Nanowire Field-Effect Transistor Sensors

Sensitive and quantitative analysis of proteins and other biochemical species are central to disease diagnosis, drug screening and proteomic studies. Research advances exploiting SiNWs configured as FETs for biomolecule analysis have emerged as one of the most promising and powerful platforms for label-free, real-time, and sensitive electrical detection of proteins as well as many other biological species. In this chapter, we first briefly introduce the fundamental principle for semiconductor NW-FET sensors. Representative examples of semiconductor NW sensors are then summarized for sensitive chemical and biomolecule detection, including proteins, nucleic acids, viruses and small molecules. In addition, this chapter discusses several electrical and surface functionalization methods for enhancing the sensitivity of semiconductor NW sensors.

Introduction
Fundamental biomedical research demands novel biosensors and assays that can fulfill the requirements of ultra-sensitivity and high-throughput [1,2]. Many semiconducting nanomaterials, such as NWs, carbon nanotubes and graphene have been studied for the electronic sensing in an effort to address these needs. Among them, SiNWs possess unique structural and chemical characteristics, including diameters similar to proteins, high surface-to-volume ratios, chemically well-defined and tailorable silicon oxide surfaces, which have enabled them to be configured as high-performance FETs for label-free, real-time, sensitive detection of proteins and other biomolecules [3][4][5].
The electrical detection of biomolecules using a NW-FET can be understood as follows. The surface of a NW-FET is functionalized with biomolecule receptors, such as monoclonal antibodies or single-strand DNA (ssDNA) probes, which can selectively bind to biomolecule targets in solution. The binding of charged biomolecules, (the sign and number of charges depend on the isoelectric point of the biomolecules and the solution pH), leads to a variation of charge or electric potential at the NW surface, in a way similar to applying an external potential to gate electrode in a conventional FET device. The charge carrier densities of the NW-FET is thus tuned and leads to an electrical conductivity change associated with the biomolecular binding events in real time. Since the NW diameters can be similar to biomolecules such as proteins and nucleic acids, these binding events can be sensitively detected by the NW-FETs. Furthermore, incorporation of a number of NW-FET elements in a single sensor chip where the NWs are functionalized with different surface receptors allows for multiplexed electrical detection in the same assay, enabling a unique and powerful platform for chemical/biological recognition [6].
In this chapter, we first briefly introduce the fundamental principles of the NW-FET sensor. Then, representative examples in which FET sensors are applied to detect chemical and biomolecule targets, including proteins, nucleic acids, viruses, and small molecules, are summarized. Furthermore, several methods for improving the sensitivity and/or capabilities of NW-FET sensors, including the use of branched NWs to enhance the capture efficiency of molecular analytes, operation of the FET in the subthreshold regime, increasing the analyte concentration by electrokinetic effects, and detection in physiological fluids, are briefly illustrated.

Fundamental Principles of Field-Effect Transistor Sensors
As previously discussed in Chap. 5, FETs are among the fundamental building blocks of today's high-density integrated circuits. In a standard planar metal-oxide semiconductor FET (Fig. 10.1a), the semiconductor substrate is connected to the gate (G), the source (S) and the drain (D) electrodes. If no gate voltage is applied (the "Off" state), the FET is equivalent to two back-to-back p-n junctions with almost no current flows. In the "On" state, when the gate bias exceeds a threshold voltage, carriers (e.g., holes for p-Si and electrons for n-Si) are induced at the semiconductor-oxide interface, the potential barrier of the channel drops, resulting in a significant current flow. Therefore, the conductance of the semiconductor channel between the source and drain regions can be switched on and off by the potential at the gate electrode. The use of planar FETs for ion-selective sensors was introduced several decades ago [7], while their opportunities as chemical and biological sensors have further been advanced in new and significant ways using NWs. Similar to planar FET, the conductance of a NW-FETs can be controlled by variations in the charge density or electric potential in the channel region. This response makes NW-FETs ideal candidates for chemical and biological sensing, as the change in electric field due to binding of a charged molecule to the NW surface, which is analogous to applying a voltage via a gate electrode, can readily change the device conductance. For example, a p-type SiNW functionalized with surface receptors that can specifically capture chemical/biomolecule targets will exhibit an increase in conductance when negatively charged molecules bind to the receptors. This increase in conductance is similar to applying a negative gate voltage and results from accumulation of charge carriers (holes) in the p-type FET ( Fig. 10.1b). Conversely, binding positively charged molecules will deplete hole carriers and reduce the conductance. Hence, NW-FETs can enable real-time label-free direct electrical readout of biological events, including binding/unbinding, enzymatic reactions and electron transfer. These detection capabilities are ideal for developing a platform system for analyzing biological samples.
Semiconductor NWs composed of Si or other materials (e.g., ZnO, SnO 2 and In 2 O 3 ) have been explored extensively as FET sensors [8][9][10]. Among these NW materials, the potential to achieve molecular-size diameters [11], high electron or hole mobility [12], and versatile surface functionalization of SiNWs [13], as well as the potential of interfacing with existing mature silicon industry processing, have made SiNWs one of the most widely studied for biomolecular sensing [3][4][5]. SiNW-FETs are transformed into nanosensors by surface functionalization with probe molecules that enable the specific recognition of chemical/biological molecule targets. Covalent binding to the native silicon oxide (SiO 2 ) layer that naturally grows on SiNWs represents one of the most robust approaches for probe attachment and takes advantage of the wealth of knowledge available from studies focused on functionalization of glass (SiO 2 ) slides [14]. A detailed surface functionalization process is described elsewhere [13]. The simplest and earliest established example of this approach is hydrogen-ion concentration detection or pH sensing [8]. In this case, The semiconductor substrate (e.g., p-Si) is connected to gate (G), source (S) and drain (D) electrodes, and can be switches between the "off" and "on" states by applying the gate voltage. b Schematic comparison of (top) a standard FET device and (bottom) a SiNW-FET sensor. The NW surface is functionalized with a receptor layer to recognize target biomolecules in a solution, which are charged and provide a molecular gating effect on SiNWs. Reproduced from [76]. Copyright 2006 Future Medicine Ltd. (color figure online) the SiO 2 layer at a p-SiNW surface is modified with 3-aminopropyltriethoxysilane (APTES), which yields amino group (-NH 2 ) termination on the NW surface ( Fig. 10.2a). The amino groups and silanol groups (Si-OH) on the unreacted regions of the oxide layer undergo protonation and deprotonation as the hydrogen-ion concentration varies, thereby changing the surface charge and the NW conductance. The NW electrical conductance shows a stepwise, discrete and stable increase, in response to increasing pH from 2 to 9 ( Fig. 10.2b). More recently, Noy and coworkers demonstrated SiNW-FETs modified with lipid bilayers with and without ligand-gated and voltage-gated ion channels to monitor the solution pH. For lipid bilayer containing ion channels, devices responded to changes in solution pH, and when the channels were blocked the device response was strongly diminished [15]. Sensing studies of several distinct classes of biological targets are discussed below.

