*Article* **The Facile Deposition of Pt Nanoparticles on Reduced Graphite Oxide in Tunable Aryl Alkyl Ionic Liquids for ORR Catalysts**

**Dennis Woitassek 1 , Swantje Lerch 2 , Wulv Jiang 3 , Meital Shviro 3, \* , Stefan Roitsch 4 , Thomas Strassner 2, \* and Christoph Janiak 1, \***

**Abstract:** In this study, we present the facile formation of platinum nanoparticles (Pt-NPs) on reduced graphite oxide (rGO) (Pt-NP@rGO) by microwave-induced heating of the organometallic precursor ((MeCp)PtMe3 in different tunable aryl alkyl ionic liquids (TAAIL). In the absence of rGO, transmission electron microscopy (TEM) reveals the formation of dense aggregates of Pt-NPs, with primary particle sizes of 2 to 6 nm. In contrast, in the Pt-NP@rGO samples, Pt-NPs are homogeneously distributed on the rGO, without any aggregation. Pt-NP@rGO samples are used as electrode materials for oxygen reduction reaction (ORR), which was assessed by cyclic voltammetry (CV) and linear sweep voltammetry (LSV). The electrochemical surface area (ECSA) and mass-specific activity (MA) increase up to twofold, compared with standard Pt/C 60%, making Pt-NP@rGO a competitive material for ORR.

**Keywords:** platinum; nanoparticles; electrochemistry; oxygen reduction reaction; reduced graphite oxide; microwave; ionic liquid; tunable aryl alkyl ionic liquid

#### **1. Introduction**

Platinum nanoparticles (Pt-NPs) are important catalysts and are often used as benchmark materials in the field of electrochemistry for the oxygen reduction reaction (ORR) [1–5], the hydrogen evolution reaction (HER) [6,7], and the methanol oxidation reaction (MOR) [8–10]. In general, the stability, catalytic activity, and chemical selectivity of Pt-NPs depend strongly on their size, shape, alloy composition, surface structure, and surface accessibility [2,5,11]. It is, therefore, important to control these parameters via the chosen synthetic method and, if possible, influence multiple parameters at the same time during NP synthesis [2,5,11].

Smaller Pt-NP sizes show higher metal-mass-based catalytic activity than larger particles or bulk-material [5,11,12]. At the same time, a crucial issue for NPs is their tendency to coalescence, which can occur as agglomeration or Ostwald ripening, increasing their size and reducing the catalytic activity, thus requiring stabilizers [13–15].

We have recently reported the wet-chemical synthesis of Ru- and Ir-NPs in tunable aryl alkyl ionic liquids (TAAILs) based on the 1-aryl-3-alkyl-substituted imidazolium motif [16]. Ionic liquids (ILs) are salts with a melting point below 100 ◦C and can be used as solvents and as stabilizing agents for M-NPs, due to their high ionic charge, polarity, dielectric constant, and electrostatic and steric interaction with the M-NPs [13–15,17,18]. All used TAAILs were suitable for the formation of very small (<5 nm) NPs, which were stable over

**Citation:** Woitassek, D.; Lerch, S.; Jiang, W.; Shviro, M.; Roitsch, S.; Strassner, T.; Janiak, C. The Facile Deposition of Pt Nanoparticles on Reduced Graphite Oxide in Tunable Aryl Alkyl Ionic Liquids for ORR Catalysts. *Molecules* **2022**, *27*, 1018. https://doi.org/10.3390/molecules 27031018

Academic Editors: Jingqi Guan and Yin Wang

Received: 17 December 2021 Accepted: 27 January 2022 Published: 2 February 2022

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months in the IL medium. An interesting observation was that the nanoparticle separation and aggregation varied strongly as a function of the aryl substituent of the TAAILs.

In addition, Supporting Materials can stabilize M-NPs, allowing for easier handling of the M-NPs and providing for additional tuning options for applications. Carbon materials as support for Pt-NPs are commonly used in the field of electrochemistry because of their high electrical conductivity, low weight, low cost, easy production, and manifold structures [6,19]. Our group has already shown the usage of (thermally) reduced graphite oxide (rGO, also known as TRGO) as M-NP support [20,21], which also offers remarkable properties as support for electrode materials [22–24]. rGO increases the electrochemical activity of Pt-NPs [19], stabilizes very small M-NPs [25,26], and offers improved protection for supported NPs against poisoning, compared with available standard carbon black materials [20]. Since rGO provides less functional groups that can interact with M-NPs than graphite oxide, the deposition of Pt-NPs on rGO (Pt-NP@rGO) in ILs remains difficult and was only successful after additional rGO functionalization with thiol groups [20] to improve the interaction between Pt-NPs and its support. Alternatively, porous organic polymers with donor atoms such as covalent triazine frameworks (CTFs) were successfully used as support [27].

In this article, we present a simple and facile way to deposit Pt-NPs in situ on rGO during a microwave-assisted reaction in ionic liquids. Novel imidazolium-ILs, which are distinguished aryl and alkyl substitutions, are used as reaction media as well as stabilizing agents. We also show that the obtained Pt-NP@rGO are electrochemically active as heterogeneous catalysts for ORR, a vital reaction for proton exchange membrane fuel cells (PEMFC).

#### **2. Results**

#### *2.1. rGO and TAAIL Presentation*

rGO was synthesized in a two-step oxidation–thermal reduction process according to procedures by Hummers [28], as described in more detail in our previous studies [20,21], with the thermal reduction occurring at 400 ◦C. CHNS elemental analysis showed a weight percentage (wt%) of 80% carbon, which indicates that functional oxygen-containing groups still remain on the surface of rGO [21]. The exact values can be found in the Supporting Information (SI), Table S2.

The synthesis and characterization of the TAAILs used here were described elsewhere [15,29,30]. TAAILs refer to 1-n-alkyl-3-arylimidazolium ILs (Scheme 1). The length of the n-alkyl chain varies here between 4 and 11 carbon atoms, and the aryl groups were 4-methoxy-, 2,4-dimethyl, 4-bromo- and 2-methylphenyl.

**Scheme 1.** Structures of utilized tunable aryl alkyl ionic liquids (TAAIL) with 1,3 disubstituted imidazolium cations and the bis(trifluoromethylsulfonyl)imide anion. The abbreviation is derived from the phenyl substitution and the alkyl chain length.

The TAAILs can be obtained on a large gram scale, and the synthesis allows manifold variations of the aryl and alkyl groups [15,29,30]. The anion purity of the TAAILs was characterized via ion chromatography and has always been found above at least 97% and more often 99%. Thermogravimetric analysis (TGA) of the TAAILs showed decomposition between 400 ◦C and 426 ◦C, which allows them to be used in the Pt-NP-forming microwave reactions at ~200 ◦C.

#### *2.2. Pt-NP Synthesis and Characterization*

Following previous research [13], (η 5 -methylcyclopentadienyl)trimethylplatinum(IV) ((MeCp)PtMe3) was used as a Pt precursor, which can be decomposed in various ways (thermal, photolytic, sonolytic) at relatively mild conditions and without any additives [13,20]. The decomposition of the precursor produces volatile side products, which do not contaminate the NP surface [31–33].

[13], (η

The general synthetic procedure of the Pt-NPs is depicted in Scheme 2. Fixed amounts of Pt precursor and rGO were suspended in the IL to achieve 1 wt% or 2 wt% Pt-NP and 0.5 wt% or 1 wt% rGO in the IL dispersion. After stirring overnight and for 3 h of sonication, the dispersion was placed in a microwave reactor, heated at 40 W to 200 ◦C, and kept at this temperature for 10 min. Black Pt-NP@rGO dispersions were reproducibly obtained.

**Scheme 2.** Reaction conditions for the synthesis of platinum nanoparticles (Pt-NPs) via microwaveinduced heating in ionic liquids (ILs). The mass of the Pt precursor was set to achieve 1 wt% or 2 wt% Pt-NP in the IL dispersion upon complete decomposition (0.5 wt% or 1 wt% reduced graphite oxide (rGO) used for Pt-NPs stabilized on rGO (Pt-NP@rGO) in brackets).

Selected samples and their respective particle size determined via transmission electron microscopy (TEM) are shown in Table 1. In the Supporting Information, Table S1, a full list of prepared samples is given. The nanocrystallinity of the Pt particles was verified by powder X-ray diffraction (PXRD) (Figure 1 and Figures S2–S6 in the Supplementary Information). The Pt-NPs form in the face-centered cubic (fcc) structure, typical for crystalline Pt. The comparison of the crystallite sizes of the NPs calculated with the Scherrer equation (Section 4.1, Equation (1)) and the Pt-NP sizes determined via TEM showed a very good agreement (Table S1, Supplementary Information). Furthermore, thermogravimetric analysis (TGA) of elected samples (Figure S28 in the Supplementary Information) gives a residual Pt mass between 41 wt% and 54 wt% or a reciprocal combined rGO and IL content between 46 wt% and 59 wt%. The mass-temperature profile in TGA did not allow a distinction between rGO and IL. From an estimate of the IL content by CHNS analysis (Table S2), the rGO mass was then approximated between 40 wt% and 54 wt% (Table S4).

Similar to Ir-NPs, Ru-NPs [15], and Pt-NPs [13], the microwave-assisted heating of the platinum metal precursor (MeCp)PtMe3 in ionic liquid dispersion results in small M-NP sizes, largely between 2 nm and 5 nm. The efficient energy uptake of ILs enables fast heating and results in rapid decomposition of the Pt precursor with a high nucleation rate of Pt-NPs, which themselves then absorb microwave radiation. This leads to "hot spots", with a further increase in localized temperature. Small particle sizes can only be achieved if the TAAIL is able to stabilize the newly formed NPs from the very beginning of the growth process. The Pt-NPs show similar sizes mostly independent of the choice of TAAIL and the wt% Pt to IL (Table S1, Supplementary Information). An exception is the TAAIL with the 4-methoxyphenyl substituent (MOP5 to MOP11), where also larger particles, up to 6 nm, were obtained.

