Detection of respiratory inflammation biomarkers in non-processed exhaled breath condensate samples using reduced graphene oxide.

In this work, we studied several important parameters regarding the standardization of a portable sensor of nitrite, a key biomarker of inflammation in the respiratory tract in untreated EBC samples. The storage of the EBC samples and electrical properties of both EBC samples and the sensor as main standardization parameters were investigated. The sensor performance was performed using differential pulse voltammetry (DPV) in a standard nitrite solution and untreated EBC samples. The storage effect was monitored by comparing sensor data of fresh and stored samples for one month at -80 °C. Results show, on average, a 20 percent reduction of peak current for stored solutions. The sensor's performance was compared with a previous EBC nitrite sensor and chemiluminescence method. The results demonstrate a good correlation between the present sensor and chemiluminescence for low nitrite concentrations in untreated EBC samples. The electrical behavior of the sensor and electrical variation between EBC samples were characterized using methods such as noise analysis, electrochemical impedance spectroscopy (EIS), electrical impedance (EI), and voltage shift. Data show that reduced graphene oxide (rGO) has lower electrical noise and a higher electron transfer rate regarding nitrite detection. Also, a voltage shift can be applied to calibrate the data based on the electrical variation between different EBC samples. This result makes it easy to calibrate the electrical difference between EBC samples and have a more reproducible portable chip design without using bulky EI instruments. This work helps detect nitrite in untreated and pure EBC samples and evaluates critical analytical EBC properties essential for developing portable and on-site point-of-care sensors.

Droplet Microfluidic Technology for the Early and Label-Free Isolation of Highly-Glycolytic, Activated T-Cells.

A label-free, fixation-free and passive sorting method is presented to isolate activated T-cells shortly after activation and prior to the display of activation surface markers. It uses a recently developed sorting platform dubbed "Sorting by Interfacial Tension" (SIFT) that sorts droplets based on pH. After polyclonal (anti-CD3/CD28 bead) activation and a brief incubation on chip, droplets containing activated T-cells display a lower pH than those containing naive cells due to increased glycolysis. Under specific surfactant conditions, a change in pH can lead to a concurrent increase in droplet interfacial tension. The isolation of activated T-cells on chip is hence achieved as flattened droplets are displaced as they encounter a micro-fabricated trench oriented diagonally with respect to the direction of flow. This technique leads to an enrichment of activated primary CD4+ T-cells to over 95% from an initial mixed population of naive cells and cells activated for as little as 15 min. Moreover, since the pH change is correlated to successful activation, the technique allows the isolation of T-cells with the earliest activation and highest glycolysis, an important feature for the testing of T-cell activation modulators and to determine regulators and predictors of differentiation outcomes.

Ionic liquid stationary phase coating optimization for semi-packed microfabricated columns.

This work highlights the effect of the stationary phase coating process on the separation efficiency of gas chromatography microcolumns. The stationary phase coating quality was characterized by three different bis(trifluoromethylsulfonyl)imide (NTf2) anion based ionic liquids. The ionic liquids containing NTf2 anion are used for gas chromatography due to their high temperature stability. In this work, the chemical and physical approaches of column deactivation as well as the temperature treatment were evaluated by separating a mixture of 20 organic components and saturated alkanes. The results show that higher oven temperature treatment provides higher efficiency while losing a bit of peak symmetry. The thermal treated 1-butylpyridinum bis(trifluoromethylsulfonyl) imide [BPY][NTf2] stationary phase at 240°C demonstrated as high as 8300 plates per meter for naphthalene. This was a 5-fold increase in separation efficiency in comparison to those of the columns treated at 200°C. Albeit being within acceptable ranges, the peak tailing degraded from 1.17 to 1.46 for naphthalene when the processing temperature for coating increased. Both chemical and physical deactivation process increased separation efficiencies and peak resolution.

Parallel Ionic Liquid Semi-Packed Microfabricated Columns for Complex Gas Analysis.