Protein Detection
The sensitive detection of proteins, especially those known as disease markers, offers substantial potential to benefit disease diagnosis and treatment. In 2001, pioneering work demonstrated real-time protein sensing with SiNW-FET device [8]. Specifically, SiNWs functionalized with biotin receptors were used to selectively detect streptavidin at concentrations down to 10 pM, substantially lower than other methods at the time. However, the strong binding affinity between biotin and streptavidin leads to effectively irreversible binding and precluded monitoring unbinding and sequential measurements at different streptavidin concentrations. To overcome this limitation, several reversible surface modifications have been explored, including biotin-monoclonal antibiotin binding and calmodulin (CaM)-Ca 2+ interaction, to investigate quantitative concentration-dependent analyses [8]. In a more recent study [16], CaM-modified SiNWs are used to detect Ca 2+ and CaMbinding proteins through the association/dissociation interaction between glutathione and glutathione S-transferase. In addition, this basic approach has been used to demonstrate successful concentration-dependent detection of cardiac troponin T [17] (a biomarker for myocardial infarction), SARS virus nucleocapsid proteins [18], and bovine serum albumin [19] in recent literature and thus further validate the efficacy of NW-FETs as protein sensors.
In genomics and proteomics research, simultaneous detection of multiple proteins is believed to be especially important for diagnosing complex diseases such as cancers [20,21]. Moreover, the availability of different biomarkers matched with different stages of diseases could allow for early detection and robust diagnosis. Early work on SiNW-FET devices [8], although powerful in detecting binding/unbinding of proteins, lacked the capability of selective multiplexed sensing. To address this issue, Zheng et al. [22] developed integrated NW sensor arrays, in which *100 individually addressable NW-FETs were functionalized with several different receptors ( Fig. 10.3a), and demonstrated several new sensing capabilities. Specifically, monoclonal antibodies for the cancer marker proteins prostate specific antigen (PSA) carcinoembryonic antigen (CEA) and mucin-1 were used to functionalize SiNW-FETs in the same device array ( Fig. 10.3b). Upon addition of buffer solutions containing different concentrations of these cancer biomarkers, Fig. 10.3 a Optical image of a NW array. b Sequential detection of PSA, CEA and mucin-1 solutions using three SiNW-FET sensors. c Complementary sensing of PSA using a p-type (NW1) and a n-type (NW2) SiNW-FET sensors in a same array. Reproduced from [22]. Copyright 2005 Nature Publishing Group changes in electrical conductance of the corresponding NW-FETs were recorded with femtomolar sensitivity, which is several orders of magnitude better than possible with the standard enzyme-linked immunosorbent assay (ELISA) [22]. This work also introduced the new concept of incorporating both p-type and n-type NWs into the same device array ( Fig. 10.3c). In so doing, the binding of a negatively charged biomarker such as PSA on the NW sensor surfaces led to an increase in conductance for p-SiNWs and a decrease for the n-SiNWs in the same sensor chip. These complementary, opposite electric signals can be used to distinguish false positive signals and enable real-time, highly sensitive and selective detection of multiplexed biomolecule targets. Similarly, Li et al. [9] reported the complementary sensing of PSA using n-type In 2 O 3 NWs and p-type carbon nanotubes. The enhanced electrical conductance for the NW sensors and the suppressed electrical signal for the carbon nanotube sensors upon the PSA addition are demonstrated with concentrations down to 5 ng/mL sensitivity at physiological buffer concentrations.
Later, an anisotropic wet-etch fabrication method was reported as an alternative 'top-down' NW device fabrication strategy for NW-FET sensors [23]. The sensitivity of these top-down fabricated SiNW devices were shown to have sub-100 fM sensitivity for biotin-streptavidin interaction, mouse immunoglobulin G (IgG), and mouse immunoglobulin A (IgA) detection.

Nucleic Acid Detection
In addition to detection of protein binding/unbinding, real-time detection of nucleic acids (e.g., DNAs and RNAs) has been successfully carried out using Si and GaN NW-FET devices [24][25][26][27]. The surface functionalization methods and detection schemes used in these studies were similar to those described above for protein sensing, where nucleic acid concentration is transduced following binding to a probe by changes in device conductance. A major difference between nucleic acid and protein detections exists in the fact that the high density of negative charges on the nucleic acid phosphate backbones requires high ionic strength buffers to screen the repulsion and allow for binding when DNA or RNA is used as the probe molecule. However, high ionic strength solutions have short Debye screening lengths (see Sect. 10.4.3), which can make difficult or preclude detection. A solution that overcomes this high ionic strength binding/screening issue involves using neutral charge peptide nucleic acids (PNAs) [28,29], which exhibit excellent binding affinity with DNA at lower ionic strengths. Indeed, modification of SiNWs with PNA probe molecules was shown to exhibit time-dependent conductance changes associated with selective binding of complimentary target DNA at concentrations as low as 10 fM. Moreover, this work showed that a DNA SiNW-FET biosensor could be used to distinguish fully complementary (wild type) versus single-base mismatched (mutant) DNA targets associated with Cystic fibrosis [25]. Additional studies using SiNWs functionalized with PNA probes in which the DNA target binding domain distance was changed exhibited a reduction in sensitivity with increasing distance between the hybridization site and the NW surface [30]. This observation is consistent with basic sensing mechanism since the 'field effect' is reduced for fixed charge as the separation from the SiNW surface increases.
An alternative approach for surface functionalization of SiNW surfaces for DNA detection involves electrostatic adsorption of the probes. For example, Bunimovich et al. [31] reported electrostatic adsorption of primary DNA probe strands onto an amine-terminated SiNW surfaces, where the *parallel orientation of the DNA probes along the NW surface reduces Debye screening effects and can thereby yield sensitive DNA detection.
More recently, detection of other nucleic acid targets, such as microRNAs (miRNAs) have been carried out using PNA-modified SiNWs. Focus on microRNAs (miRNAs), which are a large class of short, noncoding RNA molecules that regulate animal and plant genomes, is intriguing because they have been proposed as biomarkers for cancer diagnosis [32]. PNA-functionalized SiNW devices have shown the capability to detect miRNAs down to a remarkable sensitivity of 1 fM [33], ca. one order of magnitude better than reported earlier for DNA detection [30]. This phenomenon can be attributed to the higher thermal stability and melting temperature of PNA-RNA complex than that of PNA-DNA complex. The technique enabled identification of fully complementary versus one-base mismatched miRNA sequences, as well as detection of miRNA in total RNA extracted from HeLa cells, and thus offers substantial potential as a new diagnostic tool.

Virus Detection
Viruses are a major cause of infectious diseases and remain the world's leading cause of death [34]. Successful treatment of viral diseases often depends upon rapid and accurate identification of viruses at ultralow concentrations. The first demonstration of nanoFET based virus sensors involved the detection of influenza A virus using SiNW devices. By recording the electrical conductance changes upon binding/unbinding of virus particles to monoclonal antibody-modified SiNWs, the selective detection of influenza A at the single particle level was demonstrated [35]. The binding kinetics between different virus-receptor interactions were also electrically differentiated by SiNW-FETs ( Fig. 10.4). In addition, simultaneous detection of influenza A and adenovirus using independent SiNW biosensors functionalized with distinct antibodies for these two types of viruses was demonstrated [35], and more recently, SiNW-FET based selective detection of influenza A viruses down to 29 viral particles per micro-liter was achieved for breath condensate samples [36]. These achievements represent important proof-of-concept steps towards powerful viral diagnostic devices.
Another example of virus detection was the diagnosis of Dengue, a arthropod-borne viral infection [37]. In this latter work, a specific nucleic acid fragment with 69 base pairs derived from Dengus serotype 2 virus genome sequence was selected as the target DNA and amplified by the reverse transcription polymerase chain reaction (RT-PCR). The hybridization of the target DNA and PNA-functionalized SiNW-FET sensors increases the device resistance, leading to a sensitivity limit down to 10 fM.