1 wt% in TAAIL dispersion. 2 Average particle sizes obtained from transmission electron microscopy (TEM) measurements. At least 200 particles were used for size determination. See Materials and Methods for more information.

**Figure 1.** Powder X-ray diffraction (PXRD) patterns of synthesized Pt-NPs (**a**) and Pt-NP@rGO (**b**) in ILs, together with the simulation for fcc-Pt and its indexed reflections (Crystallographic open database fcc-Pt: 1011114). The -X and -X/Y numbers refer to wt% Pt (X) and wt% rGO (Y) in samples. Additional PXRD patterns can be found in Figures S2–S6 in the Supporting Information (SI).

#### *2.3. Pt-NPs Aggregation in TAAILs*

As seen before for Ru- and Ir-NPs, [15], the TAAILs allow the microwave-induced synthesis of Pt-NPs, but strong Pt-NP aggregation was observed (in the absence of rGO) for all TAAILs, independent of the alkyl chain length or the aryl substitution (Supplementary Information, Figures S7–S27), whereas the comparative IL [BMIm][NTf2] gave well-separated NPs (Figure 2 top, Figure S7). Different from the synthesis of Ru- and Ir-NPs, [15] even the 4-methoxyphenyl- and 2,4-dimethylphenyl-TAAIL did not prevent strong aggregation.

Aggregation, however, could be prevented upon deposition on rGO by performing the (MeCp)PtMe3 precursor decomposition in the presence of rGO. Representative TEM images of Pt-NPs synthesized in different TAAILs without and with rGO are shown in Figures 2 and 3. The rGO-free samples consist of highly aggregated Pt-NPs, with large, dense structures, with dimensions of several tens of nanometers. The degree of aggregation for the Pt-NPs appears independent of the TAAIL.

**Figure 2.** TEM images of Pt-NPs and Pt-NP@rGO composites obtained in the IL (from top to bottom) **Figure 2.** TEM images of Pt-NPs and Pt-NP@rGO composites obtained in the IL (from top to bottom) [BMIm][NTf2 ] (**M4-1, M4\_rGO-1/1**) and the TAAILs [Ph2,4-Me ImC9 ][NTf2 ] (**DMP9-1, DMP9\_rGO-1/1**) and [Ph4-OMe ImC5 ][NTf2 ] (**MOP5-1, MOP5\_rGO-1/1**). TEM images for Pt-NPs are shown on the left and for Pt-NP@rGO composites on the right. The rGO sheets on which the Pt-NP are deposited are clearly seen by their wrinkles at lower magnification (e.g., Figures S10, S12, S16, S17, S19, S25, and S27 in the Supplementary Information).

In previous research, we discussed the necessity of thiol functional groups on rGO to deposit Pt nanoparticles as Pt-NP@rGO when using the IL [BMIm][BF4] [20]. In contrast, with the TAAILs used in this study (and also with the [NTf2]-IL M4 (Figure S8 in the Supplementary Information)), Pt-NPs can be effectively stabilized on rGO without thiol functional groups, resulting in a rather homogenous layer of Pt-NPs on the surface of the rGO (Figures 2 and 3). Compared with the rGO-free samples, nearly no additional agglomeration of NPs can be found, which indicates that the NPs are effectively anchored to prevent aggregation.

The stability of prepared Pt-NPs and Pt-NP@rGO samples was followed by storing them dried under air at room temperature for at least 6 months. PXRD patterns and the calculated crystallite sizes (Equation (1)) from representative samples are given in Table S3 and Figures S3–S6 in the Supplementary Information. Additionally, a TEM image of an rGO-free and a Pt-NP@rGO sample after 1 year did not show significant particle growth or changes in aggregation (Figures S26 and S27 in Supplementary Information).

#### *2.4. Electrochemical Catalysis*

The electrochemical performance of obtained Pt-NP@rGO for the ORR was examined with commercial Pt/C 60% as internal benchmark material in comparison, to reduce deviations by the use of an external standard. As samples, **MP4\_rGO-1/1**, **MP5\_rGO-2/1**, **MP9\_rGO-1/0.5**, and **MOP5\_rGO-1/1** were chosen because of their low size and homogeneity, as depicted in Figure 4. The cyclic voltammogram (CV) curves were recorded in N2-saturated 0.1 M HClO4, with a sweep rate of 20 mV s −1 and, after 50 activation cycles, against reversible hydrogen electrode (RHE, Figure 4a). Afterward, ORR polarization curves (linear sweep voltammetry (LSV)) with a sweep rate of 10 mV s −1 were recorded in O2-saturated 0.1 M HClO4. These measurements were repeated three times each, and the respective combined average CVs and LSVs are shown in Figure 4a,b. The electrochemical surface area (ECSA) was determined by integrating the hydrogen underpotential deposition (Hupd) charge (Hupd charge to surface area conversion constant: 210 µC cm−2 ) in the obtained CVs, and, with the obtained LSVs, mass-specific activities (MAs) at 0.9 V were

calculated. The obtained ECSA and MA are compared in Table 2 to the results of Pt-NPs deposited on rGO via different reaction methods from previous studies of Badam et al. [34], Da¸s et al. [35], and Teran-Salgadon et al. [36].

−1 −1 **Figure 4.** Results obtained via electrochemical measurements: (**a**) averaged cyclic voltammetry (CV) in N2 -atmosphere and at 20 mV s −1 sweep rate of the four samples **MP4\_rGO-1/1**, **MP5\_rGO-2/1**, **MP9\_rGO-1/0.5**, **MOP5\_rGO-1/1**, and a Pt/C 60% standard material; (**b**) averaged oxygen reduction reaction (ORR) polarization curves (linear sweep voltammetry (LSV)) of the same samples in O2 saturated atmosphere at 10 mV s −1 sweep rate. From these data, (**c**) the electrochemical surface area (ECSA) and (**d**) the mass-specific activity (MA) at 0.9 V were calculated.

−1 **Table 2.** Electrochemical surface area (ECSA) and mass-specific activity (MA) of Pt-NP@rGO synthesized with and without IL.

1 Standard deviation obtained by three measurements (see Materials and Methods, Section 4.3).

All CVs of the materials, shown in Figure 4a, have well-pronounced underpotential hydrogen deposition–desorption areas between 0.05 V and 0.40 V, with **MP4\_rGO-1/1** and **MP5\_rGO-2/1** having a larger area than the commercial Pt/C catalyst. No contaminations or side reactions are recognizable in the CVs. The calculated ECSA, shown in Figure 4c, reveals the highest value for **MP5\_rGO-2/1**, with a 1.5-fold increase, compared with Pt/C 60%, while the other samples show similar values as the benchmark material. This shows that rGO can be effectively used as in situ carbon source, in addition to the most commonly used Vulcan XC-72R, which is only added for preparing the catalyst ink. The ORR polarization curves (Figure 4b) of the samples show better onset potentials than the benchmark material and different current density plateaus between 0.20 V and 0.60 V. The presented materials reach their respective plateau at higher voltages but at lower currents than Pt/C 60%. From the LSVs the mass-specific activity (MA) is calculated (Figure 4d). The highest MA is achieved by **MP5\_rGO-2/1**, with a promising twofold increase, compared with the Pt/C 60% benchmark, while **MP4\_rGO-1/1**, **MP9\_rGO-1/0.5**, and **MOP5\_rGO-1/1** also show an increase of more than 1.8-fold, 1.4-fold, and 1.25-fold, respectively, demonstrating a better performance of Pt-NP@rGO for ORR than Pt/C 60%. Calculated Tafel plots of the samples, shown in Figure S29 in the Supplementary Information, are similar to the Tafel plot of Pt/C 60% and indicate a four-electron pathway, with the adsorption of oxygen as a rate-limiting step [37]. The Koutecký–Levich plot calculated from LSVs of **MP4\_rGO-1/1** at different rotation speeds (Figures S30 and S31 in the Supplementary Information) also shows a dominant four-electron pathway toward the ORR process.

A comparison of previous research on Pt-NP@rGO shows the highest ECSA and MA for Pt-NP decorated on tetradecyl-methyl-imidazolium ionic liquid-treated graphene (Pt-TMIm-rGO) by Badam et al. [34], albeit with a similar ECSA to the 55 m2 gPt −1 of **MP5\_rGO-2/1**. Notably, the **MP\_rGO** samples have higher ECSA values, compared with the results of the Pt/rGO probes of Da¸s et al. [35] and Teran-Salgadon et al. [36], much higher MA values than obtained by Da¸s et al. [35] and similar to slightly smaller MA values than given by Teran-Salgadon et al. [36]. From the comparison, it becomes evident that ILs can significantly increase the catalytic activity of Pt-NP@rGO samples.

#### *2.5. Stability Testing*

**MP4\_rGO-1/1**, the most active sample for ORR, was further analyzed with an electrochemical stability test. After the material was activated via CV (20 cycles), 5k cycles between 0.5 and 1.0 VRHE were carried out. The obtained LSVs and calculated MA after activation and after 5k cycles are shown in Figure 5 and in Table 3.

**Figure 5.** Electrochemical activity of the sample **MP4\_rGO-1/1** and Pt/C 60% as reference: (**a**) obtained linear sweep voltammetry at 10 mV s −1 sweep rate after the activation and after 5k cycles; (**b**) calculated mass-specific activity at 0.9 V after activation and after 5k cycles.

**−1**

**Table 3.** Mass-specific activity at 0.9 V of the sample **MP4\_rGO-1/1** after activation and after 5k cycles.

The LSV of **MP4\_rGO-1/1** after 5k cycles shows a decrease in MA, by roughly 45%, while Pt/C 60% loses more than 60% of MA. The activity loss of the sample can be explained with the aid of TEM images obtained after the stability test, shown in Figure 6. When compared with the freshly prepared NPs (Figure 3), the Pt-NP sample lost its small-size homogeneity, with also larger NPs, for which sizes of over 20 nm were clearly visible. Since this increase in size was not observed via PXRD and TEM for dried Pt-NPs, which were stored in air at room temperature for half a year and up to one year, we assume that this NP growth is linked to the ORR.

