Graphene Field-effect Transistor Biosensor Research Articles

Enhancing graphene field-effect transistor biosensors with DNA-mediated protein orientation

Enhancing graphene field-effect transistor biosensors with DNA-mediated protein orientation

Biosensing with Surface-Charge-Modulated Graphene Field-Effect Transistors beyond Nonlinear Electrolytic Screening.

In field-effect transistor (FET) biosensors, charge screening in electrolyte solutions limits the sensitivity, thereby restricting the applicability of FET sensors. This is particularly pronounced in graphene FET (GFET) biosensors, where the bare graphene surface possesses a strongly negative charge, which impedes the high sensitivity of GFETs owing to nonlinear electrolytic screening at the interfaces between graphene and liquid. In this study, we counteracted the negative surface charge of graphene by decorating positively charged compounds and demonstrated the sensing of C-reactive protein (CRP) with surface-charge-modulated GFETs (SCM-GFETs). We integrated multiple SCM-GFETs with anti-CRP antibodies and nonfunctionalized GFETs into a chip and measured differentials to eliminate background changes to improve measurement reliability. The FET response corresponded to the fluorescence images, which visualized the specific adsorption of CRP. The estimated dissociation constant was consistent with previously reported values; this supports the conclusion that the results are attributed to specific adsorption. Conversely, the signal in GFETs without decoration was obscured by noise because of nonlinear electrolytic screening, further emphasizing the significance of surface-charge modulation. The limit of detection of the system was determined to be 2.9 nM. This value has the potential to be improved through further optimization of the surface charges to align with specific applications. Our devices effectively circumvent nonlinear electrolytic screening, opening the door for further advancements in GFET biosensor technology.

In pursuit of degenerative brain disease diagnosis: Dementia biomarkers detected by DNA aptamer-attached portable graphene biosensor.

Dementia is a brain disease which results in irreversible and progressive loss of cognition and motor activity. Despite global efforts, there is no simple and reliable diagnosis or treatment option. Current diagnosis involves indirect testing of commonly inaccessible biofluids and low-resolution brain imaging. We have developed a portable, wireless readout-based Graphene field-effect transistor (GFET) biosensor platform that can detect viruses, proteins, and small molecules with single-molecule sensitivity and specificity. We report the detection of three important amyloids, namely, Amyloid beta (Aβ), Tau (τ), and α-Synuclein (αS) using DNA aptamer nanoprobes. These amyloids were isolated, purified, and characterized from the autopsied brain tissues of Alzheimer's Disease (AD) and Parkinson's Disease (PD) patients. The limit of detection (LoD) of the sensor is 10 fM, 1-10 pM, 10-100 fM for Aβ, τ, and αS, respectively. Synthetic as well as autopsied brain-derived amyloids showed a statistically significant sensor response with respect to derived thresholds, confirming the ability to define diseased vs. nondiseased states. The detection of each amyloid was specific to their aptamers; Aβ, τ, and αS peptides when tested, respectively, with aptamers nonspecific to them showed statistically insignificant cross-reactivity. Thus, the aptamer-based GFET biosensor has high sensitivity and precision across a range of epidemiologically significant AD and PD variants. This portable diagnostic system would allow at-home and POC testing for neurodegenerative diseases globally.

The relationship between pH sensitivity and biosensitivity in graphene field effect transistor biosensors

Theoretical models have predicted that pH-responsive surface groups can reduce the sensitivity of field-effect transistor biosensors. However, attempts to prove this experimentally have shown conflicting results. In this work, a graphene field effect transistor (gFET) biosensor is used, which, without modification, is pH insensitive. The surface of the graphene is then functionalized using 1-hydroxypyrene to modulate the pH sensitivity of the gFET. A pH sensitivity ranging from 3.9 to 36.8 mV/pH was demonstrated. The biosensitivity of the gFETs was tested using streptavidin–biotin as a model system. The experimental results showed no correlation between biosensitivity and pH sensitivity. An electrochemical membrane model was used to determine the expected relationship between biosensitivity and pH sensitivity. The model results show that biosensitivity does not decrease until a certain threshold pH sensitivity is reached. This threshold is dependent on factors such as the acid dissociation constants of the surface hydroxyl groups and ion concentration. Furthermore, the differences between the simulation and experiment suggest that the effect of screening is greatly reduced when the analyte binds within a membrane.