Small Molecule Detection
Detection of small molecules that bind specifically to proteins is of vital importance to drug discovery and screening. One example of small molecule detection involves the identification of adenosine triphosphate (ATP) binding, and the small-molecule inhibition of ATP binding to the tyrosine kinase, Abl, which are proteins that mediate signal transduction in mammalian cells. Gleevec, which competitively inhibits ATP binding to Abl, has been used to monitor binding/unbinding behaviors of ATP. The gleevec concentration at fixed ATP concentration yields conductance decrease, which is consistent with reversible competitive inhibition of an agonist (ATP) with an antagonist (Gleevec) [38]. In a different direction, highly ordered flexible SiNW films, have been applied to detect NO 2 with parts-per-billion (ppb) sensitivity [39]. Other small molecules, such as ammonia (NH 3 ), acetic acid (AcOH) [40] and 2,4,6-trinitrotoluene (TNT) [41], have also been successfully detected by surface-functionalized SiNW-FET sensors. Despite numerous approaches developed for achieving highly sensitive detection of polar molecules, the detection of nonpolar volatile organic compounds (VOCs) still remains challenging, due to the weak adsorption of nonpolar VOCs on the surface of NWs and the lack of suitable nonpolar organic functionalities that can be attached to the SiNWs. To address this issue, silane monolayers with a low fraction of Si-O-Si bonds between the adjacent molecules were used to modify SiNW-FETs to enhance their sensitivity towards nonpolar VOCs [42]. In another work [43], it was demonstrated that multiple independent parameters of a specific molecularly modified SiNW-FET can provide high selectivity towards specific VOCs in both single-component and multi-component environments as well as estimating the constituent VOC concentrations.

3D Branched Nanowires for Enhanced Analyte Capture Efficiency
3D branched NWs [44][45][46][47][48][49] in which secondary NW branches are grown in a radial direction from a primary NW backbone, provide a number of unique capabilities including a substantially-enhanced surface area compared to the backbone alone. By functionally encoding at well-defined branch junctions during synthesis, rationally designed and synthesized branched NWs can provide well-controlled variations in the composition of the NW backbone and branches, and thus allow for complex electronic and photonic nanodevices. Focusing on sensing, Jiang et al. [47] developed the general synthesis of branched, single-crystalline semiconductor NW heterostructures, including Si backbones with Au branches. The Au-branched NW devices were investigated as nanoelectronic sensors for biomolecular detection ( Fig. 10.5). The Au branches, which can be modified in a highly-specific manner using thiol chemistry, can be considered as receptor-functionalized "antennae" for biomolecular analytes. The high surface area of the Au branches provides the potential for enhanced capture efficiency, and thereby can increase the overall device sensitivity. For example, a sensitivity of 80 pg/mL for PSA detection was obtained from mAb-modified p-Si/Au-branch NW-FET sensors, with high selectivity.

Detection in the Subthreshold Regime
The fundamental characteristics of NW-FET devices, such as the transconductance and noise, can have substantial effect on the ultimate detection sensitivity. Conventionally, nanoFET-based sensors are operated in the 'ON' state (above the threshold voltage), where the transconductance depends linearly on gate-voltage or surface potential. However, in the subthreshold regime it is well-known that the device conductance depends exponentially on gate-voltage [50], which could in principle lead to much higher analyte binding sensitivity. Indeed, Gao et al. [51] studied and compared the detection sensitivity of SiNW-FET sensors in the linear and subthreshold regimes (Fig. 10.6). In previous literature using SiNW-FET sensors [8], the conductance change (DG) or the resistance change (DR) of the sensor devices was used to quantify the concentration of the target molecules. However, an  absolute signal change, such as DG, does not reflect the intrinsic device sensitivity, especially when working in the subthreshold regime where device conductance is very small. To better compare sensing in different device regimes Gao et al. used a dimensionless parameter, DG/G, to characterize and compare device sensitivities. This principle is exemplified in both pH and protein sensing experiments, where the electrolyte gating is used to tune the operational mode of NW-FETs (Fig. 10.6b). These results showed that significant sensitivity enhancement could be achieved by optimization of the FET operating conditions and understanding the fundamental electrical gating property of NW-FETs. One caveat to the success of this work is that the device noise should be dominated by carrier-carrier scattering, such that the noise is also exponentially reduced in the subthreshold regime. If the noise is dominated by other scattering mechanisms, such as contact current injection and/or interface trapping/detrapping, then it may not be possible to exploit the exponential dependence of conductance on gate-voltage/surface potential in the subthreshold regime.

Reducing the Debye Screening Effect
Conventional FET sensors detect the concentration of the target species by their intrinsic charge. The charges of solution-based molecules, however, can be screened by dissolved counter ions in the solution. The Debye length, which is inversely proportional to the square root of the ionic strength of an electrolyte, represents the net or screened electrostatic effect of a charged species in ionic solution. A high ionic strength electrolyte solution leads to a short Debye length, and charges outside of the Debye length are electrically screened. For instance, the Debye length of 1 Â PBS, *0.7 nm, can screen most protein antigen charges when they bind to an antibody modified FET surface. In order to reduce the charge screening effect of electrolyte solutions, the Debye length is typically increased by using dilute buffer solutions with low ion concentrations [18,52].
Recently, several groups have reported approaches based on smaller receptors, such as aptamers [53] or antibody fragments [54] to reduce the distance between the FET surface and the receptor-bound biomolecule analyte. In one example [54], the sizes of antibody probes were reduced through common biochemical methods (Fig. 10.7), thereby improving the analyte detection capability. These studies are promising, although further studies are needed to determine how general detection is under the limit of physiological conditions (Debye length <1 nm) as the sizes of the aptamer and antibody fragment receptors are similar to or greater than this critical length scale. Zhong and co-workers [55] also reported a direct high-frequency measurement strategy for standard biological receptors, although those measurement requires significantly more complex device geometry, making difficult or precluding application to cellular and in vivo sensing.
Recently, Lieber and coworkers [56] developed a new and general strategy to overcome this challenge for NW-FET sensors that involves incorporating a biomolecule permeable polymer layer, such as polyethylene glycol (PEG), linked to the FET sensor, where the polymer increases the effective screening length near the NW-FET surface to allow for detection in high ionic strength solutions (Fig. 10.8a).
Using PSA as a model system, they showed that PEG-coated SiNW-FETs can detect PSA in phosphate buffer concentrations up to 150 mM, with a detection  Reproduced from [56]. Copyright 2015 American Chemical Society sensitivity of *10 nM and linear response range up to 1000 nM. In contrast, similar FETs without PEG functionalization can only detect PSA in buffer salt concentrations lower than 10 mM (Fig. 10.8b). This work suggests a new and general device design strategy for the FET sensor applications in physiological environments, important for in vitro and in vivo biological sensing.

Electrokinetic Enhancement
Concentration of analyte near a device surface by electrokinetic effects offers another approach for high-sensitivity protein detection [57]. In a nonuniform alternating current (AC) electric field, the dielectrophoresis (DEP) force can induce polarized particles to move in a directed manner leading to the formation of concentration enhancement and depletion regions in a microfluidic flow channel. Compared to the detection limit without AC excitation, NW sensors modified with monoclonal antibodies for PSA in an appropriate AC field exhibit close to a *10 4 fold increase in sensitivity; that is, the protein concentration at the sensor surface is increased by DEP. In addition, NW devices functionalized with other receptors for capturing cholera toxin subunit B were also demonstrated, suggesting the general applicability of this method for enhanced sensitivity detection [22,57,58]. It is important to recognize, however, the DEP enhancement, including frequency response, depends sensitively on solution ionic strength [58,59].

Frequency Domain Measurement
In addition to the conventional real time measurement device conductance to monitor sensing, frequency-dependent fluctuations in the NW-FET electric signal at equilibrium can convey additional information about the dynamics of the biomolecule-NW hybrid system through a coupling to carrier transport in the device to binding events. For example, binding and unbinding can affect the intrinsic device noise, and thus can be characterized through measurements of the frequency-dependent noise spectra (Fig. 10.9a) [60]. In a recent study, the noise spectra was used to analyze contributions from different noise sources [60]. The frequency domain spectrum of a fluctuating two-level system has the form of a Lorentzian function similar to that of a RC circuit (Fig. 10.9b and c). The 1/f noise is well-known in conventional metal-oxide semiconductor FETs (MOSFETs), and arises from electron capture/emission from trap states [61,62]. If biomolecule binding/unbinding contributes substantially to the noise, it can leads to a Lorentzian peak in addition to the 1/f background. Specifically, a p-type SiNW-FET was first modified with PSA monoclonal antibodies, then solutions with different PSA concentrations and pure buffer were sequentially delivered to the NW sensor via a microfluidic channel. In conventional time-domain measurements (Fig. 10.9d), a reliable PSA detection limit for this device was ca. 5 pM. However, the frequency domain noise spectra (Fig. 10.9e-g) from the same NW-FET device showed that the Lorentzian curve shape was still clearly observed at a PSA concentration as low as 0.15 pM, ca. 30 times better than that the same device measured in the time domain. The improved detection sensitivity was attributed to the separation of the Lorentzian characteristic frequency from the most dominant background of 1/f noise, which becomes less important at high frequencies.