**Figure 6.** TEM images of the sample **MP4\_rGO-1/1** obtained after 5k stability test. Images were measured with an FEI Titan 80–300 TEM.

We conclude that the NPs can be detached from the stabilizing rGO support during potential cycling and start to agglomerate, losing their electrochemical activity. Since small Pt-NPs remain on the GC surface and show electrochemical activity, the exact position of the NP on the rGO might have an influence on this process, with NPs attached to inner layers of the rGO being more stable. Although not visible, in images with small Pt-NPs, the resolution to depict the rGO can be difficult because stacked or strongly wrinkled rGO sheets might have been removed during ink preparation or electrocatalytic reactions. A complete detachment of Pt-NPs of the rGO would be unlikely because the activity loss would be much stronger with more NP agglomerates and no small NPs detectable.

#### **3. Conclusions**

Small Pt-nanoparticles (Pt-NPs) as dispersed in ionic liquids and in situ deposited on reduced graphite oxide (rGO) (Pt-NP@rGO) can be obtained during a fast and effective microwave-assisted thermal decomposition of (MeCp)PtMe3 without and with rGO added and tunable aryl alkyl ionic liquids (TAAILs), as solvent and stabilizer. The particle size of the obtained Pt-NPs can vary slightly, depending on the TAAIL, and lies between 2 ± 1 nm and 6 ± 2 nm. Independent of the particle size, the Pt-NPs build dense aggregates. When comparing the TAAILs as reaction media with each other, the length of the alkyl chain or the aryl group only has an insignificant effect on the obtained particle size and aggregation.

For samples containing rGO, TEM images show that Pt-NPs build a layer on top of the rGO surface and maintain a homogenous size. In addition, no particle aggregation is observable, which demonstrates that rGO effectively anchors the obtained NPs and prevents their agglomeration. Pt-NPs and Pt-NP@rGO samples show good stability over at least 12 months, with no increase in particle size and no further agglomeration observable.

Pt-NP@rGO samples on glassy carbon electrodes for the oxygen reduction reaction (ORR) exhibit an up to a twofold increase in electrochemical surface area (ECSA) and mass-specific activity (MA), compared with Pt/C 60%. **MP5\_rGO-2/1** achieved the best performance of the analyzed Pt-NP@rGO samples, which all appear promising for ORR and are worthy of further investigation. Since Pt-NP sizes between 2 nm and 3 nm are known to be the most active size range for ORR, and TAAILs make this size range readily available, their use as reaction media for Pt-NP preparation asks for further investigations. An organometallic Pt precursor and TAAILs in the presence of carbon support, in combination with microwave-induced heating, offer fast access to Pt-based electrocatalysts on the nanoscale.

#### **4. Materials and Methods**

#### *4.1. Chemicals and Equipment*

The following chemicals were received from commercial sources: Pt/C (60 wt% Pt on Vulcan XC-72R, Sigma-Aldrich), Nafion 1100 W (Sigma-Aldrich), n-butyllithium (1.6 mol L−1 in hexane, Acros organics), methyllithium (1.6 mol L−1 in diethyl ether, Sigma-Aldrich), 1,2-dibromoethane (>98%, Fluka), potassium hexachloridoplatinate (IV) (97%, BLDpharm), potassium iodide (USP, BP, Ph. Eur. pure, pharma grade, PanReac Applichem), methylcyclopentadienyl dimer (95%, Acros Organics), and perchloric acid (70%, ACS reagent, Sigma-Aldrich). All chemicals were used as received without further purification.

Thermally reduced graphite oxide (rGO) was prepared in a two-step oxidation– thermal reduction process using natural graphite (type KFL 99.5 from AMG Mining AG, former Kropfmühl AG, Passau, Germany). The graphite oxidation procedure of Hummers and Offeman [28] was followed. rGO was obtained at a reduction temperature of 400 ◦C from graphite oxide. For further information, also see our previous studies [21,25].

Tuneable aryl alkyl ionic liquids (TAAILs) were obtained in a two-step synthesis at the group of Prof. Thomas Strassner, Technische Universität, Dresden. The first step was the alkylation of the aryl imidazoles by bromoalkanes to build the IL cations. As the second step, the anion (bromide) was exchanged with LiNTf2. For detailed information, see Supporting Information in Refs. [15,30].

(η 5 -methylcyclopentadienyl)trimethylplatinum(IV) ((MeCp)PtMe3) was synthesized and characterized after a method described by Xue et al. [13,33].

Transmission electron microscopy (TEM) measurements were performed with a JEOL-2100 Plus, a Zeiss LEO912, and an FEI Titan 80–300 TEM at 200 kV, 120 kV, and 300 kV accelerating voltage, respectively. The samples were prepared using 200 µm carboncoated copper grids. Briefly, 0.05 mL of the NP/IL dispersion was dissolved in a 0.5 mL acetonitrile (ACN), and one drop of the diluted dispersion was placed on the grid. After 30 min, the grid was washed with 3 mL ACN and air dried. The images were analyzed by Gatan Microscopy Suite version 3.3, and the particle size distribution was determined from at least 200 individual particles at different positions on the TEM grid, with the same magnifications.

Powder X-ray diffractograms (PXRDs) were measured at ambient temperature on a Bruker D2-Phaser using a flat sample holder and Cu-Kα radiation (λ = 1.54182 Å, 35 kV). Diffrac.Eva V4.2 was used to evaluate PXRD data. Particle sizes were calculated with the Scherrer equation (Equation (1)) as follows:

$$L = K \times \lambda / (\Delta (2\theta) \times \cos \theta\_0) \tag{1}$$

where *L* is the average crystallite size, *K* is the dimensionless shape factor, λ is the wavelength, ∆(2*θ*) is the full width at half maximum (FWHM) in radians, and *θ* is the Bragg angle.

A CEM-Discover SP microwave setup, with a power range of 0–300 W (±30 W) was used for all microwave reactions. Thermogravimetric analysis (TGA) was conducted with a Netzsch TG 209 F3 Tarsus device, equipped with an Al crucible applying a heating rate of 5 ◦C/min under a synthetic air atmosphere.

#### *4.2. Synthesis of Pt-NPs in IL*

As a general approach to obtain Pt-NPs, (MeCp)PtMe3 was put in a 10 mL microwave vessel and dispersed in the corresponding IL. The necessary amount of Pt precursor compared with IL was determined by the wt% of obtained Pt-NPs in IL with 100% conversion, which was set between 1 wt% and 2 wt%. The general batch size was further customized with roughly 300 mg, 500 mg, and 1.00 g IL. The dispersion was stirred for at least 6 h and then heated in the microwave (200 ◦C, 40 W, 10 min holding time). Afterward, 3 mL acetonitrile was added to the black dispersion, mixed, and centrifuged. The clear liquid phase was removed. This washing process was repeated until a colorless, clean solution could be removed (mostly four times). The residue was dried in a high vacuum, to obtain pure Pt-NPs. A list of all obtained Pt-NPs can be found in Table S1 in the Supplementary Information.

For the synthesis of Pt-NPs in IL with rGO, 0.5 wt% to 1 wt% rGO was additionally added into the microwave vessel, stirred overnight, and sonicated for 3 h. The following steps were carried out as mentioned before.

#### *4.3. Electrochemical Measurements*

For all measurements, a similar setup and procedure as in Beermann et al. were used [38]. A conventional three-electrode cell, with a Pt gauze as a counter electrode (Pt furled Pt 5 × 5 cm2 mesh), a reference electrode (reversible hydrogen electrode = RHE), and a glassy carbon-working electrode (5 mm diameter), was used. The working electrode was always lowered into the electrolyte under potential control at 0.05 VRHE. For all measurements, a 0.1 M HClO4 electrolyte solution, diluted from 70% concentrated HClO4 with Milli-Q water, was used. For monitoring, BioLogics potentiostats SP-150 or SP-200 were used.

Fresh inks were prepared from the NPs as the electrochemically active material. For the Pt-NP@rGO inks, NPs containing 1 mg Pt were mixed with 0.5 mL isopropanol, 2.5 mL water, and 10 µL nafion 5% and sonicated for at least 30 min. Since the samples already contained rGO as carbon support, the catalyst ink did not contain additional carbon. Next, 10 µL of the ink was deposited onto the working electrode, to achieve a loading of 10 µgPt cm−2 , and dried for 10 min.

After the electrolyte was purged with N2 for 20 min, the electrochemical activation was performed via potential cycling between 0.050 and 0.925 VRHE, with a scan rate of 100 mV s−1 for 50 cycles under a N2-protected atmosphere. The H-adsorption-based electrochemically active surface area (ECSA) was determined, with the last cycle recorded as 20 mV s−1 , before the activity measurements were selected. The cyclic voltammetry (CV) was carried out by cycling between 0.05 and 1.0 VRHE, with a scan rate of 20 mV s−1 , under a N2 atmosphere. The charge values (QH) were calculated by integrating the respective CV between 0.05 V and 0.4 V. The measured QH values were normalized with respect to the theoretical value of QH theo = 210 µC cm−2 , which is assuming a one-electron transfer between one H atom and one Pt atom. To determine the catalytic activity of the catalysts, linear sweep voltammetry (LSV) was measured in an oxygen saturated electrolyte (after 15 min of bubbling) in a potential range between 0.05 and 1.0 VRHE, with a scan rate of 10 mV s−1 and a rotation speed of 1600 rpm. These measurements were repeated three times. The kinetic currents were calculated using (Equation (2)) as follows:

$$I\_{\mathbf{k}} = (I\_{\text{lim}} \times I) / (I\_{\text{lim}} - I) \tag{2}$$

where *I* is the measured current density, *I*lim is determined in the diffusion-limited current area, and *I*k is the calculated kinetic current density. All presented current densities are iR corrected, where the uncompensated ohmic resistance (R) was determined by potential electrochemical impedance spectroscopy (PEIS), at 0.4 VRHE. Additional rotation speeds of 225, 400, 625, 900, and 1225 rpm were measured for the sample **MP4\_rGO-1/1**.