The Application of Graphene Field-Effect Transistor Biosensors in COVID-19 Detection Technology: A Review.

Coronavirus disease 2019 (COVID-19) is a disease caused by the infectious agent of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). The primary method of diagnosing SARS-CoV-2 is nucleic acid detection, but this method requires specialized equipment and is time consuming. Therefore, a sensitive, simple, rapid, and low-cost diagnostic test is needed. Graphene field-effect transistor (GFET) biosensors have become the most promising diagnostic technology for detecting SARS-CoV-2 due to their advantages of high sensitivity, fast-detection speed, label-free operation, and low detection limit. This review mainly focus on three types of GFET biosensors to detect SARS-CoV-2. GFET biosensors can quickly identify SARS-CoV-2 within ultra-low detection limits. Finally, we will outline the pros and cons of the diagnostic approaches as well as future directions.

Label-Free Neuropeptide Detection beyond the Debye Length Limit.

Biosensors with high selectivity, high sensitivity, and real-time detection capabilities are of significant interest for diagnostic applications as well as human health and performance monitoring. Graphene field-effect transistor (GFET) based biosensors are suitable for integration into wearable sensor technology and can potentially demonstrate the sensitivity and selectivity necessary for real-time detection and monitoring of biomarkers. Previously reported DC-mode GFET biosensors showed a high sensitivity for sensing biomarkers in solutions with a low salt concentration. However, due to Debye length screening, the sensitivity of the DC-mode GFET biosensors decreases significantly during operation in a physiological fluid such as sweat or interstitial fluid. To overcome the Debye screening length limitation, we report here alternating current (AC) mode heterodyne-based GFET biosensors for sensing neuropeptide-Y (NPY), a key stress biomarker, in artificial sweat at physiologically relevant ionic concentrations. Our AC-mode GFET biosensors show a record ultralow detection limit of 2 × 10-18 M with an extensive dynamic range of 10 orders of magnitude in sensor response to target NPY concentration. The sensors were characterized for various carrier frequencies (ranging from 30 kHz to 2 MHz) of the applied AC voltages and various salt concentrations (10, 50, and 100 mM). Contrary to DC-mode sensing, the AC-mode sensor response increases with an increase in salt concentration in the electrolyte. The sensor response can be further enhanced by tuning the carrier frequency of the applied AC voltage. The optimum response frequency of our sensor is approximately 400-600 kHz for salt concentrations of 50 and 100 mM, respectively. The salt-concentration- and frequency-dependent sensor response can be explained by an electrolyte-gated capacitance model.

Magnetically controlled graphene field-effect transistor biosensor for highly sensitive detection of cardiac troponin I

Herein, we have constructed a magnetic graphene field-effect transistor biosensor (MGFETs) for highly sensitive detection of cardiac troponin I (CTNI). Graphene films transferred to ITO conductive glass as conductive channels. CTNI aptamer was immobilized onto the graphene film via 1-pyrene-butanoic acid succinimidyl ester (PBASE) to capture CTNI. Magnetic nanobeads (MBs) modified with CTNI antibody were added to the reaction chamber to form an aptamer/CTNI/antibody/magnetic nanobeads sandwich-type complex. We found that the magnetic force exerted on the complex leads to an impedance change of the graphene film. The reason for this result is that the magnetic field exerts an influence on the MBs, causing CTNI aptamer strand to bend, resulting in a change in the distance between the double conductive layers of the graphene film surface and the test solution. With periodic sampling integration, different concentrations of CTNI can be detected with high sensitivity. Due to the stringent recognition capability and high affinity between the CTNI aptamer and CTNI, MGFETs have the potential to detect various types of proteins. Furthermore, MGFETs also have the potential to be utilized for the detection of DNA or specific cells in the future.