Nanowire-Nanopore Sensors
The integrated NW-nanopore FET sensor has the potential for single-molecule DNA sequencing at low cost and with high throughput [63]. The conventional nanopore DNA sequencing technique records ionic current from nanopores [64], while NW-nanopore sensors allow for direct sequencing of DNA molecules with fast translocation rates given the much higher bandwidth of NW-FETs.
Studies have shown that nanopores can be introduced adjacent to SiNW-FETs using the focused electron beam in a transmission electron microscopy (TEM) [63] ( Fig. 10.10a). A sensor device can then be configured by attaching PDMS solution reservoir chambers above and below the silicon nitride membrane on which the SiNW-FET nanopore devices are fabricated. When the two chambers are filled with solutions of different ionic strength, FET signals corresponding to DNA translocation events can be reproducibly recorded (Fig. 10.10b and c). Notably, a 10-60 times higher signal was observed from the SiNW-FET than that of the corresponding ionic current change in these studies. This work demonstrates a new nanopore sequencing device concept with fast sequencing and large-scale integration properties.

Double-Gate Nanowire Sensors
In order to achieve higher sensitivity NW-FET sensors, extensive effort has been focused on advanced device designs prepared by top-down lithography [65,66]. For example, several groups have fabricated and explored double-gate NW-FET biosensor, with two separated gates, G1 (primary) and G2 (secondary), straddling both sidewalls of the SiNW, to enhance device sensitivity [65,66]. This work has shown that by applying the same voltage to G1 and G2, the threshold voltage (V T ) in the double gate mode is very sensitive to a small change of V G2 (the G2 voltage). Therefore, compared to a single-gate FET sensor, the sensing window of the double-gated FET is significantly broadened, especially in the subthreshold regime described earlier.

Detection of Biomolecules in Physiological Fluids
Rapid and accurate molecular analysis in physiological fluids (i.e., blood or serum) is essential for disease diagnosis and management. NW-FET sensors have routinely demonstrated ultrasensitive, real-time, multiplexed detection biomolecular species, but also have limitations with respect to sensing in complex, physiological solutions as describe in Sect. 10.4.3. To reiterate, the primary limitation for FETs is related to Debye screening effect [67] in high ionic strength blood/serum samples.
To overcome the limitation of Debye length, researchers have developed several methods to detect analytes in blood/serum samples, including simply reducing the solution ionic strength. For example, the ion concentration can be reduced by diluting a blood sample with buffer solution [68]. Dilution will reduce analyte concentration and can affect ligand-and protein-protein interactions, and thereby reduce device sensitivity. A second approach involves desalting the serum samples before detection of biomarkers [22], which can maintain or even be used to increase analyte concentrations (after dissolution in buffer). Similar to off-chip desalting using rapid size-exclusion chromatography [22], a microfluidic purification chip (MPC) can be used to pre-isolate the target molecules and then release them into a pure buffer suitable for analysis using SiNW-FET arrays [69]. A fourth method adopts a steady-state measurement instead of a real-time recording [70]. Specifically, the resistance of the SiNW is measured in a low ionic strength buffer solution after antibody functionalization. Then, the SiNW sensor is incubated with undiluted serum and subsequently washed to remove unbound proteins, followed by the measurement of the second resistance value in the buffer solution. The concentration of the target molecules can be calculated according to the resistance change before and after antibody-antigen interaction. This method is independent of the ionic strength of the sample solution, thus circumventing the Debye screening in physiological fluids; however, it is subject to variations in device properties between steps since slow changes in background conductance are not followed. Other reported methods include using smaller receptors, such as aptamers [53] or antibody fragments [54], and adding biomolecule permeable polymer layers to the FET sensor [56], as discussed in Sect. 10.4.3.
The long-term stability of the NW nanoelectronic devices in physiological studies has also been investigated [71]. Coated with a thin layer of Al 2 O 3 , SiNW-FETs yield long-term stability (>4 months) in physiological model solutions at 37°C. Notably, coating with Al 2 O 3 /HfO 2 layers has suggested that an even longer of stability of >1 year is possible for SiNW-FETs in physiological model solutions. These latter results suggest the potential of the SiNW-FETs for long-term chronic in vivo studies in animals and biomedical implants.

Future Directions and Challenges
Over the last decade, remarkable research progresses have been achieved on the design and implementation of semiconductor NW sensors. In this chapter, we have illustrated how the NW-based FET sensors modified with specific surface receptors represent a powerful chemical/biomolecule detection platform. The examples described here summarize several unique capabilities for direct, label-free, real-time, ultrasensitive and highly selective multiplexed detection of proteins, nucleic acids, viruses, and small molecules, and show clearly the potential of these materials and devices to significantly impact disease diagnosis, genetic screening, and drug discovery, as well as offering powerful new tools for research in many areas of disease diagnosis and life sciences.
Nonetheless, there are several areas of scientific study, which if addressed, could further push the limits of this technology for applications. First, one fundamental challenge to the ultrasensitive detection is to obtain well-defined receptor structures on nanodevice surfaces. In part, this reflects difficulties in characterizing receptordevice structure at the single nanodevice level and correlating such results with sensing results. One approach that could address this structural issue at the single device level would be by exploiting the substantial advances in cyro-EM [72,73], which could yield high-resolution structural information of the organic/biologic/ nanodevice interface. A second direction that could improve this critical device-receptor interface would be through exploration of highly-selective, self-limiting covalent chemistry that precisely defines distance and orientation of the receptors. Second, the real-time and multiplexed detection capabilities of nanoelectronic FET sensors for direct analyses of whole blood/serum detection could yield important advances in clinical monitoring and diagnostics. As discussed in Sects. 10.4.3 and 10.4.8, the most critical issue has been overcoming Debye screening in physiological solutions. The new strategy of modifying FET nanodevices with a permeable polymer layer to increase the effective screening length [56] is one promising strategy for achieving real-time detection, although further fundamental studies will be necessary to develop this and/or other approaches to the level of a technology. Third, almost all the nanoFET-based sensors are exclusively surface-bound devices. For many applications, one of the most impactful directions could be the transformation from on-chip signaling to the in vivo monitoring as an implant. Recent advances in the development of NW-FET arrays embedded in engineered tissue patches [74], which could be implanted, and incorporation of sensors in injectable electronics [75], which is directly implanted in specific tissue, could enable the goal of direct in vivo monitoring.
In the next decade, continued efforts to achieve the capability in controlling the mechanisms of the NW sensor arrays will move beyond current technologies and take advantage of information emerging from genomics and proteomics to improve the diagnosis and treatment of cancer and other complex diseases. We believe that these advances can be developed in simple NW sensor devices that would represent a clear application of nanotechnology and, more importantly, a substantial benefit to the society.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy:
Biology
Agricultural And Food Sciences

text:
Cellulolytic enzyme expression and simultaneous conversion of lignocellulosic sugars into ethanol and xylitol by a new Candida tropicalis strain

