were 6 × 10 9 and 5 × 10 8 molecules cm −3 for NO 2 and Br 2 respectively, of a 30 s integration period where S/N = 1. Their sensitivities were 3.1 × 10 −10 Hz/molecules/cm 3 for NO 2 and 2.4 × 10 −9 Hz/molecules/cm 3 for Br 2 . The detection limits are considerably enhanced over Frinak and Abbatt (2006) and we believe this is due in part to lessened interference from water vapor. The sensitivities of BrCl and IBr were assumed to be the same as Br 2 .

Results
The effects of phase, acidity and bromide concentration of the bulk substrate on the release of halogen gases were investigated. In particular, experiments were run over frozen solutions (248 K), desiccated salts (298 and 253 K) and humidified salts (258 and 253 K). For the humidified salt experiments, the relative humidity in the flow tube was set between the deliquescence relative humidities for NaBr (58%) and NaCl (75%) so that thermodynamics would predict that most of the NaCl will have been in the solid state and the NaBr in solution (Koop et al 2000). However, we note that we have no evidence ruling out the possibility that the surfaces had additional liquid character arising from metastable solutions.
To best quantify the release of halogens relative to OH exposure, experiments were frequently conducted as a series of runs, where a run is a 15 min period where the OH source was turned on. The amount of halogen release was then calculated from the change in signal on and signal off (see figure 1, for an example). Initially, Br 2 and Cl 2 were the two halogen species observed. In particular, Frinak and Abbatt (2006) observed an initial release of Br 2 and then later a Cl 2 release from liquid substrates. Here, Cl 2 was not observed even after prolonged (i.e. multiple hour) exposure to OH, even though the experimental configuration is the same as that of Frinak and Abbatt (2006) and so should have been observed if formed in reasonable yields. However, small signals of other species such as BrCl and IBr were observed, in addition to a large signal from Br 2 . Initially the signals rose slowly with exposure, presumably as the downstream salt surfaces became conditioned with the halogens, and then a steady signal was observed. For each run, the OH concentration was determined by the change in NO 2 signal from when the microwave signal was off and on. As demonstrated in Frinak and Abbatt (2006), all OH radicals were lost via interactions with the coated walls of the glass insert, and there is minimal gas-phase loss.
The results of the OH initiated release of gas-phase halogen species are summarized in table 1, which shows the maximum ratio of gas-phase halogen release to OH loss for each substrate and the run at which the maximum was observed. Typically 1 to 2 experiments were conducted for each set of conditions. When multiple experiments of the same substrate were performed, the mean ratios and range of values are presented. Note that IBr was not monitored in the runs with NaCl.  1 .20 ± 0.14 8 0.020 5 0.030 ± 0.003 a 'Run' refers to the run number at which the maximum ratio was observed.
The effect of pH was initially to be investigated for both desiccated salts and frozen solutions. However, after the acidified bulk solution was dried the CIMS spectrum was dominated by gas-phase HCl. Several hours of dry N 2 were required to bring the HCl concentrations down to a level where SF − 6 was again the dominant ion. Given the significant gasphase concentrations present, and because the surfaces may have been significantly modified from the high level of HCl release, no experiments were conducted with these substrates. However, the effect of pH could be investigated for the frozen sea-salt solutions where this enhanced gas-phase HCl was not observed. Presumably the HCl was sufficiently soluble in the substrate that it was not easily volatilized. Figure 2 shows that the release of Br 2 from an acidified surface was a factor of five greater than from a neutral surface. In this figure, the signals have been normalized to a constant OH concentration to enable a meaningful comparison of product yields from one experiment to another. Acidification of the frozen sea-salt substrate sustains the release of Br 2 over the entire experiment, whereas the neutral frozen seasalt substrate achieves an early maximum and then gradually decreases over the remainder of the experiment. The release of IBr and BrCl are also enhanced at lower pH and the release of BrCl occurs later than either IBr or Br 2 .
In figure 3 the effect of Br − concentration on halogen release from frozen pH neutral solutions is explored for NaCl and sea-salt substrates. As noted in section 2, although the [Cl − ]/[Br − ] = 13 700:1 for NaCl, Br 2 is the dominant gas observed. The release of BrCl was also observed but there was no evidence of gas-phase Cl 2 . For both NaCl and seasalt substrates, the observed maximum signal of gas-phase BrCl occurs after the maximum of Br 2 indicating that BrCl formation occurs once the Br − ion concentration has been depleted. The sustained production of Br 2 from the sea-salt and the higher yield of BrCl from the NaCl also reinforce this conclusion.
In figure 4, experiments were conducted with neutral seasalt substrates where the plasma was left on for ∼2.5 h to investigate how prolonged exposure to OH would affect the release of Br 2 from different phases. The release of Br 2 is dominated by an initial burst that rapidly drops off. At experiment end the release of Br 2 is an order of magnitude lower than initially. In contrast, the release of Br 2 from the frozen solution remains fairly constant throughout albeit roughly a factor of three lower than the maximum observed for  the desiccated salt. The humidified salt behavior falls between the desiccated salt and the frozen solution. The humidified salt achieves a maximum that is somewhat less pronounced and later in arriving than with the desiccated salt. The Br 2 release from the humidified salt also drops off with time, but the attenuation from its maximum is only a factor of two lower than the observed maximum.