Graphene Transistors for In Vitro Detection of Health Biomarkers

AbstractBiomarkers are primary indicators for precise diagnosis and treatment. The early identification of health biomarkers has been sustained by the evolutionary success in sensor technologies. Among them, graphene field‐effect transistor (GFET) biosensors have exhibited major advantages such as an ultrashort response time, high sensitivity, easy operation, capability of integration, and label‐free detection. Owing to the atomic thickness, graphene restricts charge carrier flow merely at the material surface and responds to foreign stimuli directly, leading to effective signal acquisition and transmission. Here, this review summarizes the latest advances in GFET biosensors in a comprehensive manner that contains the device design, working principle, surface functionalization, and proof‐of‐concept applications. It provides a comprehensive survey of GFET biosensors with regard to biomarker analysis at the single‐device level and integrated prototypes that include wearable sensors, biomimetic systems, healthcare electronics, and diagnostic platforms. Moreover, there is discussion on the long‐standing research efforts and outlook for the future development of GFET sensor systems from lab to fab.

Analytical Modelling and Simulation of Graphene Based Biosensor to Detect SARS-COV-2 from Aerosal Particles

The health sector is focusing on the wellness of the society, is advancing in the phases of diagnosis and treatment. Biosensors based devices are used to diagnose a variety of human diseases. Recently, there was a sudden hike in the human mortality rate by chronic diseases caused by mutants of SARS-COV-2, on global scale. It is important to detect these kinds of diseases on an early stage to reduce the risk of spreading. For the analysis of Covid-19 influenza, tests such as Rapid Antigen Test (RAT), True NAT, CBNAAT and the commonly done RPT PCR were utilised. This proposal describes a non-invasive, quick and practical method for sensing the at-risk or infected persons with SARS-COV-2, aiming at controlling the epidemic. The proposed method employs a breath sensing device consisting of a Graphene Field Effect Transistor biosensor which can identify disease-specific biomarkers from exhaled sniff, hence allowing speedy and precise detection. This test aids screening of large populations as it is simple and quick and emerges as a promising candidate for SARS-COV-2 tests due to a high sensitivity. This work justifies the accurate diagnosis of Severe Acute Respiratory Syndrome COV 2 from aerosol particles by GFET Biosensor.

Ultrasensitive Antibiotic Perceiving Based on Aptamer-Functionalized Ultraclean Graphene Field-Effect Transistor Biosensor.

Antibiotics are powerful tools to treat bacterial infections, but antibiotic pollution is becoming a severe threat to the effective treatment of human bacterial infections. The detection of antibiotics in water has been a crucial research area for bioassays in recent years. There is still an urgent need for a simple ultrasensitive detection approach to achieve accurate antibiotic detection at low concentrations. Herein, a field-effect transistor (FET)-based biosensor was developed using ultraclean graphene and an aptamer for ultrasensitive tetracycline detection. Using a newly designed camphor-rosin clean transfer (CRCT) scheme to prepare ultraclean graphene, the carrier mobility of the FET is found to be improved by more than 10 times compared with the FET prepared by the conventional PMMA transfer (CPT) method. Based on the FET, aptamer-functionalized transistor antibiotic biosensors were constructed and characterized. A dynamic detection range of 5 orders of magnitude, a sensitivity of 21.7 mV/decade, and a low detection limit of 100 fM are achieved for the CRCT-FET biosensors with good stability, which are much improved compared with the biosensor prepared by the CPT method. The antibiotic sensing and sensing performance enhancement mechanisms for the CRCT-FET biosensor were studied and analyzed based on experimental results and a biosensing model. Finally, the CRCT-FET biosensor was verified by detecting antibiotics in actual samples obtained from the entrances of Bohai Bay.

Real-time monitoring of cellular Ca2+ efflux with renewable graphene field effect transistor biosensor

Calcium ion (Ca2+) is not only an intracellular second messenger, but also functions as an important messenger outside cells for intercellular communications. It is significant to develop a tool for dynamically probing the release of Ca2+ in the extracellular microenvironment. Here we present a photocatalysis-induced renewable reduced graphene oxide nanosheet field effect transistor (rGON-FET) biosensor for real-time monitoring of extracellular Ca2+ release. The biosensing channel was assembled layer-by-layer with reduced graphene oxide nanosheet (rGON) and titanium dioxide-reduced graphene oxide composites (TiO2-RGO) as a sandwich conducting nanostructure. Importantly, this biosensor was capable of measuring the dynamic extracellular Ca2+ release in a real time manner after immobilization of Ca2+ specific binding molecule Fluo 4-AM on the channel interface, particularly within the nano-confined space between the sensor surface and cell bottom. It was found that this biosensor could achieve a detection limit down to 100 pM in phosphate-buffered saline (PBS), and 1 nM in fetal bovine serum (FBS), respectively. Such a functional nanosensor also showed high selectivity to Ca2+ in the presence of excessive amounts of other metal ions. By using photo-responsive self-cleaning TiO2-based nanointerface, this renewable biosensor could be recycled to detect the exocytotic Ca2+ repeatedly. These advantages allow the rGON-FET biosensor to be further used in environmentally and clinically related metal ions detection.