Background Lignocellulosic ethanol production involves major steps such as thermochemical pretreatment of biomass, enzymatic hydrolysis of pre-treated biomass and the fermentation of released sugars into ethanol. At least two different organisms are conventionally utilized for producing cellulolytic enzymes and for ethanol production through fermentation, whereas in the present study a single yeast isolate with the capacity to simultaneously produce cellulases and xylanases and ferment the released sugars into ethanol and xylitol has been described. Results A yeast strain isolated from soil samples and identified as Candida tropicalis MTCC 25057 expressed cellulases and xylanases over a wide range of temperatures (32 and 42 °C) and in the presence of different cellulosic substrates [carboxymethylcellulose and wheat straw (WS)]. The studies indicated that the cultivation of yeast at 42 °C in pre-treated hydrolysate containing 0.5 % WS resulted in proportional expression of cellulases (exoglucanases and endoglucanases) at concentrations of 114.1 and 97.8 U g−1 ds, respectively. A high xylanase activity (689.3 U g−1 ds) was also exhibited by the yeast under similar growth conditions. Maximum expression of cellulolytic enzymes by the yeast occurred within 24 h of incubation. Of the sugars released from biomass after pretreatment, 49 g L−1 xylose was aerobically converted into 15.8 g L−1 of xylitol. In addition, 25.4 g L−1 glucose released after the enzymatic hydrolysis of biomass was fermented by the same yeast to obtain an ethanol titer of 7.3 g L−1. Conclusions During the present study, a new strain of C. tropicalis was isolated and found to have potential for consolidated bioprocessing (CBP) applications. The strain could grow in a wide range of process conditions (temperature, pH) and in the presence of lignocellulosic inhibitors such as furfural, HMF and acetic acid. The new yeast produced cellulolytic enzymes over a wide temperature range and in the presence of various cellulosic substrates. The cellulolytic enzymes produced by the yeast were effectively used for the hydrolysis of pretreated biomass. The released sugars, xylose and glucose were, respectively, converted into xylitol and ethanol. The potential shown by the new inhibitor tolerant cellulolytic C. tropicalis to produce ethanol or xylitol is of great industrial significance.

using a wide variety of biomass and biocatalysts [1]. The information generated through earlier studies has aided in setting up pilot scale facilities for lignocellulosic biofuel production [2].
Notwithstanding the advancements, lignocellulosic biofuel production technologies have not been widely commercialized. The cost of producing ethanol from lignocellulosic biomass is still considered to be higher than from sugar-based feedstock mainly because the cost of processing the biomass is substantial. To minimize the risks, biomass pretreatment processes are usually designed to take in multiple or mixed feedstock which adds up to the cost of its processing [3,4]. The cost of biomass processing can, however, be controlled by selecting a process which is rapid, requires minimum catalyst (chemicals/enzymes) quantities as well as energy inputs, maximizes catalyst reuse, and results in minimal sugar degradation and inhibitor formation to achieve maximum sugar release through biomass depolymerisation [4][5][6][7][8].
The sugars released from biomass at different stages mainly consist of pentose and hexose monomers. Several industrial strains such as Saccharomyces sp. can convert hexose sugars into ethanol; albeit xylose conversion still remains challenging [9]. Although several wild-type microorganisms are known to convert pentoses into ethanol [10][11][12], a robust biocatalyst that can be utilized at the industrial levels is yet to be found. Studies from the recent past indicate the development of engineered 'pentose-to-ethanol' converting biocatalysts which may now become the way forward. As an alternative, the xylose sugars generated during biomass hydrolysis can also be fermented into a product with higher value than ethanol such as xylitol, whose co-production will reduce the cost of cellulosic ethanol production [6,13].
Another projected approach to lower the cost of lignocellulosic ethanol production is via consolidated bioprocessing (CBP) with a single microorganism which can produce cellulolytic enzymes for biomass hydrolysis and can ferment the released sugars into ethanol, all together in a single step. However, CBP for cellulosic ethanol production at even the pilot scale remains largely hypothetical mainly due to inherent problems of suboptimal cellulase/xylanase expression or impaired fermentation yields [14]. Due to these reasons, microorganisms expressing cellulolytic enzymes are still not considered as a suitable candidate for carrying out ethanol fermentations and vice versa.
In the present study, a new isolate identified as Candida tropicalis was found to express cellulases and xylanases and convert xylose into xylitol and glucose into ethanol. Preliminary studies were carried out to determine the effects of substrates on cellulase and xylanase production by the yeast. The cellulolytic enzymes produced by the yeast isolate were used for the hydrolysis of pre-treated biomass and sugar hydrolysate was fermented into ethanol. The yeast also showed the ability to aerobically convert xylose into xylitol in the presence of inhibitory compounds. The new yeast isolate is a potential candidate for producing cellulosic ethanol via CBP.

Results and discussion
A single microorganism that expresses cellulolytic enzymes for hydrolysis of biomass and ferments the released sugars into ethanol and other value added products such as xylitol is of potential interest to the industry. In this study, a new cellulolytic strain was isolated on an agar plate having CMC as the only carbon source. Microscopic examination indicated that the isolate was a yeast and 18S rRNA sequencing subsequently confirmed that it was a C. tropicalis strain. Since there are hardly any reports about cellulolytic Candida sp. the strain identity was reconfirmed by sequencing the variable D1-D2 domain of the 26S rRNA and the internal transcribed spacer (ITS) region of the 5.8S rRNA, as these have been projected as more authentic markers for strain identification than 18S rRNA [15,16]. All the three sequences of MTCC 25057 showed a complete 100 % homology to C. tropicalis.
The cellulolytic enzymes produced by the new yeast isolate were used for the hydrolysis of pre-treated wheat straw and the released glucose was converted into ethanol by the same yeast strain. After acid pretreatment of wheat straw, the xylose generated in the hydrolysate could be converted into xylitol or used for producing cellulolytic enzymes. Barring an earlier report related to endoglucanase activities in C. tropicalis cultures after prolonged incubation periods [17] and despite the knowledge about the existence of genes coding for cellulolytic enzymes [GenBank:3636270; 3637086; 8296755, etc.], no other related reports were found.

Tolerance of Candida tropicalis MTCC 25057 to inhibitory compounds
For utilizing sugars in lignocellulosic hydrolysates, the biocatalyst has to withstand the presence of inhibitory compounds such as furfural, HMF and acetic acid that are usually generated during biomass pretreatment [18]. In the present study, wheat straw procured from local sources was subjected to pretreatment with 0.2 % (v/v) phosphoric acid as optimized previously in the authors' laboratory [19]. The lignocellulosic hydrolysate contained glucose (~0.5 % w/v) and xylose (~1.5 % w/v) as the major components along with negligible quantities of inhibitory compounds such as furfural and HMF (~0.01 % w/v). Several reports indicate the formation of acetic acid during biomass pretreatment, although the same was not detected during our studies. Preliminary studies for determining the tolerance of the new yeast to higher concentrations of furfural, HMF as well as acetic acid, which might get generated during harsher pretreatments indicated that the Candida isolate tolerated furfural, HMF and acetate concentrations of 1.5, 2 and 2 g L −1 , respectively (Fig. 1a). Further increase in the levels of furfural, HMF and acetic acid resulted in a gradual lowering of biomass densities (Fig. 1a). A recent report related to the fermentative capacities of C. tropicalis isolate to produce ethanol and xylitol in hydrolysate, indicated that the yeast could not function beyond furfural, HMF and acetate levels higher than 0.23, 0.15 and 1.37 g L −1 respectively [20]. In comparison, the new yeast isolate under investigation was found to be more tolerant to commonly occurring lignocellulosic inhibitors.
In addition to ethanol toxicity, osmotic stress and pH variations [21,22], commercial scale ethanol bioprocesses also encounter increased temperatures during summer seasons during which the yeast is supposed to perform as normally as under optimal conditions [23]. We observed during our studies that cell growth of the new yeast isolate was not affected over a wide range of temperatures (32-42 °C) and pH (4-10) (Fig. 1b, c). Also, the cellulase and xylanase production by the yeast isolate remained largely unaffected over a wide range of temperatures (32-42 °C).

