Minute-scale detection of SARS-CoV-2 using a low-cost biosensor composed of pencil graphite electrodes.

COVID-19 has led to over 3.47 million deaths worldwide and continues to devastate primarily middle- and low-income countries. High-frequency testing has been proposed as a potential solution to prevent outbreaks. However, current tests are not sufficiently low-cost, rapid, or scalable to enable broad COVID-19 testing. Here, we describe LEAD (Low-cost Electrochemical Advanced Diagnostic), a diagnostic test that detects severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within 6.5 min and costs $1.50 per unit to produce using easily accessible and commercially available materials. LEAD is highly sensitive toward SARS-CoV-2 spike protein (limit of detection = 229 fg⋅mL-1) and displays an excellent performance profile using clinical saliva (100.0% sensitivity, 100.0% specificity, and 100.0% accuracy) and nasopharyngeal/oropharyngeal (88.7% sensitivity, 86.0% specificity, and 87.4% accuracy) samples. No cross-reactivity was detected with other coronavirus or influenza strains. Importantly, LEAD also successfully diagnosed the highly contagious SARS-CoV-2 B.1.1.7 UK variant. The device presents high reproducibility under all conditions tested and preserves its original sensitivity for 5 d when stored at 4 °C in phosphate-buffered saline. Our low-cost and do-it-yourself technology opens new avenues to facilitate high-frequency testing and access to much-needed diagnostic tests in resource-limited settings and low-income communities.

Laser-scribed graphene on polyetherimide substrate: an electrochemical sensor platform for forensic determination of xylazine in urine and beverage samples.

A laser-scribed graphene (LSG) device fabricated on polymeric polyetherimide (PEI) substrate is reported for sensitive electrochemical detection of xylazine (XLZ), a veterinary drug that has been associated with drug-facilitated crimes. Morphological characterization was made by scanning electron microscopy (SEM), demonstrating that the electrochemical device presents a highly porous carbonaceous structure. In addition, Raman spectroscopy, Fourier-transform infrared spectroscopy (FTIR), wettability, electrochemical impedance spectroscopy (EIS), and cyclic voltammetry (CV) measurements were used to characterize the PEI-LSG material, which showed superior electroanalytical performance after a simple anodic treatment in an alkaline medium (applying 2 V for 120 s in 0.1 mol L-1 NaOH solution). The electrochemical determination of XLZ was carried out using square wave voltammetry (SWV), which showed sensitivity and coefficient of determination (R2) equal to 0.206 ± 0.008 A L mol-1 cm-2 and 0.991, respectively. In addition, we obtained a low limit of detection (LOD) of 1.39 × 10-7 mol L-1 with a linear response in the concentration range 5.0 to 200.0 × 10-6 mol L-1. The PEI-LSG electrochemical sensor was applied to XLZ detection in commercial beverage and synthetic urine samples, providing recoveries between 96.0 and 114.8%. Collectively, our electrochemical sensor presents an easy method to manufacture reproducible (relative standard deviation (RSD) of 2.56%), low-cost ($0.12), and single-use (disposable) devices, which is a promising way for in-field determination of drug-facilitated sexual assaults and other relevant applications.

Single-Step Reagentless Laser Scribing Fabrication of Electrochemical Paper-Based Analytical Devices.

A single-step laser scribing process is used to pattern nanostructured electrodes on paper-based devices. The facile and low-cost technique eliminates the need for chemical reagents or controlled conditions. This process involves the use of a CO2 laser to pyrolyze the surface of the paperboard, producing a conductive porous non-graphitizing carbon material composed of graphene sheets and composites with aluminosilicate nanoparticles. The new electrode material was extensively characterized, and it exhibits high conductivity and an enhanced active/geometric area ratio; it is thus well-suited for electrochemical purposes. As a proof-of-concept, the devices were successfully employed for different analytical applications in the clinical, pharmaceutical, food, and forensic fields. The scalable and green fabrication method associated with the features of the new material is highly promising for the development of portable electrochemical devices.

Simple and Sensitive Paper-Based Device Coupling Electrochemical Sample Pretreatment and Colorimetric Detection.