Discussion
To summarize the findings from the results we note that (i) Br 2 is the primary halogen product for OH heterogeneous interactions with the surfaces, with smaller yields of BrCl but no observations of Cl 2 , even with prolonged exposure; (ii) frozen acidified solutions yield more halogens than neutral substrates; (iii) enhanced concentrations of Br − lead to more sustained release of Br 2 and delayed BrCl release; (iv) frozen solutions lead to more sustained release than solid salts; and (v) an iodine-containing product (IBr) is observed. To discuss these findings, potential reaction mechanisms will be presented.
In an earlier study by Frinak and Abbatt (2006), it was established that aqueous solutions also exhibit halogen release upon exposure to gas-phase OH, and that enhanced yields are observed from more acidic substrates. In that work, the CIMS sensitivity was not nearly as high as in this study so the lack of halogen products observed under neutral conditions (and the lack of observation of BrCl and IBr) may have arisen from their concentrations being below detection limits. This work indicates that halogens can indeed be released from neutral solid substrates. To determine that there were no experimental artifacts in this regard and noting that the pH of solutions may change upon freezing (Robinson et al 2006) we tested this observation by one set of experiments on frozen solutions formed from pH 9 solution and still observed halogen release.
We focus initially on pathways to form the principal product, Br 2 , and to explain the acidity enhancement (for more details on the mechanism and rate constants of specific reactions see Finlayson-Pitts (2003), or Frinak and Abbatt (2006)). In particular, although interfacial chemistry is likely to dominate, it is not known whether OH will initially react with a Br − ion on the surface or a Cl − ion. Given that the bulk concentration of Cl − dwarfs that of Br − , it is possible that the initial interaction is with Cl − : Note that the acidity dependence appears in the competition between reactions (3) and (4), relative to the loss rate back to form OH. Also, if the Br − concentration drops, then we postulate that BrCl could form via self-reaction of BrCl − . However, it is now recognized that Br − and I − ions concentrate at the surface of humidified solid salts and deliquesced solutions. This has been shown by ambient pressure electron spectroscopy and Rutherford back scattering studies (Zangmeister et al 2001, Ghosal et al 2005, Hess et al 2007. Molecular dynamics simulations indicate that polarizable species such as Br − and I − associate with the surface more than do less polarizable species such as Cl − and F − (Jungwirth and Tobias 2006).
As a result of surface enhancements, there is also the potential that the OH could first interact with a surficial Br − species: OH + Br − ↔ HOBr − followed by reactions (8) and (9) to form Br 2 . Again, an acidity dependence would arise via the competition between reactions (11) and (12). Finally, there is also the possibility of a surface reaction mechanism involving the self-reaction of short-lived OHhalide complexes but not involving protons (Oum et al 1998a, Finlayson-Pitts 2003: OH + Br − → OH·Br − Molecular dynamics studies by Roeselova et al (2003) infer that the length of time that OH is adsorbed to a surface is sufficient for heterogeneous chemistry of this type to occur in the interfacial region.
For aqueous-phase substrates, Frinak and Abbatt (2006) observed the release of Cl 2 as the signal due to Br 2 dropped, presumably due to Br − depletion. In this work, we did not see the release of Cl 2 from any of the substrates even after running experiments for as long as 12 h. Two effects might explain this. First, the phase was different in these experiments, with the vast majority of the chloride in the solid phase as either NaCl or NaCl·2H 2 O. Thus, chloride would not be as available for reaction. Also, there would be a significant enhancement of Br − at the top of the substrate, arising from the manner of substrate preparation. In particular, exclusion of ions during solution freezing may form a highly concentrated halide layer at the surface. Depletion of this layer of Br − may not occur as readily as it does in the uppermost layers of a solution that does not experience the same degree of concentration enhancement.
We note that we observe the release of IBr from the frozen solutions, even though iodide is present at only the 0.002% level in the NaCl salt. This is consistent with iodine being more easily oxidized than other halides, and with it also being more surface active (Ghosal et al 2005).
Finally, the phase of the substrate had an effect on the temporal profile of the halogen release. For the desiccated salt, although there may be a few layers of surface-adsorbed water, it is expected that both the bromide and chloride will be predominantly in the solid phase. Br 2 release was prompt from these surfaces, followed by a rapid decline in signal. This is consistent with there being depletion of surficial bromide and protons that are not readily replenished due to slow diffusion times from the bulk. In the case of the mechanism initiated by reactions (14) or (15), the formation of NaOH may poison the surface.
For frozen solutions, we note that there will be small pockets of bromide-containing brine given that the eutectic with NaBr·5H 2 O is ∼245 K (Koop et al 2000), and perhaps even the presence of metastable solutions. Thus, the mobility of bromide in the brine may sustain halogen release by replenishment of protons and halides from below the uppermost surface layers. Finally, the behavior of the humidified salts is intermediate between the desiccated salts and frozen solutions probably because the humidity was high enough to deliquesce the sodium bromide.

Conclusions
All the pH neutral substrates display the release of gasphase halogens, primarily Br 2 , upon exposure to gas-phase OH. The release was initially most pronounced with the desiccated salts due to the high concentration of Br − ions at the surface. For frozen solution substrates and humidified salts, the product release was more sustained with time, indicative of replenishment of halide ions at the surface. An acidity dependence was observed for the halogen release from the ice substrate, with the yields of all the halogens increasing when acidic solutions were used. Lastly, an oxidized iodine product, IBr was observed. From an environmental perspective, this work demonstrates that these surfaces, whether neutral or acidified, are fully able to act as sources of atmospheric halogens, principally Br 2 , when exposed to gas-phase OH.


extfieldsofstudy: Chemistry Medicine

text:
Photodegradation of Acidolysis Lignin from BCMP

A mild acidic dioxane extraction method was employed to isolate lignin from hardwood bleached chemimechanical pulp (BCMP). The isolated lignin was then purified and undergone elemental analysis. To study the photodegradation behavior, the lignin samples were impregnated onto the Whatman filter papers and irradiated with UV light for various periods. The photolyzed lignin was then recovered and analyzed by 1H-NMR spectroscopy. Phenylpropane-based formula (C9) of CMP pulp lignin and the photolyzed samples were then established with elemental analysis and 1H-NMR spectroscopy data. The results indicated that the benzaldehyde and benzoic acid type compounds were the main photodegradation products of BCMP lignin. The lignin photodegradation probably involved the degradation of phenylcoumaran units. Irradiation also increased the phenolic hydroxyl group content and decreased that of methoxyl groups, due to demethoxylation. The degrees of aromatic ring condensation were increased upon continuing the irradiation time, which imples the formation of condensed structures in photolyzed lignin.