Cellulase and xylanase production by Candida tropicalis MTCC 25057
Earlier reports indicate that the temperature of incubation and the nature of substrates mainly affect cellulolytic enzyme production [24,25]. Experiments were designed to determine cellulase (endoglucanase and exoglucanase) and xylanase production by the new yeast strain at different temperatures (32 and 42 °C), cellulosic substrates (CMC and WS) and at different concentrations (w/v) of 0.5, 1 and 2 % (Fig. 2) in pretreated lignocellulosic hydrolysate. Maximum endoglucanase activity of 98 ± 2 U g −1 ds was obtained using 0.5 % WS at 42 °C (Fig. 2a). Similarly, the exoglucanase production in hydrolysate containing 0.5 % wheat straw was maximum (114 ± 3 U g −1 ds) at 42 °C (Fig. 2b). At lower incubation temperature of 32 °C, the endo and exoglucanase levels in Fig. 1 Effect of a different inhibitors (furfural, HMF, acetate) concentration, b temperature and c initial pH on growth of Candida tropicalis. The error bar range represents standard deviation from mean, t test significance level p < 0.005 ▸ hydrolysate were lower than their corresponding levels at 42 °C with both the substrates and at all the concentrations tested (Fig. 2a, b).
Earlier studies have indicated that cellulolytic enzyme blends with approximately equal exoglucanase and endoglucanase activities are more suitable for optimal biomass hydrolysis [26]. In this work, we observed that amongst the two temperatures tested, maximum endoglucanase (Fig. 2a) and exoglucanase (Fig. 2b) activities were obtained at 42 °C, while 0.5 % WS was found to be a more suitable cellulosic substrate. The ratios of endo and exoglucanases at 42 °C were proportional and their activities were comparable to the previously reported cellulase expression levels by well-known enzyme producer strains such as Trichoderma and Aspergillus sp. [27][28][29].
Regarding xylanases, the activity obtained at 32 °C was marginally higher (825 ± 14 U g −1 ds) than that obtained at 42 °C (689 ± 17 U g −1 ds) with 0.5 % WS (Fig. 2c) in hydrolysate. The expression of xylanases with wheat straw could be attributed to the presence of xylan [30]; however, it was interesting to find xylanase production even in the presence of CMC, which is apparently xylan free. Earlier reports related to xylanase production indicate that the strains of Aspergillus sp., Penicillium sp., etc. show very high xylanase activities ranging from 5000 to 7000 U g −1 ds [31,32] along with cellulase production. It is expected that genetic improvements in the new yeast isolate may lead to increased expression of cellulolytic enzymes.

Wheat straw hydrolysis
Different acids have been used to carry out biomass pretreatments [33,34] and the use of phosphoric acid is known to generate lesser inhibitory compounds in the hydrolysate [35]. The quantity of inhibitory compounds generated during biomass pretreatment is mainly dependant on pretreatment severity. An earlier study from the author's laboratory optimized wheat straw pretreatment using 0.2 % phosphoric acid at 150 °C for 15 min [19] and the same conditions were used for biomass pretreatment in the present study. The cellulose, hemicellulose and lignin content of biomass before and after phosphoric acid pretreatment was determined (Table 1). Phosphoric acid pretreatment of wheat straw caused approx. 65 % lignin removal and 64 % hemicellulose hydrolysis. After separating the hydrolysate and neutralizing the biomass, the cellulosic biomass was subjected to hydrolysis with the enzymes produced by the new yeast at an approximate enzyme loading of 20 U g −1 of wheat straw for 48 h at 50 °C. The sugar hydrolysate generated contained about 500 mg glucose and 30 mg xylose per gram of dry wheat straw used.
It is widely acknowledged that the cost of producing cellulosic ethanol can be lowered by optimizing conversion of the released pentoses and hexoses into ethanol or by converting pentoses into an additional high value compound such as xylitol [36]. On the basis of earlier reports related to ethanol and xylitol production by Candida species [20,36], investigations were carried out to determine ethanol and xylitol fermentation capacities of the new yeast isolate.

Pentose and hexose fermentation by Candida tropicalis MTCC 25057 in synthetic media
Biomass consists of approx. 20-40 % of hemicellulose [37], which upon hydrolysis, releases xylose as the main component. The naturally occurring industrial ethanol producing yeast, Saccharomyces cerevisae, cannot utilize xylose and hence a hexose and pentose fermenting microorganism is needed. Candida sp. is a distinctive class of yeast that can ferment glucose into ethanol and xylose into xylitol and ethanol [20,[36][37][38]. Over the years, studies have shown that different strains of Candida sp. produce optimum levels of xylitol (from xylose) and ethanol (from glucose and xylose) under aerobic and anaerobic conditions, respectively [20].
During the current investigations, it was observed that Candida cell growth and ethanol production patterns were similar under both, aerobic and anaerobic conditions ( Fig. 3a, b). Ethanol production using Candida strains is essentially an anaerobic process. However, the new Candida isolate could produce comparable concentrations of ethanol under aerobic conditions although the productivities were decreased.
On culturing the yeast strain in synthetic YEPD media under anaerobic conditions, maximum ethanol levels of ~36 g L −1 were obtained within 24 h, whereas similar ethanol concentrations were attained under aerobic conditions after 48 h of incubation (Fig. 3a, b). Under anaerobic and aerobic conditions, the corresponding glucose consumption of 100 and 80 % was observed within 24 h. Under aerobic conditions, the complete consumption of the remaining 20 % glucose required extended incubation for up to 48 h (Fig. 3a). Therefore, the ethanol yields (0.36 g g −1 , i.e., ~72 % conversion) were similar while the ethanol productivities under anaerobic conditions (1.5 g L −1 h −1 ) were higher than under aerobic conditions (0.75 g L −1 h −1 ). Under both the conditions, approx. 3-4 g L −1 glycerol was detected in the medium (data not shown).
The Candida tropicalis isolate could also utilize xylose under aerobic conditions and produce xylitol (68 ± 2 g L −1 ) within 96 h of fermentation (Fig. 3c) when cultured in YEPD media. The xylitol yields and productivities obtained during aerobic fermentations were 0.67 g g −1 (67 % conversion) and 0.70 g L −1 h −1 , respectively. No consumption of xylose was observed under anaerobic conditions even after extended incubation up to 120 h (data not shown). It has been previously reported that nicotinamide adenine dinucleotide phosphate (NADPH) is required for the functioning of xylose reductase, a key enzyme involved in xylitol production [39], which is regenerated only under aerobic Since lignocellulosic hydrolysate contains a mixture of pentose and hexose sugars, it was necessary to determine the ability of the isolate to ferment both simultaneously. Therefore, in order to simulate fermentation of lignocellulosic hydrolysate containing mixed sugars (hexose and pentose), the strain of C. tropicalis was cultured in synthetic lab media in which 50 g L −1 each of xylose and glucose had been added. The anaerobic cultivation of C. tropicalis in glucose-xylose medium led to complete utilization of glucose within 24 h whereas xylose remained unutilized in the medium (Fig. 4a). The maximum ethanol concentrations obtained under anaerobic (18.8 ± 0.8 g L −1 ) and aerobic (20.4 ± 1.3 g L −1 ) conditions as well as the ethanol yields of 0.37 and 0.4 g g −1 were found to be similar. However, no xylitol was detected during anaerobic fermentations of Candida tropicalis even till the end of the fermentation (Fig. 4a). Under aerobic cultivation, xylose utilization started 48 h after complete utilization of glucose (Fig. 4b) and a xylitol yield and productivity of 0.93 g g −1 and 0.28 g L −1 h −1 , respectively, were obtained. The delayed onset of xylose utilization could be attributed either to the higher affinity of the xylose transporter for glucose or the repression of the first two genes of the xylose utilization pathway, viz. xylose reductase and xylitol dehydrogenase, as was previously reported for Candida shehatae and Pichia stipitis [41,42]. The xylitol yield obtained in case of mixed sugar fermentation in this study was higher than the previously reported values in the range of 0.49-0.84 g g −1 produced by other Candida strains [43,44]. Another important observation made during the studies was that the yield of xylitol obtained when xylose was the sole carbon source was lower (0.67 g g −1 ) than the yield (0.93 g g −1 ) obtained on using xylose and glucose in the media. The increased xylitol yield in the presence of mixed sugars might have occurred due to the utilization of glucose for biomass formation resulting in xylose being consumed only for xylitol formation and not biomass, or the sufficient availability of NADPH (essential for the optimal activity of the xylose reductase enzyme) which is regenerated best in the presence of glucose [40].