We report the development of a simple, portable, low-cost, high-throughput visual colorimetric paper-based analytical device for the detection of procaine in seized cocaine samples. The interference of most common cutting agents found in cocaine samples was verified, and a novel electrochemical approach was used for sample pretreatment in order to increase the selectivity. Under the optimized experimental conditions, a linear analytical curve was obtained for procaine concentrations ranging from 5 to 60 µmol L(-1), with a detection limit of 0.9 µmol L(-1). The accuracy of the proposed method was evaluated using seized cocaine samples and an addition and recovery protocol.

A highly efficient and portable laser-scribed graphene-based electrochemical system for forensic-oriented determination of acepromazine.

Acepromazine (ACP) is a phenothiazine derivative drug commonly used as a tranquilizer veterinary medication due to its sedative properties. Benefiting from sedative properties, ACP has emerged as a drug of abuse and has been associated with drug-facilitated sexual assaults. Herein, we report, for the first time, the electrochemical behavior of ACP using a miniaturized and environmentally friendly laser-scribed graphene-based (LSG) sensor fabricated on a polyetherimide (PEI) substrate. The LSG device presented high porosity, as demonstrated by scanning electron microscopy (SEM). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements of the PEI-LSG electrode confirmed the enhanced electroactive area (3.1-fold increase) caused by the rough surface and revealed a low charge transfer resistance of the electrode material, with a heterogeneous electron transfer rate constant (k0) of 8.66 × 10-3 cm s-1 for potassium ferricyanide redox probe. A simple and accurate method was applied to quantify ACP by using square wave voltammetry (SWV) under optimized experimental conditions, which exhibited high sensitivity (0.686 ± 0.008 A L mol-1 cm-2) and a low limit of detection (LOD) of 7.43 × 10-8 mol L-1, with a linear concentration ranging from 0.5 to 100 µmol L-1 ACP. Aiming for on-site analysis, the PEI-LSG sensor was integrated with a miniaturized potentiostat controlled by using a smartphone and applied as proof of applicability to ACP detection in commercial beverage and synthetic urine samples. These studies demonstrated adequate recoveries, ranging from 95.1% to 115.8%. The analytical parameters highlight the robustness and reliability of the proposed method for analyses of ACP directly at a potential crime scene.

Laser-scribing fabrication of a disposable electrochemical device for forensic detection of crime facilitating drugs in beverage samples.

A portable and disposable laser-scribed graphene (LSG) device was fabricated on polyetherimide (PEI) substrate for electrochemical detection of benzodiazepines (BZ) drugs such as diazepam (DZ) and midazolam (MZ) in commercial beverage samples. Morphological characterizations of the LSG material recorded by scanning electron microscopy (SEM) revealed the porous nature of the proposed electrochemical device, which contributed to the enhancement of the electroactive area. Besides, the structural and electrochemical characterizations performed by Raman spectroscopy, cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) measurements revealed that the PEI-LSG material presents highly disordered graphene-like structures and high electron transfer features, respectively. The electrochemical detection of DZ and MZ was carried out by Square Wave Voltammetry (SWV), whose analytical curves exhibited two linear intervals in concentrations ranging from 2.5 µmol L-1 to 25.0 µmol L-1 and from 25.0 µmol L-1 to 100.0 µmol L-1 for both BZ. We obtained limits of detection (LOD) and quantification (LOQ) of 0.66 and 2.18 µmol L-1 for DZ and 0.61 µmol L-1 and 2.01 µmol L-1 for MZ, respectively. The developed sensor was applied to detect DZ and MZ in commercial beverages such as juice, whisky, and sugarcane spirit samples to mimic potential forensic evidence of drug-facilitated crimes. The recoveries ranged from 97.1% to 117.2% for DZ and from 92.2% to 114.3% for MZ. In addition, the proposed method presented high manufacturing reproducibility (relative standard deviation (RSD) = 2.18% for DZ and RSD = 3.82% for MZ, n = 8 sensors) and adequate selectivity, highlighting the potential of PEI-LSG sensor as an excellent alternative method for forensic detection of crime facilitating drugs in commercial beverage samples.

Silicone glue-based graphite ink incorporated on paper platform as an affordable approach to construct stable electrochemical sensors.