Introduction
Lignin-rich papers made from mechanical pulps turn yellow when exposed to light and this phenomenon is a major barrier for using mechanical pulps in papers that require long-term brightness stability. So their utilization is usually limited to making newsprint and short-life advertising papers [1,2].
The photoyellowing process of mechanical pulp is believed to be initiated by chromophoric groups found in lignin, including catechols [3], aromatic ketones, coniferaldehyde, stilbenes, and conjugated phenolics [4]. These functional units absorb near UV light and initiate a series of complex chemical reactions that ultimately yield visible chromophores, which discolor the pulps. Recently dibenzodioxocin structures which include a large part of the biphenyl structures of lignins [5][6][7], were also shown to be photoreactive [8,9], due to the presence of both photoreactive α-O-4 and β-O-4 linkages.
The photodegradation mechanisms have not completely been elucidated, but three main reaction pathways have been identified, namely, the phenoxy pathway, the phenacyl pathway and the ketyl pathway. Phenoxy radicals, the key intermediates, are formed through these pathways. Colored species like O-benzoquinones, formed by oxidation of phenoxy radicals [10] and the hydroquinone/ benzoquinone redox systems have been considered in the coloration process of mechanical pulps [11,12].However, the overall contribution to the photoyellowing of mechanical pulps by these functionalities, characterization of photodegraded lignin products, and chromophoric structures of photoaged mechanical pulps, still remain as active fields of investigation.
Mixtures of hardwoods are usually used in Iran to produce high yield pulp with a chemimechanical pulping (CMP) process. Recently high pressure size exclusion chromatography (HPSEC) analyses have been used to determine molecular weight changes and distribution of reaction products formed during irradiation of bleached CMP lignin [13]. In order to monitor exactly the light-induced changes in lignin structure, this study was designated to examine the photodegradation of lignin isolated from hardwood bleached CMP pulp using 1 H-NMR spectroscopy and elemental analysis. This study will increase our basic knowledge about hardwood bleached CMP pulp photochemical behaviors and would pave the way to develop a practical solution to retard photoyellowing.

H-NMR spectroscopy
The 1 H-NMR spectra of acidolysis dioxane lignin isolated from BCMP and irradiated lignin samples for 4 h and 120 h are shown in Figure 1. The interpretations of spectra are based on data which reported elsewhere [14][15][16]. The related signals assignments in chemical shift ranges are summarized in Table 1. All spectra normalized to 100. The integration of the signal areas of the spectra is given in Table 2.
Broadening of signals which is obvious from the appearance of the lignin spectra, is thought to be due to a tendency toward rigidity caused by cross linking and large rings in the macromolecular structure of the lignins and to spin-spin splitting effects.
The 1 H-NMR spectra of the irradiated lignin samples exhibited sharp peaks in the aldehyde proton region (δ = 9.87-9.58 ppm) and on the broad signal in the aromatic proton region (δ = 8.0-6.28 ppm). The peaks were probably due to monomeric aromatic aldehyde and carboxylic acids resulting from the light-induced degradation of the lignin. Vanillin and vanillic acid have been found to be the main lowmolecular degradation products formed on irradiation of lignin-rich paper made of softwoods [17]. However, since BCMP pulp has been produced from hardwoods, then oxidation products of hardwood lignin such as syringaldehyde and syringic acid [18] may consist part of the photodegraded products. However no direct measurements were conducted to determine the photodegraded products.  Besides the aldehyde signal of vanillin and syringaldehyde, the formyl proton signal in cinnamaldehyde type units could be observed in the region δ = 9.87-9.58 ppm [14,15] of the spectra of the irradiated samples. Since the signals all appear to be singlet, then it excludes cinnamaldehyde type groups and are probably due to different benzaldehyde type units and/or monomers. Long-term 24h and 120h irradiation caused to a relative intensity increase within the aromatic part of δ=8.0-7.4 ppm region of the spectra. In this region mainly H-2 and H-6 in aromatic rings with an αcarbonyl group as well as H-α in cinnamaldehyde-cinnamic acid type units absorb [16]. The largest contribution to this intensity increase then probably comes from the signals of H-2 and H-6 in benzaldehyde and benzoic acid type units, besides other α-carbonyl group containing species.
Intensities of signals at δ = 5.8-5.2 ppm assigned to H-α in β-5/α-O-4 (phenylcoumaran) units, decreased in the spectra of the lignin samples photolyzed up to 4 h irradiation. The decrease is probably a result of α-O-4 cleavage which could be followed by formation of stilbene. This is in agreement with others' findings [19]. However, with proceeding of irradiation up to 24 h and 120 h, the intensities of signals at this region increased which could be attributed to the high formation rate of condensed structures like phenylcoumaran unit than its degradation at long-term irradiation.
A decrease of the intensities of the signals of H-α in β-O-4 units at δ = 4.9-4.5 ppm was also observed in spectra of irradiated samples. This indicates that α-CHOH groups are transformed, probably by oxidation to carbonyl groups. This is supported by the intensity increase of low-field part of the aromatic signals especially in spectra of long-term irradiated lignin.