Pentose and Hexose fermentation by Candida tropicalis MTCC 25057 in hydrolysate
Although wheat straw pretreatment with 0.2 % phosphoric acid has been optimized in our laboratory due to minimal inhibitor generation, here we have used harsher pretreatment conditions (4 % phosphoric acid) so as to achieve maximal xylose generation in the hydrolysate as this yeast isolate has the capability to withstand high inhibitor concentrations. Interestingly, even after the use of such highly acidic conditions for pretreatment, the amount of inhibitors produced was minimal-1 g L −1 furfural and 0.075 g L −1 HMF, reaffirming the use of phosphoric acid as an effective yet gentle pretreatment agent. The pretreated hydrolysate generated was fermented initially using the new yeast isolate after supplementation with minimal salts only. However, no ethanol or xylitol production was observed (data not shown). Subsequently, to meet the nutritional deficiencies, an additional nitrogen source (1 % yeast extract) was added to the hydrolysate which resulted in increased xylose utilization. Almost 60 % of the xylose was consumed within the first twelve hours of fermentation itself and no residual xylose was observed beyond 72 h of fermentation (Fig. 5a). Xylitol production reached maximum levels of ~15 ± 1 g L −1 in 72 h with an average yield of ~32 %. The hydrolysate also contained some amount of glucose (10 g L −1 ) which was converted into ethanol (~4.5 g L −1 ) in the initial 24 h.
In a similar study reported earlier, fermentation of concentrated corn cob hydrolysate (without nutritional supplementation) using a hydrolysate adapted Candida strain resulted in a xylitol yield of approx. 14.81 % with most of the xylose being diverted for biomass formation which affected the final yields [45]. In our study, a xylitol yield of ~32 % was achieved using a wild-type Candida isolate; although here too, most of the sugar was being used for cell growth instead of xylitol production. Further improvements in xylitol titers and yields may be achieved by strain adaptation, optimization of the cultivation conditions, enriching the lignocellulosic hydrolysate for better cell growth, etc.
The sugar hydrolysate obtained post-enzymatic saccharification of the pretreated wheat straw was also fermented using the same yeast strain. This hydrolysate has more hexoses than pentoses as it is derived from the cellulose rich biomass. In this case also, supplementation of the hydrolysate with additional N source led to greater diversion of the sugars to cell growth than to product formation. The yeast was able to convert ~25 g L −1 glucose to 7.3 ± 1.1 g L −1 ethanol with a net yield of 0.28 g g −1 (Fig. 5b), which was slightly lower than the ethanol yield observed in case of mixed sugar fermentation in synthetic medium. The sugar hydrolysate also contained ~7.5 g L −1 of xylose that was converted to xylitol in 24 h with a yield of 0.31 g g −1 . The ethanol yield and productivity could be enhanced further by fine tuning the addition of nutritional supplements so as to ensure maximum sugar fermentation into ethanol production. A recently published review by Jouzani and Taherzadeh [46] comprehensively covers almost all the wild-type single microbial strains, microbial co-cultures as well as genetically engineered microorganisms with cellulolytic activities and ethanol producing abilities that showed potential for converting biomass into bioethanol via CBP. In comparison, the strain isolated in the present study also showed cellulolytic as well as ethanol fermenting abilities. In addition, the yeast strain also exhibited significant inhibitor tolerance and produced xylitol from hemicellulosic sugars. To establish the strain's potential for CBP applications, detailed investigations into the metabolic pathways are needed.
Generally, ethanol producing yeast such as Saccharomyces, Kluyveromyces naturally lack cellulase expression and hence cellulolytic fungi such as Trichoderma and Aspergillus are used for producing cellulolytic enzymes. However, yeast cells are widely preferred as the hosts for recombinant enzyme production due to their robust nature, rapid growth rates and higher protein secretion [47][48][49]. In view of the advantages offered by yeast cells, research efforts have focussed on imparting cellulolytic abilities for their development as CBP-suitable hosts [50][51][52]. To develop cellulolytic yeast, multiple genes coding for various cellulases have to be cloned and their gene products need to be expressed in a stable manner, at a certain proportion and in higher quantities for achieving hydrolytic action on cellulose in a synergistic manner. The insertion of δ-retrotransposons regulates simultaneous expression of multiple genes [53,54], whereas the synthetic promoter and terminator sequences improve the stability as well as activity of the mRNA [55,56] while hybrid transcriptional activators enhance the secretion of cellulolytic enzymes [57]. Earlier studies have also focussed on increasing the extracellular enzyme secretion by manipulating the expression of proteins such as SNAREs, karyopherins and superoxide dismutase, which have been implicated in the regulation of intracellular membrane transport [58,59]. However, all these approaches in yeast engineering have met with limited success since the cellulolytic enzyme yields were much lower than those produced by the established strains such as Aspergillus, Trichoderma etc. [60][61][62].
An ideal CBP operation for lignocellulosic bioethanol production would depend upon higher expression of hydrolytic enzymes for optimum biomass hydrolysis and the rapid fermentation of released sugars, all occurring within a relatively narrow range of process parameters. In actuality, since the temperature optima for enzyme production (30-32 °C), biomass hydrolysis (50 °C) and fermentation (30-32 °C) vary, the processes are still carried out in a successive manner. During our studies, the inhibitor tolerant strain of Candida exhibited maximal enzyme production (42 °C and pH 6-7) and biomass hydrolysis (at 50 °C and pH 5) within a relatively narrower range of process parameters, thereby indicating its potential for CBP applications.
Further improvements in the new yeast isolate can include overexpressing native cellulases by inserting multiple gene copies and the development of recombinants by inserting genes from conventional enzyme producers such as Aspergillus or Trichoderma. The thermostability, substrate specificity, etc. of native cellulases produced by C. tropicalis isolate can also be improved via protein engineering. Ongoing investigations in the authors' laboratory also focus upon adaptation of the yeast to nutritionally deficient environments such as hydrolysate as well as imparting the ability to co-utilize hexose and pentose sugars for increased productivity during mixed sugar fermentations.

Conclusions
Till now, several wild-type bacteria and filamentous fungi along with their recombinant strains and a few genetically engineered yeast were considered as potential CBP candidates for lignocellulosic ethanol production. The current work is the first definitive report describing a new strain of Candida tropicalis with ability to produce cellulolytic enzymes, ethanol and xylitol, thereby making it a prospective candidate for consolidated bioprocessing. The new yeast isolate also exhibited high tolerance to inhibitors such as furfural, HMF and acetic acid, found commonly in lignocellulosic hydrolysate. In addition, it produced cellulases and xylanases over a wide range of temperatures and in the presence of diverse cellulosic substrates. However, in order to carry out efficient hydrolysis of pre-treated biomass, the expression and activities of the cellulolytic enzymes will have to be significantly improved by the over-expression of heterologous cellulases and accessory proteins. Also, the ethanol and xylitol titers obtained during fermentations in hydrolysates were relatively lower despite the strain's tolerance to inhibitors and this could be attributed to the existing nutritional deficiencies in hydrolysates. The fermentative performance of the new yeast strain and the co-utilization of mixed sugars can be further improved by strain adaptation to nutritionally deficient conditions and genetic engineering.