Stability measurements were performed via potential cycling between 0.5 and 1.0 VRHE, with a scan rate of 50 mV s−1 , in N2-saturated 0.1 M HClO4, with 0 rpm. In total, 5k cycles were carried out. Before and after each stability protocol, three CV cycles between 0.05 and 1.0 VRHE at a scan rate of 100 mV s−1 were recorded, followed by oxygen bubbling (15 min) and LSV, in a potential range between 0.05 and 1.0 VRHE, with a scan rate of 10 mV s−1 and a rotation speed of 1600 rpm.

**Supplementary Materials:** The following supporting information can be downloaded. S1. Pt-NP and Pt-NP@rGO character-ization; S2. Powder X-ray diffractograms (PXRDs); S3. Transmission electron microscopy (TEM) images of Pt-NP and Pt-NP@rGO samples; S4. TGA measurements of selected Pt@rGO samples; S5. Electrocatalytic investigations of Pt@rGO samples.

**Author Contributions:** Conceptualization, C.J. and D.W.; methodology, D.W.; validation, D.W. and M.S.; formal analysis, D.W.; investigation, D.W., S.L., W.J., M.S. and S.R.; resources, C.J. and T.S.; data curation, D.W.; writing—original draft preparation, D.W.; writing—review and editing, C.J., M.S. and T.S.; visualization, D.W.; supervision, C.J.; project administration, C.J.; funding acquisition, C.J. and T.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** The research of C.J. was supported by a joint National Natural Science Foundation of China–Deutsche Forschungsgemeinschaft (NSFC-DFG) project (DFG JA466/39-1). T.S. is grateful for funding by the Deutsche Forschungsgemeinschaft (SPP 1708, STR 526/20-1/2).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** We thank Rolf Mülhaupt and his group for providing the rGO material. We are grateful to Harry Biller for helpful discussions and Dennis Woschko for the TGA measurements. Access to the infrastructure at the Ernst Ruska-Centre, Forschungszentrum Jülich is gratefully acknowledged.

**Conflicts of Interest:** The authors declare that they have no known competing financial interest or personal relationship that could have appeared to influence the work reported in this paper.

#### **References**


**20. Radiological methods to predict prognosis**

Due to the complexity of CF lung disease, multiple modalities are needed to fully assess the extent of disease severity, including radiological imaging. Several imaging techniques have been employed and studied in CF; with plain radiograph, CT, and MRI all having varying abilities to predict prognosis, as well as correlate with clinical condition, and a number of scoring systems exist to qualify the extent of pulmonary involvement. High-resolution CT imaging of the lungs is currently the most sensitive method of assessing the structural changes in CF. HRCT permits airway thickness measurements and the extent of bronchiectactic changes, and these changes closely correlate with FEV1 and often adult patients with CF show more acute changes in HRCT abnormalities than decline in spirometric results [125]. The progression of bronchiectasis also correlates with CFTR genotype and levels of neutrophilic inflammation [126], while also significantly predicting exercise capacity in CF patients [127].

Newer methods to image the lungs in CF include hyperpolarised helium magnetic resonance imaging (He3-MRI) [8] and flurodeoxyglucose positron emission tomography (FDG-PET) imaging [5-7]. Both modalities correlate closely with abnormalities seen on HRCT and improve with antibiotic treatment, making them both sensitive useful tools in the acute setting. He3- MRI may identify early ventilatory changes in paediatric disease before identifiable changes on HRCT [128] and may better correlate with spirometry in assessing response after exacer‐ bation treatment [8]. These modalities are not currently in widespread use due to both cost and availability, however, He3-MRI may become more routine in the future as life expectancy increases and the risk associated with increased cumulative exposure to ionising radiation associated with CT and FDG-PET [129].

Bactericidal/permeability-increasing protein (BPI) is one of the most potent endogenously produced antibacterials secreted by neutrophils, and autoantibodies against this protein are present in up to 90% of CF patients [120]. Levels of BPI-ANCA correlate strongly with markers of lung disease and it may indicate a pathogenic role in CF [121] and a recent study has shown that the presence of BPI-ANCA correlated significantly with a poor outcome, being death or requiring a lung transplantation. Furthermore, in patients colonised with PA the outcome was

While no single biomarker of disease severity exists from either sputum or serum, there are several potential biomarkers that need further study and more robust results to support their

One biomarker that has recently re-emerged as a useful index of disease severity is the measurement of CFTR function. This may be measured by nasal potential difference (NPD) and sweat chloride concentration. It is important as a biomarker as it has been shown that CFTR and sweat chloride concentration at diagnosis predicts long term prognosis [123]. NPD is now reproducible in a more robust manor than previously, as it was difficult to accurately to so in the past [124]. The use of this biomarker has been further established in the recent clinical trials of CFTR modulators and correctors such as ivacaftor, with a change in NPD and sweat chloride seen as marker of improvement and correlating with improvements in lung function and BMI, whether this will be a long-term prognostic indictor remains to be seen [4].

Due to the complexity of CF lung disease, multiple modalities are needed to fully assess the extent of disease severity, including radiological imaging. Several imaging techniques have been employed and studied in CF; with plain radiograph, CT, and MRI all having varying abilities to predict prognosis, as well as correlate with clinical condition, and a number of scoring systems exist to qualify the extent of pulmonary involvement. High-resolution CT imaging of the lungs is currently the most sensitive method of assessing the structural changes in CF. HRCT permits airway thickness measurements and the extent of bronchiectactic changes, and these changes closely correlate with FEV1 and often adult patients with CF show more acute changes in HRCT abnormalities than decline in spirometric results [125]. The progression of bronchiectasis also correlates with CFTR genotype and levels of neutrophilic inflammation [126], while also significantly predicting exercise capacity in CF patients [127]. Newer methods to image the lungs in CF include hyperpolarised helium magnetic resonance imaging (He3-MRI) [8] and flurodeoxyglucose positron emission tomography (FDG-PET) imaging [5-7]. Both modalities correlate closely with abnormalities seen on HRCT and improve with antibiotic treatment, making them both sensitive useful tools in the acute setting. He3- MRI may identify early ventilatory changes in paediatric disease before identifiable changes on HRCT [128] and may better correlate with spirometry in assessing response after exacer‐ bation treatment [8]. These modalities are not currently in widespread use due to both cost and availability, however, He3-MRI may become more routine in the future as life expectancy

significantly worse if associated with this biomarker [122].

**20. Radiological methods to predict prognosis**

routine use in clinical practice.

18 Cystic Fibrosis in the Light of New Research

A number of different radiological scoring systems have been developed to assess disease burden in CF. Older scoring systems were based on chest radiograph changes, and these included the Brasfield [130], National Institutes of Health (NIH) chest radiograph [131], and the Royal Children's Hospital (RCH) chest radiograph score [132]. While these were somewhat useful, they correlated poorly with spirometry and with clinical condition [133]. With the continual improvement in CT imaging modalities, HRCT has become the gold standard for assessing structural abnormalities, and several robust validated scoring systems exist for CT abnormalities. These include the Brody II score [134] and the CF-CT score [135], while one of the older original scoring tools, the Bhalla score, for bronchiectasis is still widely used in modified versions [107]. The Bhalla scoring system is an objective measure incorporating all aspects of CF structural abnormalities and has been validated and correlates with spirome‐ try[136], clinical condition [127, 137], and has some correlation with health related quality of life [138], however, there is little correlation between these scores and other biomarkers of inflammation [106].

Radiological scoring systems are useful in assessing the extent of structural changes in CF, and correlate with certain aspects of the disease, however, they alone are not sufficient to predict prognosis and are likely more beneficial when used as part of composite clinical prediction tools.


**Microbial Enzymes with Special Characteristics for Biotechnological Applications**

#### **Poonam Singh Nigam**

**Abstract:** This article overviews the enzymes produced by microorganisms, which have been extensively studied worldwide for their isolation, purification and characterization of their specific properties. Researchers have isolated specific microorganisms from extreme sources under extreme culture conditions, with the objective that such isolated microbes would possess the capability to bio-synthesize special enzymes. Various Bio-industries require enzymes possessing special characteristics for their applications in processing of substrates and raw materials. The microbial enzymes act as bio-catalysts to perform reactions in bio-processes in an economical and environmentally-friendly way as opposed to the use of chemical catalysts. The special characteristics of enzymes are exploited for their commercial interest and industrial applications, which include: thermotolerance, thermophilic nature, tolerance to a varied range of pH, stability of enzyme activity over a range of temperature and pH, and other harsh reaction conditions. Such enzymes have proven their utility in bio-industries such as food, leather, textiles, animal feed, and in bio-conversions and bio-remediations.

Reprinted from *Biomolecules*. Cite as: Nigam, P.S. Microbial Enzymes with Special Characteristics for Biotechnological Applications. *Biomolecules* **2013**, *3*, 597-611.

#### **1. Enzymes from Microbial Sources**

Enzymes are the bio-catalysts playing an important role in all stages of metabolism and biochemical reactions. Certain enzymes are of special interest and are utilized as organic catalysts in numerous processes on an industrial scale. Microbial enzymes are known to be superior enzymes obtained from different microorganisms, particularly for applications in industries on commercial scales. Though the enzymes were discovered from microorganisms in the 20th century, studies on their isolation, characterization of properties, production on bench-scale to pilot-scale and their application in bio-industry have continuously progressed, and the knowledge has regularly been updated. Many enzymes from microbial sources are already being used in various commercial processes. Selected microorganisms including bacteria, fungi and yeasts have been globally studied for the bio-synthesis of economically viable preparations of various enzymes for commercial applications [1].