Rapid self-test of unprocessed viruses of SARS-CoV-2 and its variants in saliva by portable wireless graphene biosensor

We have developed a DNA aptamer-conjugated graphene field-effect transistor (GFET) biosensor platform to detect receptor-binding domain (RBD), nucleocapsid (N), and spike (S) proteins, as well as viral particles of original Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus and its variants in saliva samples. The GFET biosensor is a label-free, rapid (≤20 min), ultrasensitive handheld wireless readout device. The limit of detection (LoD) and the limit of quantitation (LoQ) of the sensor are 1.28 and 3.89 plaque-forming units (PFU)/mL for S protein and 1.45 and 4.39 PFU/mL for N protein, respectively. Cognate spike proteins of major variants of concern (N501Y, D614G, Y453F, Omicron-B1.1.529) showed sensor response ≥40 mV from the control (aptamer alone) for fM to nM concentration range. The sensor response was significantly lower for viral particles and cognate proteins of Middle East Respiratory Syndrome (MERS) compared to SARS-CoV-2, indicating the specificity of the diagnostic platform for SARS-CoV-2 vs. MERS viral proteins. During the early phase of the pandemic, the GFET sensor response agreed with RT-PCR data for oral human samples, as determined by the negative percent agreement (NPA) and positive percent agreement (PPA). During the recent Delta/Omicron wave, the GFET sensor also reliably distinguished positive and negative clinical saliva samples. Although the sensitivity is lower during the later pandemic phase, the GFET-defined positivity rate is in statistically close alignment with the epidemiological population-scale data. Thus, the aptamer-based GFET biosensor has a high level of precision in clinically and epidemiologically significant SARS-CoV-2 variant detection. This universal pathogen-sensing platform is amenable for a broad range of public health applications and real-time environmental monitoring.

Detection of Glial Fibrillary Acidic Protein in Patient Plasma Using On-Chip Graphene Field-Effect Biosensors, in Comparison with ELISA and Single-Molecule Array.

Glial fibrillary acidic protein (GFAP) is a discriminative blood biomarker for many neurological diseases, such as traumatic brain injury. Detection of GFAP in buffer solutions using biosensors has been demonstrated, but accurate quantification of GFAP in patient samples has not been reported, yet in urgent need. Herein, we demonstrate a robust on-chip graphene field-effect transistor (GFET) biosensing method for sensitive and ultrafast detection of GFAP in patient plasma. Patients with moderate–severe traumatic brain injuries, defined by the Mayo classification, are recruited to provide plasma samples. The binding of target GFAP with the specific antibodies that are conjugated on a monolayer GFET device triggers the shift of its Dirac point, and this signal change is correlated with the GFAP concentration in the patient plasma. The limit of detection (LOD) values of 20 fg/mL (400 aM) in buffer solution and 231 fg/mL (4 fM) in patient plasma have been achieved using this approach. In parallel, for the first time, we compare our results to the state-of-the-art single-molecule array (Simoa) technology and the classic enzyme-linked immunosorbent assay (ELISA) for reference. The GFET biosensor shows competitive LOD to Simoa (1.18 pg/mL) and faster sample-to-result time (<15 min), and also it is cheaper and more user-friendly. In comparison to ELISA, GFET offers advantages of total detection time, detection sensitivity, and simplicity. This GFET biosensing platform holds high promise for the point-of-care diagnosis and monitoring of traumatic brain injury in GP surgeries and patient homes.

A Sensitivity-Enhanced Electrolyte-Gated Graphene Field-Effect Transistor Biosensor by Acoustic Tweezers.