Elemental analysis and developing C 9 -based empirical formulas
The isolated and purified BCMP lignin was also characterized by elemental analysis. These analytical data and the results of the semiquantitative analysis of the 1 H-NMR spectra of unirradiated and irradiated samples were used to establish empirical formula calculated on phenyl propane (C 9 ) basis. These formulas serve to lend emphasis to the fact that the monomeric units comprising lignin have C 9 carbon skeletons and to underscore the significance of the methoxyl group for both identifying the lignin type and for indicating the contributions of component monomer units [20].
A method developed by Ludwig et al. [15] used to determine the number of hydroxyl groups using 1 H-NMR data of acetate derivatives and elemental analysis data. Aliphatic acetoxyl and aromatic acetoxyl signals permit the estimation of the number of hydroxyl groups. However, one should bear in mind that the aromatic acetoxyl groups in 5-5' bonded dimeric units appear in the range of aliphatic acetoxyl groups and are not considered in the estimation of the phenolic groups [21].  Table 3 were then developed in accordance with the procedure described elsewhere [15]. As can be seen from the empirical formulas, the phenolic hydroxyl groups of the photolyzed lignin samples increased, especially after long-term irradiation. The photodegradation of lignin caused the scission of β-O-4 structures resulting in the formation of free guaiacyl and syringyl phenolic units. It seems that some of the quenching of these phenoxy free radicals proceeds via hydrogen abstraction yielding new phenolic units. However, formation of such units is slower than the rate of cleavage of β-O-4 bonds [22]. The formation of new phenolic units is not remarkable given the extreme reactivity of phenoxy radicals in reactions other than simple hydrogen abstraction. Because phenoxy radicals are the products of hemolytic bond scission taking place within the β-O-4 structures, they may further condense or disproportionate to yield chromophoric structures. In addition, the opening of the hydrofuran ring by cleavage of α-O-4 bond in phenylcoumaran units and formation of stilbene structures during irradiation could lead to the formation of new phenolic hydroxyl groups.
The methoxyl group content of bleached CMP acidolysis lignin supports the fact that the isolated lignin is a syringyl-guaiacyl type. The main photodegradation process of phenolic structural elements of lignin is demethoxylation of the guaiacyl or syringyl rings; however, simple phenols might be photooxidized into quinones and muconic acids [11]. Among phenols, guaiacyl elements are more photodegraded than syringyl units. Syringyl elements give very stable phenoxy radicals [23] with a structure related to hindered phenols such as BHT [24]. Guaiacoxy radicals are prone to be transformed in o-quinones by demethoxylation. The observed loss of methoxyl groups during photolysis of BCMP lignin has been also noted by examining wood, lignin, and mechanical pulps [25,26]. Our results are consistent with these findings.

Determination of the degree of condensation
Condensed structure may arise from radical coupling reactions during irradiation. As the aromatic rings condense in various manners such as 5-5', β-5, 4-O-5, or other types of linkages, the number of aromatic protons will decrease. Therefore, the condensation degree of the lignin can be calculated from the 1 H-NMR spectra and the methoxyl contents. For each C 9 unit, a maximum of four aromatic hydrogen atoms is expected, since the carbons at positions 1 and 4 are linked to the propyl chain and to the phenolic hydroxyl/ether linkage, respectively. The aromatic positions which are occupied by methoxyl groups can be obtained from the C 9 formula. Therefore, the difference between the four hydrogen atoms and the methoxyl group content gives the theoretical number of aromatic protons. By comparing the actual number of aromatic protons which is obtained from the 1 H-NMR spectra with this theoretical value, the degree of condensation can be estimated [27,28]. The degrees of condensation of isolated BCMP acidolysis lignin and the irradiated samples are shown in Table 4. The results indicated that irradiation led to the formation of condensed structures. Increasing phenolic condensed structure with irradiation time has also been reported by Li and Ragauskas [25].

Conclusions
Our results indicate that photodegradation of acidolysis lignin isolated from hardwood BCMP is accompanied by a decrease in intensity of a part of the aliphatic proton NMR resonanances, while new signals apperar in the aromatic-olefinic and the aldehyde regions, suggesting that C α -C β bond cleavage and formation of benzaldehyde and benzoic acid type compounds could be dominant reactions in the light-induced degradation of BCMP lignin. The formation of stilbenes via opening the hydrofuran ring of phenylcoumaran units may also contribute to the discoloration. In addition, the irradiation caused the number of aromatic ring protons to decrease due to condensation reactions or other mechanisms such as oxidation of aromatic rings to nonaromatic structures. Loss of methoxyl groups was another structural change observed during the photo-degradation of BCMP acidolysis lignin.

Materials
All reagents and solvents were commercially purchased and used as received. A commercial hardwood CMP bleached with hydrogen peroxide received from Mazandaran Pulp and Paper Ltd. was used in all experiments. The pulp properties were as follows: brightness 71 % ISO; yellowness 26.8 %; yield 83%. Whatman No. 1 filter paper was used as cellulose matrix.

Isolation of Lignin
Isolation of lignin from BCMP was carried out in accordance with Wang et al. [29] and Li et al. [25] with slight changes. Prior to isolation of the lignin, the hardwood BCMP was Soxhlet-extracted with acetone for 24 h and the extracted pulp then left to air-dry overnight. Dry pulp (75 g) was then refluxed in a 0.01 N HCl dioxane-water solution (1.5 L, 8.5:1.5) under an argon atmosphere for 1 h. This pulp was filtered and washed with fresh dioxane (100 mL×3). The combined aliquots were neutralized with powdered NaHCO 3 , filtered and concentrated under reduced pressure at 40°C. The concentrated solution was added to 0.006 N aqueous HCl (500 mL) and the precipitated lignin was twice washed with acidified water (pH 2-3) and then freeze-dried. The lyophilized lignin was subsequently dried under high vacuum at room temperature. Mild acidolysis lignins like the ones used in this study are representative of those usually found in mechanical pulp fibers. The lignin preparations were dissolved in a solution of acetic acid-water (90:10 v/v, 1 g of lignin/10 mL of solvent) and then precipitated in ether. After filtration, the precipitated lignin preparations were washed with extra ether and dried under vacuum with P 2 O 5 .