This study describes the development of electrochemical paper-based analytical devices (ePADs) using carbon-based paste combining silicone glue and graphite powder. The ePADs were manufactured using the screen-printing technique, which consisted of depositing the conductive ink on a screencast on the paper surface. In addition, an alternative electrical connector was designed and 3D-printed to make the detection method cheaper, portable and reproducible. The morphological, structural, and electrochemical properties of the conductive material developed were investigated through scanning electron microscopy (SEM), Raman spectroscopy, electrochemical impedance spectroscopy (EIS), and cyclic voltammetry (CV) measurements. The ePADs combined with the alternative connector revealed high repeatability, reproducibility, and stable responses considering a well-known redox probe ([Fe(CN)6]4-/3-). In addition, the proposed ePAD provided a linear response for standard solutions of ascorbic acid (AA) in the concentration range between 0.1 and 2.0 mmol L-1. The achieved limit of detection was 4.0 µmol L-1. As proof of applicability, the ePADs were evaluated for AA analysis in synthetic biofluids (blood plasma and urine), vitamin C tablets, and food (gelatine and orange juice) samples. The analytical parameters of the proposed device were compared with other reports in the literature and exhibited similar or even superior performance, highlighting its feasibility for sensing applications.

Electrochemical Paper-Based Nanobiosensor for Rapid and Sensitive Detection of Monkeypox Virus.

Monkeypox virus (MPXV) infection was classified as a public health emergency of international concern by the World Health Organization (WHO) in 2022, being transmitted between humans by large respiratory droplets, in contact with skin lesions, fomites, and sexually. Currently, there are no available accessible and simple-to-use diagnostic tests that accurately detect MPXV antigens for decentralized and frequent testing. Here, we report an electrochemical biosensor to detect MPXV antigens in saliva and plasma samples within 15 min using accessible materials. The electrochemical system was manufactured onto a paper substrate engraved by a CO2 laser machine, modified with gold nanostructures (AuNS) and a monoclonal antibody, enabling sensitive detection of A29 viral protein. The diagnostic test is based on the use of electrochemical impedance spectroscopy (EIS) and can be run by a miniaturized potentiostat connected to a smartphone. The impedimetric biosensing method presented excellent analytical parameters, enabling the detection of A29 glycoprotein in the concentration ranging from 1 × 10-14 to 1 × 10-7 g mL-1, with a limit of detection (LOD) of 3.0 × 10-16 g mL-1. Furthermore, it enabled the detection of MPXV antigens in the concentration ranging from 1 × 10-1 to 1 × 104 PFU mL-1, with an LOD of 7.8 × 10-3 PFU mL-1. Importantly, no cross-reactivity was observed when our device was tested in the presence of other poxvirus and nonpoxvirus strains, indicating the adequate selectivity of our nanobiosensor for MPXV detection. Collectively, the nanobiosensor presents high greenness metrics associated with the use of a reproducible and large-scale fabrication method, an accessible and sustainable paper substrate, and a low volume of sample (2.5 µL), which could facilitate frequent testing of MPXV at point-of-care (POC).

A bacterial cellulose-based and low-cost electrochemical biosensor for ultrasensitive detection of SARS-CoV-2.

COVID-19 has led to over 6.8 million deaths worldwide and continues to affect millions of people, primarily in low-income countries and communities with low vaccination coverage. Low-cost and rapid response technologies that enable accurate, frequent testing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are crucial for outbreak prevention and infectious disease control. Here we produce and characterize cellulose fibers naturally generated by the bacterium Gluconacetobacter hansenii as an alternative biodegradable substrate for manufacturing an eco-friendly diagnostic test for COVID-19. Using this green technology, we describe a novel and label-free potentiometric diagnostic test that can detect SARS-CoV-2 within 10 min and costs US$3.50 per unit. The test has bacterial cellulose (BC) as its substrate and a carbon-based electrode modified with graphene oxide and the human angiotensin-converting enzyme-2 (ACE2) as its receptor. Our device accurately and precisely detects emerging SARS-CoV-2 variants and demonstrates exceptional sensitivity, specificity, and accuracy for tested clinical nasopharyngeal/oropharyngeal (NP/OP) samples.

Rapid and accurate detection of herpes simplex virus type 2 using a low-cost electrochemical biosensor.