In conventional catalytic reactions using biocatalysts the use of enzymes, either in free or in immobilized forms, is dependent on the specificity of enzyme. In recent advances of biotechnology, according to the requirements of a process, various enzymes have been and are being designed or purposely engineered. Various established classes of enzymes are specific to perform specialized catalytic reactions and have established their uses in selected bio-processes. A large number of new enzymes have been designed with the input of protein-engineering, biochemical-reaction engineering and metagenomics. Various molecular techniques have also been applied to improve the quality and performance of microbial enzymes for their wider applications in many industries [2]. As a result, many added-value products are being synthesized in global market with the use of established bioprocess-technology employing purposely engineered biocatalyst-enzymes.

Most of the commercially applicable proteases are alkaline and are bio-synthesized mainly by bacteria such as *Pseudomonas*, *Bacillus*, and *Clostridium*, and some fungi are also reported to produce these enzymes [3]. The xylanases with significant applications in bio-industries are produced by the fungal species belonging to genera *Trichoderma*, *Penicillium* and *Aspergillus*; the xylanases produced by these microorganisms have been found to possess high activity over a wide range of temperatures (40–60 °C) [4].

#### **2. Enzymes with Special Characteristics**

Special characteristics of microbial enzymes include their capability and appreciable activity under abnormal conditions, mainly of temperature and pH. Hence, certain microbial enzymes are categorized as thermophilic, acidophilic or alkalophilic. Microorganisms with systems of thermostable enzymes that can function at higher than normal reaction temperatures would decrease the possibility of microbial contamination in large scale industrial reactions of prolonged durations [5–7]. The quality of thermostability in enzymes promotes the breakdown and digestion of raw materials; also the higher reaction temperature enhances the penetration of enzymes [8]. The complete saccharification and hydrolysis of polysaccharides containing agricultural residues requires a longer reaction time, which is often associated with the contamination risks over a period of time. Therefore, the hydrolytic enzymes are well sought after, being active at higher temperatures as well as retaining stability over a prolonged period of processing at a range of temperatures. The high temperature enzymes also help in enhancing the mass-transfer and reduction of the substrate viscosity [9,10] during the progress of hydrolysis of substrates or raw materials in industrial processes. Thermophilic xylanase are considered to be of commercial interest in many industries particularly in the mashing process of brewing. The thermostable plant xerophytic isoforms of laccase enzyme are considered to be useful for their applications in textile, dyeing, pulping and bioremediation [1,4].

#### **3. Enzymes with Special Characteristics in Biotechnology**

#### *3.1. Protease*

Though the hydrolytic enzymes belong to the largest group of enzymes and are the most commercially-applicable enzymes, among the enzymes within this group the microbial proteases have been extensively studied [11–16]. Proteases prepared from microbial systems are of three types: acidic, neutral and alkaline. Alkaline proteases are efficient under alkaline pH conditions and consist of a serine residue at their active site [15]. Alkaline serine proteases have the largest applications in bio-industry. Alkaline proteases are of particular interest being more suitable for a wide range of applications, since these possess high activity and stability in abnormal conditions of extreme physiological parameters. Alkaline proteases have shown their capability to work under high pH, temperature and in presence of inhibitory compounds [15–18].

Vijayalakshmi *et al*. [16] have optimized and characterized the cultural conditions for the production of alkalophilic as well as a thermophilic extracellular protease enzyme from *Bacillus*. This bacteria named *Bacillus* RV.B2.90 was found to be capable of producing an enzyme preparation possessing special characteristics such as being highly alkalophilic, moderately halophilic, thermophilic, and exhibiting the quality of a thermostable protease enzyme. Alkaline proteases possess the property of a great stability in their enzyme activity when used in detergents [16,18,19]. The alkaline protease produced from *Bacilli* and proteases from other microorganisms have found more applications overall in bio-industries such as: washing powders, tannery, food-industry, leather processing, pharmaceuticals, for studies in molecular biology and in peptide synthesis [1,3].

#### *3.2. Keratinases*

Keratin is an insoluble and fibrous structural protein that is a constituent of feathers and wool. The protein is abundantly available as a by-product from keratinous wastes, representing a valuable source of proteins and amino acids that could be useful for animal feeds or as a source of nitrogen for plants [20]. However, the keratin-containing substrates and materials have high mechanical stability and hence are difficult to be degraded by common proteases. Keratinases are specific proteolytic enzymes which are capable of degrading insoluble keratins. The importance of these enzymes is being increasingly recognized in fields as diverse as animal feed production, textile processing, detergent formulation, leather manufacture, and medicine. Proteolytic enzymes with specialized keratinase activity are required to degrade keratins and for this purpose the keratinases have been isolated and purified from certain bacteria, actinomycetes, and fungi [20,21].

Keratinases have been classified as serine- or metallo-proteases. Cloning and expression of keratinase genes in a variety of expression systems have also been reported [22]. A higher operation temperature is required in the degradation of materials like feathers and wool, which would be possible using a thermostable keratinase. This aspect is of added advantage in achieving a higher reactivity due to lower diffusional restrictions and hence a higher reaction rate would be established. The enhanced stability of keratinase would increase the overall process yield due to the increased solubility of keratin and favorable equilibrium displacement in endothermic reactions.

Baihong *et al*. [23] have reported the enhanced thermostability of a preparation of keratinase by computational design and empirical mutation. The quadruple mutant of *Bacillus subtilis* has been characterised to exhibit the synergistic and additive effects at 60 °C with an increase of 8.6-fold in the t1/2 value. The N122Y substitution also led to an approximately 5.6-fold increase in catalytic efficiency compared to that of the wild-type keratinase.

An alkalophilic strain of *Streptomyces albidoflavus* has been reported to produce extracellular proteases [24]. This particular type of protease was capable of hydrolyzing keratin. The biosynthesis of this specific enzyme was optimized in submerged batch cultures at highly alkaline pH 10.5 and the enzyme yield was stimulated by using an inducer substrate containing keratin in the form of white chicken feathers. An enhanced (six-fold) protease production could be achieved with modified composition of culture-medium containing inducer at the concentration of 0.8% in the fermentation medium. The novelty of this crude enzyme has been reported to be its activity and stability in neutral and alkaline conditions. The maximum activity has been obtained at pH 9.0 and in the temperature range of 60–70 °C. This type of protease (keratinase- hydrolyzing keratins) is of particular significance for its application in industries since the crude enzyme showed its tolerance to the detergents and solvents tested [24]. Liu *et al*. [25] have studied the expression of extreme alkaline, oxidation-resistant keratinase from *Bacillus licheniformis* into the recombinant *Bacillus subtilis* WB600 expression system. The alkaline keratinase was characterized for its application in the processing of wool fibers.

#### *3.3. Amylase*

Amylases are significant enzymes for their specific use in the industrial starch conversion process [26]. Amylolytic enzymes act on starch and related oligo- and polysaccharides [27]. The global research on starch hydrolyzing enzymes based on the DNA sequence, structural analysis and catalytic mechanism has led to the concept of one enzyme family—the alpha amylase. The amylolytic and related enzymes have been classified as glycoside hydrolases. The enzymes have been produced by a wide range of microorganisms and substrates [28–30] and categorized as exo-, endo-, de-branching and cyclodextrin producing enzyme. The application of these enzymes has been established in starch liquefaction, paper, food, sugar and pharmaceutical industries. In the food industry amylolytic enzymes have a large scale of applications, such as the production of glucose syrups, high fructose corn syrups, maltose syrup, reduction of viscosity of sugar syrups, reduction of turbidity to produce clarified fruit juice for longer shelf-life, solubilisation and saccharification of starch in the brewing industry [31]. The baking industry uses amylases to delay the staling of bread and other baked products; the paper industry uses amylases for the reduction of starch viscosity to achieve the appropriate coating of paper. Amylase enzyme is used in the textile industry for warp sizing of textile fibers, and used as a digestive aid in the pharmaceutical industry [28].

Li *et al*. [32] have recently isolated, characterized and cloned a themotolerant isoamylase. For this purpose the enzyme was bio-synthesized using a thermophilic bacterium *Bacillus* sp. This novel enzyme has been reported to display its optimal activity at a remarkably high temperature of 70 °C, as well as being active in the alkaline range. This thermophilic enzyme has also been found to be thermo-stable between 30 and 70 °C, and its activity has been reported to be stable within a pH range of 5.5 to 9.0.

Gurumurthy *et al*. [33] completed the molecular characterization of an extremely thermostable alpha-amylase for industrial applications. This novel enzyme was produced by a bacterium *Geobacillus* sp. which was isolated from the thermal water of a geothermal spring. This isolated bacterium showed the characteristics of thermo-tolerance and alkali-resistance. A purified preparation of amylase suitable for application was obtained using a DEAE-cellulose column and Sephadex G-150 gel filtration chromatography. The enzyme is a novel alpha-amylase due to its optimum activity at a very high temperature of 90 °C and an alkaline pH 8.0. However, this purified preparation enzyme was found to be stable only for 10 min at 90 °C.

#### *3.4. Xylanase*

Hemicellulose is one of main constituents of agricultural residues and plants along with cellulose, lignin and pectin [34]. Xylan is the major component of hemicellulose consisting of ȕ-1,4-linked D-xylopyranosyl residues. The hydrolysis of xylan in plant materials is achieved by the use of a mixture of hydrolytic enzymes including endo-ȕ-1,4-xylanase and ȕ-D-xylosidase [35]. The importance of xylanase has tremendously increased due to its biotechnological applications for pentose production, fruit-juice clarification, improving rumen digestion and the bioconversion of lignocellulosic agricultural residues to fuels and chemicals [34]. Collins *et al*. [36] have extensively studied the xylanase enzyme and its families as well as the special xylanases possessing extremophilic characteristics. Xylanases have established their uses in the food, pulp, paper and textile industries, agri-industrial residues utilization, and ethanol and animal feed production [37,38].