Filter paper impregnation
The BCMP lignin was dissolved in a 9:1 dioxane-water solution (3 mL) and applied homogeneously to both sides of Whatman filter paper using a syringe. The impregnated test-sheets were dried overnight under partial vacuum over P 2 O 5 . Approximately 21% (based on the weight of the sheet) lignin was impregnated on the filter paper. This amount simulates the lignin content of the hardwood BCMP pulp.

UV irradiation of lignin treated filter papers
For accelerated exposure, lignin-impregnated sheets were placed in custom-made photoreactor. This reactor was maintained at 30°C and equipped with four 350 nm black lights. Each side of the paper was photolyzed for half of the desired irradiation time.

Extraction of the photolyzed lignin
The photolyzed filter papers were wrapped to fit into test tubes and were extracted with 9:1 dioxane-water mixture (25 mL × 4). The extract was filtered and then concentrated under reduced pressure at 40°C. The photolyzed lignin samples were freeze-dried and then further dried at room temperature using higher vacuum. This procedure yielded 80-92% of the lignin content of test sheets.

Acetylation of lignin
According to Lundquist [14], acetylation of lignin samples (100 mg) was performed with acetic anhydride-pyridine (2 mL, 1:1, v/v) at room temperature overnight in a 50 mL flask. Ethanol (25 mL) was added, and after 30 min, the solvents were removed by evaporation under reduced pressure at 40 °C. Addition and removal of ethanol was repeated more than ten times to ensure complete removal of acetic acid and pyridine from the sample.

Purification of the lignin acetate
The acetate lignin was dissolved in chloroform (2 mL) and the solution was added dropwise to magnetically stirred ether (100 mL). The precipitated acetate was filtered, with ether and then dried under vacuum with P 2 O 5 .

Quantitative 1 H-NMR spectroscopy
The quantitative 1 H-NMR spectra of acetylated lignin samples were recorded on a Bruker Avance 400 MHz spectrometer at room temperature. The samples (60 mg) were accurately weighed and dissolved in CDCl 3 (0.6 mL). Conditions for analysis included a 90° pulse width, a 1.3 s acquisition time, and a 7 s pulse delay (d1). A relatively high relaxation delay of 7 s was applied to ensure complete relaxation of aldehyde protons, and a total of 128 scans were collected. The spectra were calibrated from the signal of residual chloroform (7.3 ppm).

Elemental analysis
The carbon and hydrogen determinations of BCMP lignin performed in a CHN-O-Rapide, Elementar analyzer. The oxygen content was calculated by the difference with respect to the total sample. The sulfur content was neglected because it was negligible.


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NO2 Detection Using Microcantilever Based Potentiometry

A highly sensitive and novel sensor platform for gases and volatile chemicals using microcantilever based potentiometry is reported. A resonant cantilever is used to detect the changes in surface work functions of functionalized substrates caused by adsorption of target gas molecules. Surface work function (SWF) changes were measured for different functionalization layers made of transition metal oxide thin films with the flow of NO2. The rate of change in SWF for In2O3 and SnO2 were found to be ∼80 and ∼100 μV/sec, respectively, for 70 ppm NO2. A sensitivity of 64 μV/sec for SWF change was also found for 70 ppm NO2 concentration for isolated clusters of ZnO nanowires, indicating that this technique is applicable even for nano-clusters of sensing materials where amperometric detection is impossible due to material discontinuity. NO2 detection as low as 400 ppb was possible using highly insulating In2O3 and SnO2 thin films (resistivity > 1 TΩ/□. Two different forms of nano scale graphite were compared with the transition oxide based functionalization layer for sensing sub-ppm NO2 sensing. It was observed that nanostructured graphite (NG) shows much higher sensitivity and lower response time than transition metal oxides.