Herpes simplex virus type 2 (HSV-2) infection, which is almost exclusively sexually transmitted, causes genital herpes. Although this lifelong and incurable infection is extremely widespread, currently there is no readily available diagnostic device that accurately detects HSV-2 antigens to a satisfactory degree. Here, we report an ultrasensitive electrochemical device that detects HSV-2 antigens within 9 min and costs just $1 (USD) to manufacture. The electrochemical biosensor is biofunctionalized with the human cellular receptor nectin-1 and detects the glycoprotein gD2, which is present within the HSV-2 viral envelope. The performance of the device is tested in a guinea pig model that mimics human biofluids, yielding 88.9% sensitivity, 100.0% specificity, and 95.0% accuracy under these conditions, with a limit of detection of 0.019 fg mL-1 for gD2 protein and 0.057 PFU mL-1 for titered viral samples. Importantly, no cross-reactions with other viruses were detected, indicating the adequate robustness and selectivity of the sensor. Our low-cost technology could facilitate more frequent testing for HSV-2.

Detection of SARS-CoV-2 with RAPID: A prospective cohort study.

COVID-19 has killed over 6 million people worldwide. Currently available methods to detect SARS-CoV-2 are limited by their cost and need for multistep sample preparation and trained personnel. Therefore, there is an urgent need to develop fast, inexpensive, and scalable point-of-care diagnostics that can be used for mass testing. Between January and March 2021, we obtained 321 anterior nare swab samples from individuals in Philadelphia (PA, USA). For the Real-time Accurate Portable Impedimetric Detection prototype 1.0 (RAPID) test, anterior nare samples were tested via an electrochemical impedance spectroscopy (EIS) approach. The overall sensitivity, specificity, and accuracy of RAPID in this cohort study were 80.6%, 89.0%, and 88.2%, respectively. We present a rapid, accurate, inexpensive (<$5.00 per unit), and scalable test for diagnosing COVID-19 at the point-of-care. We anticipate that further iterations of this approach will enable widespread deployment, large-scale testing, and population-level surveillance.

Debulking different Corona (SARS-CoV-2 delta, omicron, OC43) and Influenza (H1N1, H3N2) virus strains by plant viral trap proteins in chewing gums to decrease infection and transmission.

Because oral transmission of SARS-CoV-2 is 3-5 orders of magnitude higher than nasal transmission, we investigated debulking of oral viruses using viral trap proteins (CTB-ACE2, FRIL) expressed in plant cells, delivered through the chewing gum. In omicron nasopharyngeal (NP) samples, the microbubble count (based on N-antigen) was significantly reduced by 20 µg of FRIL (p < 0.0001) and 0.925 µg of CTB-ACE2 (p = 0.0001). Among 20 delta or omicron NP samples, 17 had virus load reduced below the detection level of spike protein in the RAPID assay, after incubation with the CTB-ACE2 gum powder. A dose-dependent 50% plaque reduction with 50-100 ng FRIL or 600-800 µg FRIL gum against Influenza strains H1N1, H3N2, and Coronavirus HCoV-OC43 was observed with both purified FRIL, lablab bean powder or gum. In electron micrographs, large/densely packed clumps of overlapping influenza particles and FRIL protein were observed. Chewing simulator studies revealed that CTB-ACE2 release was time/dose-dependent and release was linear up to 20 min chewing. Phase I/II placebo-controlled, double-blinded clinical trial (IND 154897) is in progress to evaluate viral load in saliva before or after chewing CTB-ACE2/placebo gum. Collectively, this study advances the concept of chewing gum to deliver proteins to debulk oral viruses and decrease infection/transmission.

Enhanced performance of pencil-drawn paper-based electrodes by laser-scribing treatment.