The enzyme used for the purpose of bio-bleaching of wood pulp should be active in the conditions of alkaline pH, high temperature and at the same time it is desirable that this enzyme is stable at high reaction temperatures. Xylanase preparations used for wood processing in the paper industry should be free of cellulose activity. Cellulase-free xylanase preparations have applications in the paper industry to provide brightness to the paper due to their preferential solubilisation of xylans in plant materials and selective removal of hemicelluloses from the kraft-pulp. Kohli *et al*. [39] have studied the production of a c cellulase free extracellular endo-1,4-ȕ-xylanase at a higher temperature of 50 °C and at pH 8.5 employing a selected microorganism: *Thermoactinomyces thalophilus*. The enzyme preparation was found to be thermostable at 65 °C, retaining its activity at 50% after 125 min of incubation at 65 °C. The crude enzyme preparation showed no cellulase activity and the optimum temperature and pH for maximum xylanase activity was found to be 65 °C and 8.5–9.0, respectively. A thermotolerant and alkalotolerant xylanase has been reported to be produced by *Bacillus* sp. [40]. To make the applications of xylanase viable on commercial scales, heterologous systems of *Escherichis coli*, *Pichia pastoris* and *Bacillus* sp. have been used to express xylanase activity [41,42]. The thermophilic microorganism *Humicola* spp. has been studied for its capability of bio-synthesising an alkali-tolerant ȕ-mannase xylanase [43]. Acidophilic xylanases stable under acidic conditions of reaction are reported to be produced by an acidophilic fungus *Bispora* [44], in contrast a xylanase active under conditions of alkaline pH has been studied by Mamo *et al*. [45] for the mechanism of their high pH catalytic adaptation.

Recently three novel xylanases thermophilic in nature (XynA,B,C) have been characterized by Yanlong *et al*. [46], these were produced by *Humicola* sp. for their potential applications in the brewing industry. One xylanase gene, XynA, has been found to adapt to alkaline conditions and stability at higher temperatures. This XynA also possessed higher catalytic efficiency and specificity for a range of substrates. Yanlong *et al*. [46] have reported the application of three xylanases, XynA-C, in simulated mashing conditions in the brewing industry and found the better performance of 37% on filtration acceleration and 13% reduction in viscosity of substrate in comparison to the performance of a commercial trade enzyme, Ultraflo, a product from Novozyme.

#### *3.5. Laccase/Ligninase*

Ligninolytic enzymes are applicable in the hydrolysis of lignocellulosic agricultural residues, particularly for the degradation of the complex and recalcitrant constituent lignin. This group of enzymes is a mixture of synergistic enzymes, hence they are highly versatile in nature and can be used in a range of industrial processes [47–49]. The complex enzyme system consists of three oxidative enzymes: lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase. These enzymes have established their applications in bio-remediation, pollution control and in the treatment of industrial effluents containing recalcitrant and hazardous chemicals such as textile dyes, phenols and other xenobiotics [50–53].

The paper and pulp industry requires a step of separation and degradation of lignin from plant material, where the pretreatment of wood pulp using ligninolytic enzymes is important for a milder and cleaner strategy of lignin removal compared to chemical bleaching. Bleach enhancement of mixed wood pulp has been achieved using co-culture strategies, through the combined activity of xylanase and laccase [54]. The ligninolytic enzyme system is used in bio-bleaching of craft pulp and in other industries such as for the stabilization of wine and fruit juices, denim washing [49], the cosmetic industry and biosensors [1,34]. Fungi are the most potent producers of lignin degrading enzymes. White rot fungi have been specifically studied for the production of these enzymes by Robinson *et al*. [50–52]. For the economical production of ligninolytic enzymes, agricultural residues have been used as the substrate in microbial production of lignin degrading enzymes [34].

Thermophilic laccase enzyme is of particular use in the pulping industry. Recently, Gali and Kotteazeth [55] reported the biophysical characterization of thermophilic laccase isoforms. These were initially isolated from the xerophytic plant species *Cereus pterogonus* and *Opuntia vulgaris* and showed thermophilic property [56–58]. In order to prepare laccase enzymes with special characteristics, several studies have been conducted to provide a scientific basis for the employment of laccases in biotechnological processes [59–62]. Forms of laccase with unusual properties have been isolated from the basidiomycetes culture of *Steccherinum ochraceum* [63], *Polyporus versicolor* [64] and a microbial consortium [65].

#### *3.6. Cellulase*

Cellulase enzymes are the third most important enzyme for industrial uses: world-wide research has been focused on the commercial potential of cellulolytic enzymes for the commercial production of glucose feedstock from the agricultural cellulosic materials [1]. The significance of cellulose hydrolyzing thermophilic enzymes in various industries includes the production of bio-ethanol and value-added organic compounds from renewable agricultural residues [66]. Cellulose is the most abundant natural resource available globally for bioconversion into numerous products in bio-industry on a commercial scale. For efficient bioconversion a strategy of efficient saccharification using cellulolytic enzymes is required. Hardiman *et al*. [66] used the approach of thermophilc directed evolution of a thermophilic ȕ-glucosidase.

Cellulase is complex of three important enzymes which work synergistically owing to the crystalline and amorphous complex structure of cellulose. These enzymes, acting synergistically, hydrolyse cellulose to cello-biose, glucose and oligo-saccharides. Endoglucanase enzyme is the first one acting on amorphous cellulose fibers, attacking the glucose-polymer chain randomly, which releases small fibers consisting of free-reducing and non-reducing ends. The free-ends of the chain are then exposed to the activity of exoglucanase enzyme, which produces cellobiose. The third component of cellulase is ȕ-glucosidase, which hydrolyses the cellobiose, producing the glucose as the final product of cellulose sacharification.

Thermostability is an important technical property for cellulases: since the saccharification of cellulose is faster at higher temperatures, the stability of enzyme activity is necessary to be maintained for the completion of the process. Though the enzymes have been prepared using thermophilic microorganisms, these enzyme preparations are not necessarily heat-stable. The activity profile for the thermal activation and stability of cellulases derived from two *Basidiomycetes* cultures was studied by Nigam and Prabhu [67]. The results proved that the prior heat-treatment of enzyme preparation caused activation of exo- and endo-glucanase activities, and improved the stability of enzymes over a period of reaction time. Therefore, the efficiency of cellulolytic enzymes may be increased by heat-treatment, by incubating buffered enzyme preparations without cellulose or substrates prior to the saccharification process [67].

Cellulolytic enzymes have been produced by a range of microorganisms including bacteria and fungi. The studies have been performed for the biosynthesis of a high-activity preparation in high yields [68–70]. Researchers have cultivated microorganisms to achieve cellulases of desired quality under submerged and solid state fermentation conditions for the economical production of enzyme using waste agricultural residues [1].

#### *3.7. Miscellaneous Enzymes in Biotechnology*

Various enzymes other than those described above have a significant place in the list of microbial enzymes, which have established their applications in bio-industries. Lipases have been widely studied for their properties and utilization in many industries [71–75]. Pectinases have established their role in the fruit and juice industries [76]. Certain enzymes are specifically required in pharmaceutical industry for diagnostic kits and analytical assays [77–80].

Bornscheuer *et al*. [81] have currently mentioned that in all the research and developments so far in the field of biocatalysis, the researchers have contributed in three waves of outcomes. These innovations have played an important role in the establishment of current commercially successful level of bio-industries. As a result recent bioprocess-technology is capable of meeting future challenges and the requirements of conventional and modern industries, for example Trincone [82] has reviewed the options for unique enzymatic preparation of glycosides. Earlier enzymatic process were performed within the limitations of an enzyme, whereas currently with the knowledge of modern techniques, the enzyme can be engineered to be a suitable biocatalyst to meet the process requirement. Riva [83] has identified the scope of a long-term research in biocatalysis, since there are underlying problems in the shift from classical processes to bio-based processes for commercial market.

Table 1 summarizes some enzymes produced by microorganisms possessing special characteristics useful in various bio-processes. There is a tremendous scope for research and development to meet the challenges of third generation biorefeneries [83], for the production of numerous chemicals and bio-products from renewable biomasses [34]; or by the new glycoside hydrolases [82]; or new enzymes found in marine environments [84]. Although the research for the hemicellulases as important biorefining enzymes has not well established, biocatalysis for xylan processing is slowly progressing and a wide range of hemicellulases have been isolated and characterized [85]. Specifically about the biobased glycosynthesis, Trincone [82] has mentioned that the new prospects are open for the use of pentose sugars as main building blocks for engineered pentosides to be used as non-ionic surfactants or as the ingredients for prebiotic food and feed preparations.

**Table 1.** A summarized overview of some microbial enzymes with special characteristics of industrial importance.

**Table 1.** *Cont.*

#### **4. Conclusions**

Biotechnology is utilizing a wide range of enzymes synthesized on a commercial scale employing purposely screened microorganisms. Selected microorganisms have been characterized, purposely designed and optimized to produce a high-quality enzyme preparation on large scales for industrial applications. Different industries require enzymes for different purposes; hence microbial enzymes have been studied for their special characteristics applicable in various bio-processes. Recent molecular biology techniques have allowed to tailor a specific microorganism, to produce not only the high yields of an enzyme, but also enzyme with desired special characteristics such as

thermostability, tolerance at high temperature and its stability in acidic or alkaline environment, and retaining the enzyme activity under severe reaction conditions such as in presence of other metals and compounds.

### **Conflicts of Interest**

The author declares no conflict of interest.

#### **References**


*2.1.5 Influence of failure on production loss (PL)*

The PL indicator is due to the failure of components, subsystems, or systems, which will cause changes in product operation, cause standby products to be put into operation, and cause production problems such as drilling stoppages, which will cause economic losses in the enterprise. In combination with the production of offshore oil and gas, in the case of production loss caused by failure, the PL index is divided into six levels, and the specific scoring standards are shown in **Table 6**.