Introduction
In the recent past, a great deal of research has been directed towards fast, accurate and highly sensitive gas sensing devices that are important for various applications including providing precaution against toxic gases that create health hazards, for monitoring and reducing pollutants in the environment, early detection of nerve gases in defense and military applications, and for monitoring pollutants emitted from vehicular exhaust system. Such sensors should be capable of monitoring the concentrations of particular gases continuously with accuracy and selectivity [1]. Nitrogen oxides (mainly NO and NO 2 , together referred to as NO x ) are among the six most common air pollutants defined by EPA (Environmental Protection Agency), along with particle pollution, ground-level ozone, carbon monoxide, sulfur oxides and lead [2]. During NO x emission, NO 2 is produced by the further oxidation of NO and the resulting concentrations of NO 2 are rather complex. NO 2 plays a major role in atmospheric reactions that produce ground-level ozone, a major component of smog, and it is also a precursor to nitrates, which contribute to increased respiratory problems. Continued or frequent exposure to NO 2 concentrations higher than the EPA air quality standard (53 ppb) may cause increased incidence of acute respiratory illnesses in children [2]. NO 2 is also one of the main toxic components emitted from vehicular exhaust and also a main component of emissions from indoor appliances. In addition, it transforms in the air to form gaseous nitric acid and toxic organic nitrates, hence contributing to the production of acid rain [3]. Therefore a selective and accurate NO 2 sensor is of extreme importance for continuous monitoring of emission processes. Existing gas detector devices use non-selective techniques based on responses that detects the change in electronic or mechanical property of the chemoselective sensing materials due to adsorption of target molecules. One of the most common methods for detection of gases is based on the change in current flow in a semiconductor device (called an amperometric technique, since it is based on the change in current conduction due to surface adsorption upon exposure to the target gases). Examples of other available detection techniques include chem-Field Effect Transistor (FET) type devices that are based on the change in current flow [4], potentiometric sensors where changes in surface work function are measured [5,6], microcantilever based sensors where changes in static deflection [7] or frequency shifts of the cantilever are detected [8], and chemi-capacitive sensors that are based on changes in the permittivity of the dielectric medium [9]. Although widely used for detection of various gases and vapors, one of the drawbacks of the mentioned amperometric technique is that the sensing response depends on the bulk electronic properties as well as the thickness of the sensor material coating. The extent of current change, and hence the sensitivity, depends on the bulk mobility of the carriers (which in turn depends on the material properties such as the barrier height of the grain boundary [10]), as well as the thickness of the sensing film. Thus, a large variation in sensitivity and stability for different films is possible [6].
Recently, several reports have shown that microcantilever based detection is an attractive alternative to improve detection sensitivity [7,8]. These include micro(nano)cantilevers as sensor elements for specific chemicals [7,8,11,12] and biological molecules [13,14] exploiting their high sensitivity toward changes in surface stress. A compelling feature of microcantilever sensors is that they can be operated in air, vacuum, or liquids, which facilitates the selective and sensitive detection of airborne, waterborne and pathogenic substances [15]. The microcantilever surface is usually functionalized (coated with an appropriate selective layer) to facilitate adsorption of target molecules which changes the surface stress. However, functionalization of cantilevers has several drawbacks that hinder wide commercial application of cantilever based sensors. Firstly, surface stress is extremely sensitive to uniformity and chemical integrity of the coating that can lead to sensitivity variation from cantilever to cantilever. Secondly, fabrication of series of functionalized cantilever arrays on micron scale is quite expensive and inconvenient. Thirdly, there are several molecules which adsorb on the sensing layers through exothermic or endothermic reactions creating a resultant temperature change on the cantilever [5]. This can result in unwanted heat induced expansion/contraction thereby introducing erroneous results.
As sensing materials, semiconducting transition metal oxides have been widely used for NO 2 detection. Researchers have successfully reported NO 2 detection using a wide range of transition metal oxides including In 2 O 3 [16], SnO 2 [17] and WO 3 [18]. NO 2 target molecules accept electrons from the transition metal oxides which creates changes in the electrical properties of the material. Carbon has also showed itself to be an ideal sensing material due to its excellent molecular adsorption property [19]. Carbon based sensors, especially those utilizing carbon nanotubes (CNT), have been the focus for gas sensing during the last decade because of their high surface-to-volume ratio, excellent mechanical stiffness and hollow structure [20][21][22][23]. However, there are still significant challenges in large scale production and deployment of CNT based sensors. Carbon based sensors have been developed and researched almost exclusively using CNTs until the discovery of graphene in late 2004 [24,25]. Graphene based functionalization layers have been shown to have molecular level sensitivity where one electron conductance change is reported to be detected with the adsorption of single NO 2 molecule [26]. However, the search for a highly sensitive carbon based functionalization layer that is easy to fabricate and economical to commercialize have still not concluded because of the lack of film control for graphene preparation.
In this article, we demonstrate a highly sensitive microcantilever based detection technique that is generally applicable for the detection of gases and volatile chemicals, and eliminates the need for modifying the cantilever itself with a selective layer. The technique is based on adsorption induced surface work function (SWF) change of the sensing layers; however unlike other SWF based sensors [27] it uses a microcantilever which is resonated very near to a functionalized substrate with a setup similar to Kelvin probe technique ( Figure 1) [5,28]. In this work detailed analyses of the operation and sensitivity of our microcantilever based detection technique is presented. SWF changes of different transition metal oxide thin films (Section 3.1) and carbon based layers (Section 3.2) in response to trace NO 2 are also compared. Simultaneous potentiometric and amperometric measurements on nanostructured graphite (NG) layers are discussed at the end of the article in Section 3.3.

Experimental
Microcantilever based potentiometric measurements were carried out using a commercial Atomic Force Microscope (AFM) setup (Autoprobe M5, originally made by Thermomicroscope, now a part of Veeco Metrology Group) [5]. The topographic signal was used for monitoring the cantilever oscillation. A Si microcantilever (purchased from Mikromasch, model CSC12/tipless) was excited at its resonance frequency,  0 in non-contact mode using the piezo-actuator. The cantilever is excited by an external AC bias with a frequency  which is close to  0 , so that beats are formed in the oscillation amplitude, and the topography feedback signal is sinusoidal ( Figure 1). The amplitude of the sinusoidal topography signal [having frequency ( ~  0 ) and representing the movement of the piezo actuator in response to the ac excitation] was recorded using the system software, which varied with change in surface work function as the target molecules get adsorbed on the functionalization layers. The amperometric measurements were performed using a current preamplifier (DL Instruments, model 1211), with metal press contacts established at the two ends of the nanostructured graphite film. The resonance frequency of the cantilevers used was ~20 kHz with a quality factor of ~35. A tipless cantilever is used to enhance the capacitive interaction between cantilever and surface. For our detection experiments, the desired NO 2 concentrations were prepared by intermixing calibrated commercial NO 2 with purified N 2 at specific ratios using mass flow controllers (MKS Instruments). The test gas was passed at the rate of ~200 sccm using a gas flow fixture with cross-sectional area of 1.5 cm 2 which was positioned within a few mm of the cantilever. The cantilever-sample (electrode) distance during measurements was kept ~10 m during measurements. The measurement set up with an external ac bias, piezo-actuator, and the gas flow tubes are shown schematically in Figure 1.
For sensing experiments, the transition metal oxide (In 2 O 3 and SnO 2 ) thin films were deposited on a conducting Si wafer by the Successive-Ionic-Layer-Adsorption-and-Reaction (SILAR) technique followed by high temperature annealing. The SILAR process we use for depositing thin films has been described in greater detail elsewhere [29]. ZnO nanowires (the method of preparation is described in ref 30) were also investigated that were deposited on Si substrate after sonication. Clusters of nanowires were formed on the substrate after the solution dried out [ Figure 3(d)]. The NG functionalization layers were prepared by mechanical exfoliation of highly pure graphite (purchased from Carbone of America Ltd.) by simple abrasion on insulating substrates. An insulating ceramic substrate was chosen to perform simultaneous amperometric and potentiometric measurements. TEM images of NG particles were obtained using high resolution Transmission Electron Microscope (Hitachi, model H-8000). The TEM samples of the mechanically exfoliated NG particles were sonicated in an acetone solution and subsequently transferred on to copper TEM grid.