Low-abundance biomolecule detection is very crucial in many biological and medical applications. In this paper, we present a novel electrolyte-gated graphene field-effect transistor (EGFET) biosensor consisting of acoustic tweezers to increase the sensitivity. The acoustic tweezers are based on a high-frequency bulk acoustic resonator with thousands of MHz, which has excellent ability to concentrate nanoparticles. The operating principle of the acoustic tweezers to concentrate biomolecules is analyzed and verified by experiments. After the actuation of acoustic tweezers for 10 min, the IgG molecules are accumulated onto the graphene. The sensitivities of the EGFET biosensor with accumulation and without accumulation are compared. As a result, the sensitivity of the graphene-based biosensor is remarkably increased using SMR as the biomolecule concentrator. Since the device has advantages such as miniaturized size, low reagent consumption, high sensitivity, and rapid detection, we expect it to be readily applied to many biological and medical applications.

Fabrication and microfluidic analysis of graphene-based molecular communication receiver for Internet of Nano Things (IoNT)

Bio-inspired molecular communications (MC), where molecules are used to transfer information, is the most promising technique to realise the Internet of Nano Things (IoNT), thanks to its inherent biocompatibility, energy-efficiency, and reliability in physiologically-relevant environments. Despite a substantial body of theoretical work concerning MC, the lack of practical micro/nanoscale MC devices and MC testbeds has led researchers to make overly simplifying assumptions about the implications of the channel conditions and the physical architectures of the practical transceivers in developing theoretical models and devising communication methods for MC. On the other hand, MC imposes unique challenges resulting from the highly complex, nonlinear, time-varying channel properties that cannot be always tackled by conventional information and communication tools and technologies (ICT). As a result, the reliability of the existing MC methods, which are mostly adopted from electromagnetic communications and not validated with practical testbeds, is highly questionable. As the first step to remove this discrepancy, in this study, we report on the fabrication of a nanoscale MC receiver based on graphene field-effect transistor biosensors. We perform its ICT characterisation in a custom-designed microfluidic MC system with the information encoded into the concentration of single-stranded DNA molecules. This experimental platform is the first practical implementation of a micro/nanoscale MC system with nanoscale MC receivers, and can serve as a testbed for developing realistic MC methods and IoNT applications.

Ultrasensitive Detection of Dopamine, IL-6 and SARS-CoV-2 Proteins on Crumpled Graphene FET Biosensor.

Universal platforms for biomolecular analysis using label‐free sensing modalities can address important diagnostic challenges. Electrical field effect‐sensors are an important class of devices that can enable point‐of‐care sensing by probing the charge in the biological entities. Use of crumpled graphene for this application is especially promising. It is previously reported that the limit of detection (LoD) on electrical field effect‐based sensors using DNA molecules on the crumpled graphene FET (field‐effect transistor) platform. Here, the crumpled graphene FET‐based biosensing of important biomarkers including small molecules and proteins is reported. The performance of devices is systematically evaluated and optimized by studying the effect of the crumpling ratio on electrical double layer (EDL) formation and bandgap opening on the graphene. It is also shown that a small and electroneutral molecule dopamine can be captured by an aptamer and its conformation change induced electrical signal changes. Three kinds of proteins were captured with specific antibodies including interleukin‐6 (IL‐6) and two viral proteins. All tested biomarkers are detectable with the highest sensitivity reported on the electrical platform. Significantly, two COVID‐19 related proteins, nucleocapsid (N‐) and spike (S‐) proteins antigens are successfully detected with extremely low LoDs. This electrical antigen tests can contribute to the challenge of rapid, point‐of‐care diagnostics.

Silk fibroin hydrogel encapsulated graphene filed-effect transistors as enzyme-based biosensors

Graphene field-effect transistor (GFET) is a useful device which could be designed as an ultrasensitive and high-throughput biosensor in biological sensing. However, bioprobes like enzymes are easy to lose activity after immobilizing on the graphene. In addition, measurement of biomarkers in aqueous solutions with high ionic concentration and multiple interfering components is still a challenge. Here, the enzymatic cross-linked silk fibroin (SF) hydrogel is selected as a carrier for glucose oxidase and horseradish peroxidase to encapsulate GFET biosensor functionally, in order to detect glucose in a physiological buffer solution. The SF hydrogel showed the effects of prolonging the lifetime of these enzymes and enabled the reduction of nonspecific adsorption of the GFET. The SF hydrogel encapsulated GFET exhibited high sensitivity for glucose sensing with a wide detection range of 1 µM to 10 mM and a limit of detection (LOD) down 200 nM, as well as a high specificity over other carbohydrates such as sucrose and trehalose. Moreover, the biosensor has exhibited renewability, which maintained a consistent sensing response after 10 times of regenerative cycles, with a variation less than 4.9%. These results demonstrate that the strategy for encapsulating GFET using the SF hydrogel is valid, suggesting the potential application of the SF hydrogel in biosensors.