**1. Introduction**

To date, nanotechnology is the operation of matter on an atomic and molecular scale. Generally speaking, the size scale in nanotechnology including materials, devices, and other structures is at least from 1 to 100 nanometers in one dimension. The revolution of the nanotechnology is taking a crucial effect on the different fields, such as commercial sectors, engineering, science, drug delivery, sensors, and the construction industry. Nanostructures in such size have made steadily increasing attraction because of their attractive and captivating properties, same as their fascinating applications complementary to the materials in bulk. The interesting properties of materials in nanoscale (both physical and chemical) can make the efficacy enhanced distinctively in mechanical strength, (photo)catalysis, optical sensitivity, and (thermal and electrical) conductivity which enable applications such as improved materials with higher properties, storage devices of the electronic and energy, sensors, and catalysts [1–8].

**50**

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According to the dimensions in nanometer scale size, nanostructures can be classified into the following three groups:

In comparison with 0D nanostructures, it is easier to investigate the relationship between the mechanical properties, optical and electronic transport, and the confinement of the size and dimensionality for 1D nanostructures. Moreover, 1D semiconductor nanomaterials have an extremely crucial effect of the active components and interconnect in the nanoscale electronic and photonic devices fabrications.

Up to now, 1D nanostructure is generally used to describe large aspect ratio rods, wires, tubes, and belt and tubes and has been the key point of investigation due to its attractive physical and technological significance in fabricating nanoscale devices. When the radial diameter of the 1D nanostructure is lower than some lengths (the path of the phonon mean free, the light of wavelength, Bohr radius, etc.), the effect of the quantum mechanics will be crucial. Owing to the large surface-to-volume ratio and the confinement of two dimensions, nanowires possess the definitely attractive electronic, magnetic, and optical properties. In addition, because nanowires' aspect ratio is extremely large, the quantum particles (photons, phonons, electrons, etc.) can be conducted directly easily to make the nanowires as the ideal candidate for the energy transport materials to enhance the relevant technique applications [9–17].

Today, numerous approaches have been researched to synthesize 1D nanostructures. Two fundamental steps are essentially involved in the evolution: nucleation and growth. A lot of solid materials with 1D nanostructures in nature are controlled by the bonding in the structure of crystallography in the highly anisotropic. The materials need common growth conditions including chemical vapor deposition (CVD), wet-chemical routs, and template-assistant methods. In classification, all the contemporary approaches are divided into bottom-up and top-down methods. The most important issue for developing a new synthetic method is to control the dimensions, morphology, and uniformity of nanostructures. When making a method to synthesize the nanostructures by the synthetic effects, it is definitely important to control the related morphology (or shape), dimensions, and uniformity simultaneously. To obtain 1D growth nanostructures, several chemical methods were generated. The current common six different strategies are (1) the reduction of a 1D microstructure in size, (2) 0D nanostructure self-assembly, (3) by a capping reagent kinetic control, (4) a template usage for direction, (5) a liquid droplet confinement as in the vapor-liquid-solid process, and (6) the control of a solid with the anisotropic crystallographic structure.

Generally, there are four popular mechanisms for understanding the synthesis of 1D nanostructure materials. They are the mechanism of vapor-liquid-solid (VLS), the mechanism of oxide-assisted growth (OAG), the mechanism of vapor-solid (VS), and the mechanism of solution-liquid-solid (SLS).

### **1.1 Vapor-liquid-solid (VLS)**

In the VLS mechanism, a liquid metal cluster or catalyst, such as Au, Fe, Ni, or Co, is taken as the energetically favorable point of the gas-phase reactant absorption.

**53**

dimensions.

*Methodologies for Achieving 1D ZnO Nanostructures Potential for Solar Cells*

metal catalyst that can be achieved under equilibrium conditions [18–21].

The reactant supersaturates and segregates from the cluster and then grows into a 1D structure of the material, the diameter of which is limited by the size of the liquid

This kind of the synthesized technique, in which oxides replaced by metals have a crucial effect on the nucleation induction and the nanowire growth, can produce the high-purity 1D nanomaterials in the large quantities, and the metal catalysts do not need any more. The 1D nanomaterial synthesis with the mechanism of the oxide-assisted growth is the extension of the traditional vapor-liquid-solid method with the metal catalyst. Moreover, it can be taken to make the nanowires by other

For the mechanism of the vapor-solid (VS), the size of the nucleation site is critical for defining the rod diameter when the vapor supersaturation is appropriately controlled. Metal catalysts are not necessary. Three stages can be summarized as the illustration: (i) The source forms vapor phase, (ii) the vapor is transported by the carrier gas and deposits on the substrate to form crystalline nuclei, and (iii) the defects of the nuclei become the growth points, and the reactive vapor molecules

Solution-liquid-solid (SLS) phases are involved in the nanowire growth that is in fact an analogy to the conventional whisker growth via vapor-liquid-solid (VLS) mechanism. The difference is that the vapor phase involved in the VLS growth is now substituted by a solution phase in the SLS mechanism. In turns out, however, the nanowires prepared by the SLS mechanism have a varying diameter ranging

Up to now, it is still a challenge to accurately characterize the property of 1D nanostructures due to the constrains of the current measuring techniques, such as (1) the size of 1D nanostructures is too small to adopt the well-established testing techniques and (2) 1D nanostructures different from bulk materials are hard to pinpoint at the desired location. Therefore, the relevant techniques should be explored

1D nanomaterials behave qualitatively different from the conventional bulk materials when the size reduces to nanoscale. It is well known that the increments of yield stress and the hardness of a polycrystalline material are consistent with the decrement of the grain size to the micrometer scale, and this significant phenomenon is defined as the Hall-Petch effect. For the single-crystalline 1D nanostructures, their property is extremely higher than that of the counterparts in the larger

*DOI: http://dx.doi.org/10.5772/intechopen.83618*

**1.2 Oxide-assisted growth (OAG)**

materials than silicon [22–26].

further grow into nanostructures [27, 28].

from 10 to 150 nm, which is not uniform [29, 30].

**2. Property of one-dimensional (1D) nanomaterials**

to detect the property of 1D nanostructures accurately.

**1.4 Solution-liquid-solid (SLS)**

**2.1 Mechanical property**

**1.3 Vapor-solid (VS)**

The reactant supersaturates and segregates from the cluster and then grows into a 1D structure of the material, the diameter of which is limited by the size of the liquid metal catalyst that can be achieved under equilibrium conditions [18–21].


**2. Use of reference intervals, consequence of misclassification and selection of a reference population**

#### **2.1. The use of reference intervals**

304 Blood Cell – An Overview of Studies in Hematology

PARAMETER GRADE 1

NEUTROPHILS

clarification August 2009.

assumes a value of zero.

MILD

1.0 –

1.3 x 109 cells/L

abnormal values, how to grade the severity of the abnormality.

GRADE 2 MODERATE

HEMOGLOBIN 10.0 – 10.9 g/dL 9.0 – 9.9 g/dL 7.0 – 8.9 g/dL < 7.0 g/dL

0.999 x 109cells/L

Adult and pediatric values for age >57 days, HIV-negative from the DAIDS toxicity tables version 1.0, December 2004;

**Table 1.** Examples of DAIDS criteria of estimating severity grading based on laboratory parameters.

Reference values, in general, refer to the value or test result obtained by the observation or measurement of a particular type of quantity on an adequate number of persons (reference sample group) selected to represent the general population. Reference values are usually presented as reference intervals which refer to the interval between, and including two reference limits i.e., from the lower reference limit to the upper reference limit defined by a specific percentage (usually 95%). In certain parameters such as absolute counts of monocytes, eosinophils and basophils, only one reference limit (decision limit), more often the upper reference limit is of biological significance hence the lower reference limit

Reference values go hand in hand with toxicity grading or decision limits, which can be defined as specific levels of the analyte that correspond to mild to life threatening clinical situations. Toxicity grading is particularly useful in the decision-making process of interpreting a measured value and assessing the health status of the subject being tested. For this reason reference values or toxicity grading are routinely used in clinical trials at enrollment to determine eligibility, establish baseline measures, and also during the course of the trial to monitor the participants' health. Moreover, several analytes are used either as markers for the possible presence of a disease or as direct evidence for that disease. Reference values, especially hematological and immunologic indices, are influenced by such factors as genetics, dietary patterns, pregnancy, gender, age, ethnic origin and prior exposure to environmental pathogens. Thus, it is important to consider these factors when

0.75 –

GRADE 3 SEVERE

0.5 – 0.749 x 109 GRADE 4 POTENTIALLY LIFE-THREATENING

cells/L

cells/L < 0.5 x 109

Historically, clinical studies as well as routine clinical patient management in most African countries have relied on European-generated automated instrument values, US established reference intervals or the U.S. NIH division of AIDS (DAIDS) toxicity grading tables in assessing clinical parameters in study participants. The US-established reference intervals are obtained from the Massachusetts General Hospital reference values and serve as the standard reference interval comparison for most studies [6]. The DAIDS toxicity tables, also derived from a Caucasian population, are used for grading the severity of adult and pediatric adverse events, whether or not they are considered to be related to the study intervention [7]. DAIDS provides guidelines for estimating severity of adverse events using specific reference intervals (Table 1) as criteria for determining what is 'normal' and among

> Reference intervals are useful both in the clinical and research environment. Medical laboratory reference intervals are primarily used for clinical purposes. They can be used as an indicator of good health. Alternatively, reference intervals/limits can be used to screen for physiological or pathological conditions hence important in routine health assessment,

particularly for screening of anemia, blood disorders and diseases of the immune system. Reference intervals are important for accurate interpretation of laboratory data and provide assistance to the clinician in creating a more comprehensive clinical perspective for diagnosis and management of patients [10]. Of particular importance is the use of reference values as surrogate markers for monitoring disease progression and response to antiretroviral therapy in HIV-infected individuals [11]. For example, decisions to initiate, continue, or change antiretroviral therapy regimens are determined using CD4+ Tlymphocyte cell (CD4) counts, while drug toxicity is monitored using liver function tests, renal function tests, and full blood counts (FBC) [12, 13]. The hemoglobin concentration is used as a marker of anemia. As part of the management of anemia, the clinician conducts additional tests to identify a reversible etiology for anemia (eg, iron deficiency, infection) and if present treats it appropriately. However, in the clinical environment, the statistical definition of reference intervals may not allow certain clinical uses. Because these reference intervals have been derived statistically from a healthy population, they may not be used to rule in or rule out specific medical conditions. The statistically derived 95% reference interval would mean that 5% of normal subjects would have abnormal laboratory values. This is erroneously interpreted that 95% of diseased individuals would fall outside the derived reference interval. It is recommended that the number of diseased individuals who fall outside the defined 95% reference intervals be determined through a study of the distribution of such persons with the target condition [14]. Thus, it is necessary to confirm the validity of the proposed reference intervals with clinicians using a particular test to manage patients.