Results and Discussion
The total force between the cantilever and the sensing surface consists of an electrostatic component and a capacitive component [28]. If we ignore the fixed charges on the surface the electrostatic force component can be considered zero. Therefore the total force between cantilever and the sensing surface will reduce to the capacitive component. So mathematically, if the external applied ac and dc bias is V ac sint and V dc respectively, and  is the surface work function difference between the cantilever and sensing surface then the total force is: F 0 , F  and F 2 are the dc,  and 2 components of the total force respectively and z C   is the capacitance gradient of the cantilever. The expression of the oscillation amplitude is given by [31], Here, k and Q are the spring constant and quality factor of the cantilever. As  is very close to the resonant frequency of the cantilever, -component of the oscillation will be dominant due to the high quality factor of cantilever at resonant frequency. The oscillation is converted using optical transduction system and the amplitude of the -component is extracted by a lock-in amplifier. So the obtained electrical signal is proportional to: Clearly, the oscillation amplitude depends on the capacitance gradient (which in turn depends on the distance between cantilever and the substrate z), quality factor, spring constant, and the ac voltage amplitude. To analyze the sensing technique of our sensing measurements we determined the dc and ac sensitivity of the cantilever sensor.
The sensitivity data for uncoated Si cantilevers is shown in Figure 2(a), in which the amplitude of beat oscillations is plotted against dc bias for ac biases of 2, 4, and 6 V. Figure 2(b) shows the sensitivity plotted against the applied ac bias. The sensitivity is calculated as the rate of change of amplitude with dc bias, and expressed in nm/mV. From Figure 2(b), we see that the sensitivity increases linearly with the ac bias which directly follows from Eq. 1. The rms noise (calculated as the standard deviation of the beat amplitude as a function of time for constant ac and dc biases) corresponding to this sensitivity was found to be less than 20 nm. The maximum sensitivity obtained for our present setup was 360 nm/mV. So the minimum change in surface work function that can be resolved is calculated as 55 V [= (20/360) mV].
We also found that the rms noise does not have any correlation with the ac bias, and varies almost randomly in the range of 8-20 nm, possibly due to the ambient noise. Increase in sensitivity with ac bias, indicates that the voltage resolution can be enhanced by increasing the ac bias. It also follows from Eq. 1 that reducing the cantilever-sample distance or spring constant or increasing Q of the cantilever will also results in increased sensitivity.

Transition metal oxides
Measurements were performed on In 2 O 3 and SnO 2 thin films to investigate the surface work function responses with the adsorption of NO 2 molecules. Figure 3(a) shows the surface work function (SWF) transients measured on In 2 O 3 and SnO 2 thin films when 70 ppm NO 2 flow was turned on and off for two cycles. The oxide thin films were highly insulating (resistivity > 1 TΩ/ ) which is believed to be due to the presence of high defects in the material. Such films were unsuitable for any amperometric measurements; however they showed significant response in the SWF where the initial rates of change in SWF of ~80 and ~100 V/sec, respectively, were found for In 2 O 3 and SnO 2 [ Figure  3(a)]. Figures 3(b) and 3(c) are the SEM images of the surface of the In 2 O 3 and SnO 2 thin films which show that they had fairly even surfaces. However, the In 2 O 3 films show presence of tiny bubble shaped morphology with average diameter of around 3.5 m [ Figure 3(b)] that might have grown due to emission of any gaseous species during high temperature annealing. However, any error that is incorporated due these spots can be neglected since the dimension of the cantilever is relatively large (350m  35m) with respect to these bubbles. To further investigate the efficacy of the potentiometric technique, we performed the NO 2 sensing measurements on ZnO nanoclusters. ZnO has been proved as sensing material for various gases [32]; recently ZnO nanowires FET has also been demonstrated as an efficient NO 2 sensor in sub-ppm range [33]. The ZnO nanowires we used for our experiments were deposited on Si substrate by sonication. Figure 3(d) shows a 5 mm 7 mm optical image of the sample produced. It can be seen in this figure that the nanowires are clustered together making small islands of material which are unsuitable as sensing materials for amerometric measurements due to the material discontinuity. However, significant SWF change was observed with flow of 70 ppm NO 2 as the ZnO clusters showed initial rise rate of 64V/sec [ Figure 3(a)]. This shows that the potentiometric technique is equally efficient for sensing in isolated structures.  The observed changes in SWF for SnO 2 thin film are ~105, 62, and 35 mV, respectively, for 18, 1 and 0.4 ppm NO 2 , passed for 500 secs [ Figure 4(a)]. The inset of Figure 4(a) shows the calibration curve for maximum SWF change plotted against the concentration of NO 2 measured on SnO 2 thin film. In Figure 4(b) it can be seen that after 500 secs of 18, 1 and 0.4 ppm NO 2 flow, In 2 O 3 thin film showed SWF changes of ~95, 68 and 23 mV. As evident from the figures the SWF responses of In 2 O 3 and SnO 2 thin films with the flow of NO 2 were very much comparable. Since the work function measurement resolution for this measurement system is ~55 V [5], it is also possible to further improve the NO 2 detection sensitivity with wide area metal oxides.