Detection of Bacterial Metabolic Volatile Indole Using a Graphene-Based Field-Effect Transistor Biosensor.

The existence of bacteria is a great threat to food safety. Volatile compounds secreted by bacteria during their metabolic process can be dissected to evaluate bacterial contamination. Indole, as a major volatile molecule released by Escherichia coli (E. coli), was chosen to examine the presence of E. coli in this research. In this work, a graphene field-effect transistor (G-FET) was employed to detect the volatile molecule-indole based on a π-π stacking interaction between the indole and the graphene. The exposure of G-FET devices to the indole provokes a change in electrical signal, which is ascribed to the adsorption of the indole molecule onto the graphene surface via π-π stacking. The adsorption of the indole causes a charge rearrangement of the graphene-indole complex, which leads to changes in the electrical signal of G-FET biosensors with a different indole concentration. Currently, the indole biosensor can detect indole from 10 ppb to 250 ppb and reach a limit of detection of 10 ppb for indole solution detection. We believe that our detection strategy for detecting bacterial metabolic gas molecules will pave a way to developing an effective platform for bacteria detection in food safety monitoring.

Graphene FET Sensors for Alzheimer's Disease Protein Biomarker Clusterin Detection.

We report on the fabrication and characterisation of graphene field-effect transistor (GFET) biosensors for the detection of Clusterin, a prominent protein biomarker of Alzheimer’s disease (AD). The GFET sensors were fabricated on Si/SiO2 substrate using photolithographic patterning and metal lift-off techniques with evaporated chromium and sputtered gold contacts. Raman Spectroscopy was performed on the devices to determine the quality of the graphene. The GFETs were annealed to improve their performance before the channels were functionalized by immobilising the graphene surface with linker molecules and anti-Clusterin antibodies. Concentration of linker molecules was also independently verified by absorption spectroscopy using the highly collimated micro-beam light of Diamond B23 beamline. The detection was achieved through the binding reaction between the antibody and varying concentrations of Clusterin antigen from 1 to 100 pg/mL, as well as specificity tests using human chorionic gonadotropin (hCG), a glycoprotein risk biomarker of certain cancers. The GFETs were characterized using direct current (DC) 4-probe electrical resistance (4-PER) measurements, which demonstrated a limit of detection of the biosensors to be ∼ 300 fg/mL (4 fM). Comparison with back-gated Dirac voltage shifts with varying concentration of Clusterin show 4-PER measurements to be more accurate, at present, and point to a requirement for further optimisation of the fabrication processes for our next generation of GFET sensors. Thus, we have successfully fabricated a promising set of GFET biosensors for the detection of Clusterin protein biomarker. The developed GFET biosensors are entirely generic and also have the potential to be applied to a variety of other disease detection applications such as Parkinson’s, cancer, and cardiovascular.

Graphene field effect transistor for ultrasensitive label-free detection of ATP and Adenosine

Because of unique electrical and structural properties, graphene has attracted widespread attention in biosensing applications. In this paper, a single layer of graphene was grown by chemical vapor deposition (CVD). Using graphene as the electric channel, a graphene field effect transistor (G-FET) biosensor was fabricated and used to detect adenosine triphosphate (ATP) and adenosine. Compared with traditional methods, the G-FET biosensor has the advantages of higher sensitivity and better stability. The sensor showed high performance and achieved a detection limit down to 0.5 pM for both ATP and adenosine. Moreover, the G-FET biosensor showed an excellent linear electrical response to ATP concentrations in a broad range from 0.5 pM to 50 μM. The developed graphene biosensor has high sensitivity, simple operation, and fast analysis speed, which may provide a new feasible direction to detect ATP and adenosine. Healthy sexually mature male laboratory Wistar rats, weighing 180-200 gr (“FSUE “Nursery of laboratory animals “Rappolovo”) and having been placed under quarantine not less than for 14 days, were selected for the experiment.