Laboratory Reference Intervals in Africa 307

these challenges, there is a need to establish accurate, locally derived reference intervals for the target population. Within the last decade, several studies in sub-Saharan Africa have attempted to establish hematological and biochemical reference intervals for use in clinical

The selection of a reference population is as per described in the Clinical Laboratory Standards Institute (CLSI, Wayne, PA, USA) guidelines [21]. The guidelines state that reference individuals selected for the determination of reference intervals should closely resemble the patient population undergoing medical examination and should be of similar age to be clinically significant [21]. The reference individuals should not be hospital or clinic patients unless absolutely necessary. The guidelines describe two selection methods for a reference population: *a priori* and *a posteriori*. *A priori* sampling method involves selection of reference individuals based on well-defined exclusion and partition criteria. The entire selection process takes place before any blood sample is drawn and a sufficient number of reference individuals are targeted to provide statistical validity. *A posteriori* sampling method involves selection of the reference population after the analyte has been tested. The CLSI guidelines recommend a minimum of 120 individuals to allow 90% confidence limits to be non-parametrically calculated for the reference limits [22]. Partitioning of reference intervals either by gender or age is recommended if clinically useful or physiologically well grounded. Even though 120 samples remains the recommended standard, an efficient laboratory, by considering the CLSI revised guideline strategies [8], can determine reference intervals using fewer samples [23]. Alternatively, a laboratory can adopt reference intervals established from another laboratory if the values are verified using the procedures set out in the guidelines.

In our study [9] of adolescents and adults living in rural western Kenya, all participants were screened by a review of medical history, a physical examination, tested for HIV and pregnancy (for females), and treated for any illnesses diagnosed. Participants were included if they were a permanent resident of the study area, between 13 and 34 years of age and able to provide informed consent or assent if a minor. Participants were excluded if they were HIV-seropositive, pregnant, exhibiting febrile symptoms or on any medication. Blood samples were therefore obtained from clinically healthy participants selected to generate hematologic and biochemical reference intervals. Data were partitioned by age (<18 years of age as adolescents and ≥18 as adults) and gender; median and 95th percentile intervals were calculated. The lower 95% reference limit was defined as the 2.5th percentile while the upper limit was defined as the 97.5th percentile. A Wilcoxon rank-sum test was used to test for age and gender differences. We compared our data against reference intervals from the Massachusetts General Hospital (MGH-USA) [6] and the U.S. NIH Division of AIDS (DAIDS) toxicity tables [7] to determine the number of study participants with values outside the MGH ranges or who had any adverse event as graded by the DAIDS criteria. However, while the CLSI guidelines recommend a description of the population from which reference intervals are derived, the DAIDS and Massachusetts General Hospital reference

monitoring and patient management.

**2.3. Selection of a reference population**

values do not provide such information.

In the research environment, however, the aim is to define a reference population that is as similar as possible to that for which a particular test will be applied with the exception of the presence of the disease. During clinical trials, reference intervals relevant to the study of interest are required to interpret normal values of standard laboratory test results from the target population [15]. This is particularly important during phase I/II safety trials where healthy individuals are assessed without a control group [15-17]. Moreover, clinical reference intervals are necessary in order to accurately assess potential adverse events observed during the course of clinical trials.

#### **2.2. Consequences of misclassification**

A majority of clinically healthy participants have been excluded from several clinical trials in Africa because laboratory hematological and biochemical parameters are classified as abnormal [18-20]. Unnecessary exclusion of potential participants generally results in increased cost for study recruitment to achieve the target sample size. Accurate reference intervals are required for monitoring adverse events during vaccine and drug trials to limit misclassification that might otherwise lead to discontinuation of such trials or erroneous conclusions that the trial interventions are associated with adverse events. A study documented that the expense of adverse event investigation and reporting accounted for at least one-third of the study cost, irrespective of the adverse event grade [18]. To overcome these challenges, there is a need to establish accurate, locally derived reference intervals for the target population. Within the last decade, several studies in sub-Saharan Africa have attempted to establish hematological and biochemical reference intervals for use in clinical monitoring and patient management.

#### **2.3. Selection of a reference population**

306 Blood Cell – An Overview of Studies in Hematology

manage patients.

observed during the course of clinical trials.

**2.2. Consequences of misclassification**

particularly for screening of anemia, blood disorders and diseases of the immune system. Reference intervals are important for accurate interpretation of laboratory data and provide assistance to the clinician in creating a more comprehensive clinical perspective for diagnosis and management of patients [10]. Of particular importance is the use of reference values as surrogate markers for monitoring disease progression and response to antiretroviral therapy in HIV-infected individuals [11]. For example, decisions to initiate, continue, or change antiretroviral therapy regimens are determined using CD4+ Tlymphocyte cell (CD4) counts, while drug toxicity is monitored using liver function tests, renal function tests, and full blood counts (FBC) [12, 13]. The hemoglobin concentration is used as a marker of anemia. As part of the management of anemia, the clinician conducts additional tests to identify a reversible etiology for anemia (eg, iron deficiency, infection) and if present treats it appropriately. However, in the clinical environment, the statistical definition of reference intervals may not allow certain clinical uses. Because these reference intervals have been derived statistically from a healthy population, they may not be used to rule in or rule out specific medical conditions. The statistically derived 95% reference interval would mean that 5% of normal subjects would have abnormal laboratory values. This is erroneously interpreted that 95% of diseased individuals would fall outside the derived reference interval. It is recommended that the number of diseased individuals who fall outside the defined 95% reference intervals be determined through a study of the distribution of such persons with the target condition [14]. Thus, it is necessary to confirm the validity of the proposed reference intervals with clinicians using a particular test to

In the research environment, however, the aim is to define a reference population that is as similar as possible to that for which a particular test will be applied with the exception of the presence of the disease. During clinical trials, reference intervals relevant to the study of interest are required to interpret normal values of standard laboratory test results from the target population [15]. This is particularly important during phase I/II safety trials where healthy individuals are assessed without a control group [15-17]. Moreover, clinical reference intervals are necessary in order to accurately assess potential adverse events

A majority of clinically healthy participants have been excluded from several clinical trials in Africa because laboratory hematological and biochemical parameters are classified as abnormal [18-20]. Unnecessary exclusion of potential participants generally results in increased cost for study recruitment to achieve the target sample size. Accurate reference intervals are required for monitoring adverse events during vaccine and drug trials to limit misclassification that might otherwise lead to discontinuation of such trials or erroneous conclusions that the trial interventions are associated with adverse events. A study documented that the expense of adverse event investigation and reporting accounted for at least one-third of the study cost, irrespective of the adverse event grade [18]. To overcome The selection of a reference population is as per described in the Clinical Laboratory Standards Institute (CLSI, Wayne, PA, USA) guidelines [21]. The guidelines state that reference individuals selected for the determination of reference intervals should closely resemble the patient population undergoing medical examination and should be of similar age to be clinically significant [21]. The reference individuals should not be hospital or clinic patients unless absolutely necessary. The guidelines describe two selection methods for a reference population: *a priori* and *a posteriori*. *A priori* sampling method involves selection of reference individuals based on well-defined exclusion and partition criteria. The entire selection process takes place before any blood sample is drawn and a sufficient number of reference individuals are targeted to provide statistical validity. *A posteriori* sampling method involves selection of the reference population after the analyte has been tested. The CLSI guidelines recommend a minimum of 120 individuals to allow 90% confidence limits to be non-parametrically calculated for the reference limits [22]. Partitioning of reference intervals either by gender or age is recommended if clinically useful or physiologically well grounded. Even though 120 samples remains the recommended standard, an efficient laboratory, by considering the CLSI revised guideline strategies [8], can determine reference intervals using fewer samples [23]. Alternatively, a laboratory can adopt reference intervals established from another laboratory if the values are verified using the procedures set out in the guidelines.

In our study [9] of adolescents and adults living in rural western Kenya, all participants were screened by a review of medical history, a physical examination, tested for HIV and pregnancy (for females), and treated for any illnesses diagnosed. Participants were included if they were a permanent resident of the study area, between 13 and 34 years of age and able to provide informed consent or assent if a minor. Participants were excluded if they were HIV-seropositive, pregnant, exhibiting febrile symptoms or on any medication. Blood samples were therefore obtained from clinically healthy participants selected to generate hematologic and biochemical reference intervals. Data were partitioned by age (<18 years of age as adolescents and ≥18 as adults) and gender; median and 95th percentile intervals were calculated. The lower 95% reference limit was defined as the 2.5th percentile while the upper limit was defined as the 97.5th percentile. A Wilcoxon rank-sum test was used to test for age and gender differences. We compared our data against reference intervals from the Massachusetts General Hospital (MGH-USA) [6] and the U.S. NIH Division of AIDS (DAIDS) toxicity tables [7] to determine the number of study participants with values outside the MGH ranges or who had any adverse event as graded by the DAIDS criteria. However, while the CLSI guidelines recommend a description of the population from which reference intervals are derived, the DAIDS and Massachusetts General Hospital reference values do not provide such information.


# **Artisanal and Small-Scale Gold Mining (ASGM) Related Environmental and Health Problems**

Edited by

Masayuki Sakakibara, Win Thiri Kyaw and José Luis Rivera Parra Printed Edition of the Special Issue Published in *International Journal of Environmental Research and Public Health*

www.mdpi.com/journal/ijerph