Electrochemical Paper-based Analytical Devices (ePADs) are an alternative to traditional portable analytical techniques due to features such as low-cost, easy surface modification with different materials, and high sensitivity. A fast and simple method to fabricate enhanced ePADs using pencil-drawing which involves the CO2 laser treatment of the carbon surface deposited on paper is described. The electrochemical performances of the devices were evaluated using cyclic voltammetry (CV) with different redox probes and electrochemical impedance spectroscopy (EIS). The electrochemical results show that a treated surface presents a lower resistance to charge transfer and changes the approach of the probe and the overlap of its orbitals with the electrode. To investigate the effects of the laser treatment process, chemical and structural characteristics were evaluated using scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and Raman spectroscopy. These results indicated that laser treatment promoted the restoration of carbon-carbon double bonds and removed a thin layer of nanodebris present in commercial pencils, resulting in an improvement of the electrochemical kinetics. As a proof-of-concept, the Pencil-Drawing Electrode (PDE) was used for the detection and quantification of furosemide (FUR) in a sample of synthetic urine, exhibiting a limit of detection (LOD) of 2.4 × 10-7 mol L-1. The percentages of recovery of the FUR added to the samples A and B were 95% and 110%, respectively. The analysis using CO2 laser-treated PDE resulted in a fast, simple, and reliable method for this doping agent.

Disposable and low-cost electrochemical sensor based on the colorless nail polish and graphite composite material for tartrazine detection.

A new method to manufacture electrochemical devices based on the graphite and colorless nail polish (N-grap) film was developed for tartrazine (Tz) detection. Scanning Electron Microscopy (SEM) demonstrates that the composite material presents a high porous carbon structure. Cyclic voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS) were employed to electrochemically characterize the electrode material, which corroborates the porous structure of the N-graph due to the enhanced electroactive area (5.4-fold increase) and presented a heterogeneous electron transfer rate constant (k0) of 5.82 × 10-3 cm s-1 for potassium ferricyanide. The electrochemical determination of the Tz was carried out using square-wave voltammetry (SWV), under the optimized experimental conditions, which showed high sensitivity (0.793 A L mol-1) and a lower limit of detection (LOD) of 2.10 × 10-8 mol L-1 with a linear concentration ranging from 2.0 to 50.0 µmol L-1. The developed sensor was applied for the analysis of Tz in sports drink samples and the result obtained by N-grap device was statistically compared with a spectrophotometric method demonstrating good accordance and the accuracy of the proposed method. Based on these results, we believe that this new fabrication method to produce disposable and low-cost electrochemical devices can be an alternative method for in-field analysis of dye in commercial sport drink samples and other relevant applications.

Low-cost biosensor for rapid detection of SARS-CoV-2 at the point of care.

SARS-CoV-2, the virus that causes COVID-19, has killed over 3 million people worldwide. Despite the urgency of the current pandemic, most available diagnostic methods for COVID-19 use RT-PCR to detect nucleic acid sequences specific to SARS-CoV-2. These tests are limited by their requirement of a large laboratory space, high reagent costs, multistep sample preparation, and the potential for cross-contamination. Moreover, results usually take hours to days to become available. Therefore, fast, reliable, inexpensive, and scalable point-of-care diagnostics are urgently needed. Here, we describe RAPID 1.0, a simple, handheld, and highly sensitive miniaturized biosensor modified with human receptor angiotensin-converting enzyme-2. RAPID 1.0 can detect SARS-CoV-2 using 10 µL of sample within 4 min through its increased resistance to charge transfer of a redox probe measured by electrochemical impedance spectroscopy. The sensitivity and specificity of RAPID for nasopharyngeal/oropharyngeal swab and saliva samples are 85.3% and 100% and 100% and 86.5%, respectively.

A "turn-off" fluorescent sensor based on electrospun polycaprolactone nanofibers and fluorene(bisthiophene) derivative for nitroaromatic explosive detection.

The preparation of fluorene(bisthiophene)-based fluorescent nanofibers for nitroaromatic explosive detection provides a convenient rapid and low-cost strategy aiming at forensic applications. Polycaprolactone (PCL) and fluorene(bisthiophene) derivative (FBT) nanofibers were obtained by electrospinning technique as a free-standing mat and characterized by SEM, FTIR, thermal analysis and fluorescence spectroscopy. The PCL/FBT nanofibers presented high sensitivity towards 2,4,6-trinitrotoluene (TNT) and picric acid (PA), with fluorescence quenching (turn-off mechanism), and selectivity to another kind of explosives. The free-standing mats were used as a cloth strip that was swiped on surfaces contaminated with TNT traces allowing its visual detection under UV light source. These findings are particularly important for the development of a facile and promising strategy to assembly portable optical devices for nitroaromatic explosive detection.