Carbon based nano-materials
The potentiometric sensor was also applied to investigate the sensitivity of carbon based nanoscaled materials for detection of NO 2 . NG layer was deposited on insulating substrates using mechanical exfoliation technique. Insulating substrates were chosen over metallic or semiconducting surfaces to perform simultaneous amperometric and potentiometric measurements. However, the sensitivity of the potentiometric technique was found to be independent of the substrate conductivity. ppb NO 2 using nanocrystalline graphite (NCG) and nanostructured graphite (NG). NG shows higher sensitivity than NCG. Figure 5(a) shows a transmission electron microscope (TEM) image of a representative NG film that we used for our sensing experiments. The image shows the presence of multi-layered graphite resembling mica-flakes, whose lateral dimensions are of the order of a few tenths of a micron. These nanostructures can significantly increase the number of the molecular adsorption sites on the surface resulting in the high sensitivity observed in our experiments. The sensitivity of the NG layers was also compared with nanocrystalline graphite (NCG) [34] layers which were prepared by Chemical Vapor Deposition (CVD) from graphitization of SiC wafers [35]. Figure 5(b) (taken from [35]) is the image of the surface morphology of NCG taken by Atomic Force Microscope (AFM) which reveals ~20 nm grain size of the surface. However, the rms surface roughness is found to be ~3 nm [35]. Surface work function changes of NCG and NG layers with the flow of 400 ppb NO 2 were compared in Figure 5(c).
The changes in SWF are found to be ~25 and ~11 mV (in 100 secs) for NG layers and NCG respectively. These responses are higher than the response we got for traditional metal oxides for subppm NO 2 (Figure 4). We believe the increased sensitivity of NG over NCG is most likely due to the increased surface roughness which increases the NO 2 adsorption on the surface. The potentiometric sensing technique is also applied for sub-ppm NO 2 detection using NG layers. Figure 6 shows the SWF changes with flow of 200, 100 and 60 ppb NO 2 . The initial rise rates for the SWF changes in response to 3 different concentrations are found to be 80, 12 and 5 V/sec respectively. Therefore with a noise resolution of ~55 V [5], we infer that 60 ppb NO 2 can be sensed in less than 12 secs. We believe proper optimization of this sensing technique and better preparation of NG can improve the sensitivity to even lower concentration.

Simultaneous Amperometric and Potentiometric Measurements
The main advantage of the potentiometric technique is that it is entirely based on the surface properties of the sensing materials. On the other hand, the amperometric technique (which detects the conductance change of the material with adsorption of target molecules) relies on the adsorption process as well as the diffusive property of molecules through porous graphite. In case of molecules which do not create significant conductance change, potentiometric measurements can be a better tool since the orientation of the molecule can allow significant change in SWF. Figure 7 shows the comparison for simultaneous current and SWF measurements on NG layer with 3 cycles of 100 ppm NO 2 flow. Carbon is a highly conductive material, so to realize the amperometric measurements, very thin layer (< 1 m) of NG was deposited on insulating material such as ceramic.

Figure 7:
Simultaneous conductance (right axis) and surface work function (left axis) changes measured using NG layer when exposed to 100 ppm NO 2 .
The SWF changes quickly up to ~240 mV within 20 secs of NO 2 exposure; in contrast, the current increases slowly even when the SWF change had already reached saturation (Figure 7). These instances of complete surface state occupation can be distinguished as slope changes in G/G (happening after ~15 secs for each cycle). We believe that the slope change signals the end of the fast surface adsorption, which is followed by slow diffusion through the porous material and adsorption at sites deeper inside, resulting in a slow increase in current [36]. After discontinuing the NO 2 exposure, we find that the SWF reverts to almost the original value as the molecules leave the surface, even though conductance continues to decrease slowly due to much slower desorption kinetics of the molecules from inside the porous NG material. The slow desorption causes an increase in the residual adsorbed NO 2 molecules in successive cycles and therefore increases the minimum current reached at the end of each cycle. During the decay transient the SWF changes to ~70 mV in ~2.5 mins which is the same for all the 3 cycles observed. This is an indication that the surface molecules getting desorbed at the same rate during all the decay transients. However, there is less possibility of the diffused molecules to come out of the material at the same rate as the molecules getting desorbed from the surface. This is clearly observed in Figure 7 as the values of the percentage conductance changes reached 0.83, 1.3 and 1.67 respectively at the end of 3 decay transients indicating a residual increase in conduction. Figure 7 also indicates that both the SWF and conductance increases due to NO 2 adsorption. This is possible if the graphene layer has an overall p-type nature, as has been observed in earlier studies [21]. Since NO 2 has an electron-withdrawing nature, its adsorption enhances the p-type conductivity of the NG layer.

Conclusions
We have investigated the sensing behavior of various sensing materials for NO 2 detection using a highly sensitive potentiometric technique. The potentiometric measurements indicate that the transition metal oxides such as In 2 O 3 and SnO 2 have almost similar SWF response for NO 2 detection. The technique was found to be also sensitive for isolated structures and thin films with high resistance; these structures are unsuitable for amperometric detection. It has been found out that carbon based materials such as nanocrystalline graphite and nanostructured graphite exhibits better sensitivity than traditional transition metal oxides. The enhanced sensitivity of NG over NCG is attributed to its structural non-uniformity and increased number of adsorption site. Simultaneous SWF and conductance measurement is performed to compare the sensitivity of potentiometric technique over the amperometric one. NO 2 detection as low as 60 ppb was possible using this technique on nanostructured graphite layers.