Microorganism and culture conditions
Soil samples were collected from Sri Chamarajendra Zoological Gardens, Mysore, India and stored in sterile containers at 4 °C until subsequent use. Approximately, 1 g of soil sample was inoculated into yeast extract peptone dextrose broth (YEPD) and incubated at 32 °C for 48 h. On obtaining growth, the liquid culture was serially diluted in sterile saline and plated onto agar plates with 1 % CMC which were incubated at 32 °C for 48 h. The colonies obtained were sub-cultured till pure isolates were obtained. Upon microscopic observations, one of the isolated colonies was found to be a yeast. The isolate was maintained on YEPD agar slants at 4 °C. Liquid cultures of the isolate were obtained by inoculating a loopful of culture from YEPD slants into YEPD broth and incubating at 32 °C for 24 h at 150 rpm.

Identification of new strain
Strain identification of the new yeast isolate was done by sequencing the 18S, 5.8S and 26S rRNA and the isolate was found to be C. tropicalis. The primers used for strain identification spanned the 18S rRNA, the variable D1-D2 domain of the 26S rRNA and the internal transcribed spacer region of the 5.8S rRNA. The primer sequences were as follows: 18S_ fwd 5′-TCCTCCGCTTATTGATATGC-3′, 18S_rev 5′-GAAGTAAAAGTCGTAAC AAG G-3′,26S_f wd 5′GCATATCAATAAGCGGAGGAAAAAG-3′, 26S_rev 5′-GGTCCGTGTTTCAAGACG-3′, ITS1 5′TCCGTAGG TGAACCTGCGG-3′ and ITS4 5′-TCCTCCGCTTATTG ATATGC-3′. The PCR conditions used were: initial denaturation at 95 °C for 5 min, followed by 30 cycles of denaturation, annealing and extension at 95, 52 and 72 °C, respectively, for 30 s or 1 min. A final product extension at 72 °C for ten minutes was also done before analysing the PCR products by gel electrophoresis. The PCR products were purified using the GeneJET gel extraction kit (Thermo Fisher Scientific) before sequencing. The strain was submitted to Microbial Type Culture Collection (MTCC), India and assigned the accession number -MTCC 25057.

Biomass pretreatment and enzymatic hydrolysis
Wheat straw procured from local sources around Bangalore was stored and used throughout the study. Wheat straw was subjected to phosphoric acid (0.2 % v/v) pretreatment as described previously [63]. The cellulose, hemicellulose and lignin composition of the wheat straw was determined using standard NREL procedures [64]. The liquid hydrolysate obtained was neutralized to pH 6-7 using ammonia and used for cellulolytic enzyme production. The solid biomass obtained after phosphoric acid pretreatment was washed with distilled water, dried at 60 °C and used for enzymatic hydrolysis. Briefly, 5 g of pretreated biomass, 20 U g −1 ds enzyme and 50 mL sodium citrate buffer (50 mM, pH 4.8) were taken in 250 mL Erlenmeyer flasks and incubated at 50 °C and 150 rpm shaking for 24-48 h. The sugar hydrolysate was separated by centrifugation at 15,000 rpm for 10 min and the reducing sugar content was determined by uHPLC.

Cellulase and xylanase production under submerged fermentation
Submerged fermentation was carried out by inoculating 0.2 mL of the exponential phase MTCC 25057 culture to 250 mL Erlenmeyer flasks containing 20 mL of wheat straw hydrolysate supplemented with minimal salts. The composition of minimal salts (g L −1 ) was-NaNO 3 : 2.5, KH 2 PO 4 : 1, MgSO 4 ·7H 2 O: 0.5, KCl: 0.5. To these flasks, cellulosic substrates (CMC or pretreated WS) at different concentrations (0.5 % w/v, 1 % w/v and 2 % w/v) were added. The cultures were incubated on a rotary shaker (150 rpm) at different temperatures (32 and 42 °C) and for varying periods (24,48,72 and 96 h). The crude supernatant obtained by centrifuging the culture broth at 8000 rpm for 10 min was used for determining enzyme activity.

Enzyme assays
Crude endoglucanase activity was determined by the CMC method [65]. The substrate was prepared by solubilizing carboxymethyl cellulose (Sigma) (2 % w/v) in 0.05 M sodium citrate buffer at pH 4.8. Briefly, 0.5 mL of the crude enzyme supernatant (diluted appropriately) was added to 0.5 mL of the substrate and incubated at 50 °C for 30 min. The reducing sugars liberated were estimated by the DNS method [66]. Crude exoglucanase activity was determined by the filter paper assay method [67]. The substrate used was 50 mg of Whatman No. 1 filter paper in 0.05 M sodium citrate buffer at pH 4.8. The crude enzyme supernatant (0.5 mL) was added to 1 mL of the buffer having 50 mg filter paper and incubated at 50 °C for 60 min and the reducing sugars liberated were estimated by the DNS method. Xylanase activity was determined by the beechwood xylan assay method [65]. Substrate preparation was done by adding beechwood xylan (Sigma) (1 % w/v) to 0.05 M sodium citrate buffer, pH 4.8. The appropriately diluted crude enzyme supernatant (0.1 mL) was added to 0.5 mL of the substrate and 0.4 mL of buffer and incubated at 50 °C for 15 min. The reducing sugars released were measured by the DNS method. All the estimations were carried out in triplicates. The enzyme activity was expressed in terms of International units (IU) for endoglucanase and xylanase and filter paper unit (FPU) for exoglucanase, respectively, or as units per gram of dry substrate (gds), i.e., U g −1 ds. One IU or FPU is defined as the amount of enzyme activity required to release one micromole of reducing sugar from a cellulosic substrate (CMC, xylan, filter paper, etc.) per mL in a minute under the suitable assay conditions.

Fermentation conditions
The C. tropicalis MTCC 25057 strain was grown in 50 mL of yeast extract-peptone broth (20 g L −1 peptone and 10 g L −1 yeast extract) supplemented with either 10 % glucose, 10 % xylose or a mixture of 5 % glucose and 5 % xylose in 250 mL Erlenmeyer flasks in an incubator shaker maintained at 32 °C and 150 rpm. Samples were collected at appropriate intervals and the metabolites produced were analysed using UHPLC. Anaerobic condition was maintained by flushing nitrogen initially through the headspace of the flask and then incubating at 32 °C as mentioned previously. For inhibitor tolerance studies, furfural, 5-hydroxymethylfurfural or acetic acid (Himedia Laboratories) was added to the yeast extract-peptone broth with 1.5 % xylose and 0.5 % glucose (to simulate pretreated hydrolysate), in concentrations ranging from 0.5 to 3 g L −1 and grown at 32 °C for 24 h. The yeast was grown in YEPD, i.e., yeast extract peptone broth with 20 g L −1 glucose medium maintained at different pH (4,5,6,7,8,9,10) and at different temperatures (32,37,42,45,50 °C) for 24 h for determining the pH and temperature tolerance of the strain.
The pretreated and sugar hydrolysates were supplemented either with minimal salts (as mentioned previously) or with 10 g L −1 yeast extract for ethanol/xylitol production studies. The yeast was inoculated from a fresh plate into 50 mL hydrolysate and incubated at 150 rpm at 32 °C in 250 mL Erlenmeyer flasks. The samples were collected at suitable intervals and analysed.

Analytical methods
The soluble sugars or metabolites content in the culture samples were determined using a 1290 Infinity series UHPLC system (Agilent) equipped with a Hiplex H anion exchange column (Agilent). Filtered and degassed 5 mM H 2 SO 4 was used as the mobile phase at a flow rate of 0.6 mL/min. The column was maintained at 45 °C in a thermostat chamber, while the refractive index (RI) detector was maintained at 55 °C. The concentrations of glucose, xylose, ethanol and xylitol were estimated using appropriate calibration curves. The biomass or cell density of the culture samples was estimated by measuring the optical density at 600 nm using a Perkin Elmer Lambda 35 UV/visible spectrophotometer.