Environmentally Friendly Manufacturing of Flexible Graphite Electrodes for a Wearable Device Monitoring Zinc in Sweat.

Electrochemical sensors based on graphite and polymers have emerged as powerful analytical tools for bioanalytical applications. However, most of the fabrication processes are not environmentally friendly because they often involve the use of toxic reagents and generate waste. This study describes an alternative method to produce flexible electrodes in plastic substrates using graphite powder and thermal laminating sheets by solid-solid deposition through hot compression, without the use of hazardous chemical reagents. The electrodes developed through the proposed approach have successfully demonstrated flexibility, robustness, reproducibility (relative standard deviation around 6%), and versatility. The electrodes were thoroughly characterized by cyclic voltammetry, electrochemical impedance spectroscopy, Raman spectroscopy, and scanning electron microscopy. As a proof of concept, the electrode surfaces were modified with bismuth and used for zinc analysis in sweat. The modified electrodes presented linearity (R2 = 0.996) for a wide zinc concentration range (50-2000 ppb) and low detection limit (4.31 ppb). The proposed electrodes were tested using real sweat samples and the achieved zinc concentrations did not differ statistically from the data obtained by atomic absorption spectroscopy. To allow wearable applications, a 3D-printed device was fabricated, integrated with the proposed electrochemical system, and fixed at the abdomen by using an elastic tape to collect, store, and analyze the sweat sample. The matrix effect test was performed, spiking the real sample with different zinc levels, and the recovery values varied between 85 and 106%, thus demonstrating adequate accuracy and robustness of the flexible electrodes developed based on the proposed fabrication method.

Portable and low-cost colorimetric office paper-based device for phenacetin detection in seized cocaine samples.

An office paper-based colorimetric device is proposed as a portable, rapid, and low-cost sensor for forensic applications aiming to detect phenacetin used as adulterant in illicit seized materials such as cocaine. The proposed method uses white office paper as the substrate and wax printing technology to fabricate the detection zones. Based on the optimum conditions, a linear analytical curve was obtained for phenacetin concentrations ranging from 0 to 64.52µgmL‒1, and the straight line was in accordance with the following equation: (Magenta percentage color) = 1.19 + 0.458 (CPhe/µgmL‒1), R2 = 0.990. The limit of detection was calculated as 3.5µgmL‒1 (3σ/slope). The accuracy of the proposed method was evaluated using real seized cocaine samples and the spike-recovery procedure.

Portable analytical platforms for forensic chemistry: A review.

This current review article focuses on recent contributions to on-site forensic investigations. Portable and potentially portable methods are presented and critically discussed about (bio)chemical trace analysis and studies performed outside the controlled laboratory environment to rapidly help in crime scene inquiries or forensic intelligence purposes. A wide range of approaches including electrochemical sensors, microchip electrophoresis, ambient ionization on portable mass spectrometers, handheld Raman and NIR instruments as well as and point-of-need devices, like paper-based platforms, for in-field analysis of latent evidences, controlled substances, drug screening, hazards, and others to assist in law enforcements and solving crime more efficiently are highlighted. The covered examples have successfully demonstrated the huge potential of portable devices for on-site applications. Future investigations should consider analytical validation to compete equality and even replace current gold standard methods.

Wearable salivary uric acid mouthguard biosensor with integrated wireless electronics.

This article demonstrates an instrumented mouthguard capable of non-invasively monitoring salivary uric acid (SUA) levels. The enzyme (uricase)-modified screen printed electrode system has been integrated onto a mouthguard platform along with anatomically-miniaturized instrumentation electronics featuring a potentiostat, microcontroller, and a Bluetooth Low Energy (BLE) transceiver. Unlike RFID-based biosensing systems, which require large proximal power sources, the developed platform enables real-time wireless transmission of the sensed information to standard smartphones, laptops, and other consumer electronics for on-demand processing, diagnostics, or storage. The mouthguard biosensor system offers high sensitivity, selectivity, and stability towards uric acid detection in human saliva, covering the concentration ranges for both healthy people and hyperuricemia patients. The new wireless mouthguard biosensor system is able to monitor SUA level in real-time and continuous fashion, and can be readily expanded to an array of sensors for different analytes to enable an attractive wearable monitoring system for diverse health and fitness applications.