The paper presents a parallel micro gas chromatography approach using three ionic liquid semipacked columns. Switching from single column to multiple parallel columns with different selectivity enhances the power of compound identification without increasing the analysis time. The columns are fabricated using microelectromechanical systems (MEMS) technology containing an array of microfabricated pillars. The columns are 1 m-long and 240 µm-deep with four pillars per row. All columns were functionalized with ionic liquid stationary phases using a modified static coating technique and demonstrated the number of theoretical plates between 5000 and 8300 per meter. The chip performance was investigated with four different samples: (1) a mixture of C7-C30 saturated alkanes, (2) a multianalyte mixture consisting of 20 compounds ranging from 80 to 238 °C in boiling point, (3) a mixture of five organic chemicals with varying degrees of polarity, and (4) 46-compounds mixture containing all the chemicals in the first three samples. The individual columns separated 75%-100% of the first three samples but failed to distinguish all 46 compounds due to coeluting analytes; however, the parallel configuration provided more retention time information by which all the compounds in all samples were fully determined.

A portable battery powered microfluidic impedance cytometer with smartphone readout: towards personal health monitoring.

We present a portable system for personalized blood cell counting consisting of a microfluidic impedance cytometer and portable analog readout electronics, feeding into an analog-to-digital converter (ADC), and being transmitted via Bluetooth to a user-accessible mobile application. We fabricated a microfluidic impedance cytometer with a novel portable analog readout. The novel design of the analog readout, which consists of a lock-in-amplifier followed by a high-pass filter stage for subtraction of drift and DC offset, and a post-subtraction high gain stage, enables detection of particles and cells as small as 1 µm in diameter, despite using a low-end 8-bit ADC. The lock-in-amplifier and the ADC were set up to receive and transmit data from a Bluetooth module. In order to initiate the system, as well as to transmit all of the data, a user friendly mobile application was developed, and a proof-of-concept trial was run on a blood sample. Applications such as personalized health monitoring require robust device operation and resilience to clogging. It is desirable to avoid using channels comparable in size to the particles being detected thus requiring high levels of sensitivity. Despite using low-end off-the-shelf hardware, our sensing platform was capable of detecting changes in impedance as small as 0.032%, allowing detection of 3 µm diameter particles in a 300 µm wide channel. The sensitivity of our system is comparable to that of a high-end bench-top impedance spectrometer when tested using the same sensors. The novel analog design allowed for an instrument with a footprint of less than 80 cm2. The aim of this work is to demonstrate the potential of using microfluidic impedance spectroscopy for low cost health monitoring. We demonstrated the utility of the platform technology towards cell counting, however, our platform is broadly applicable to assaying wide panels of biomarkers including proteins, nucleic acids, and various cell types.

Toward point-of-care management of chronic respiratory conditions: Electrochemical sensing of nitrite content in exhaled breath condensate using reduced graphene oxide.

We present a portable non-invasive approach for measuring indicators of inflammation and oxidative stress in the respiratory tract by quantifying a biomarker in exhaled breath condensate (EBC). We discuss the fabrication and characterization of a miniaturized electrochemical sensor for detecting nitrite content in EBC using reduced graphene oxide. The nitrite content in EBC has been demonstrated to be a promising biomarker of inflammation in the respiratory tract, particularly in asthma. We utilized the unique properties of reduced graphene oxide (rGO); specifically, the material is resilient to corrosion while exhibiting rapid electron transfer with electrolytes, thus allowing for highly sensitive electrochemical detection with minimal fouling. Our rGO sensor was housed in an electrochemical cell fabricated from polydimethyl siloxane (PDMS), which was necessary to analyze small EBC sample volumes. The sensor is capable of detecting nitrite at a low over-potential of 0.7 V with respect to an Ag/AgCl reference electrode. We characterized the performance of the sensors using standard nitrite/buffer solutions, nitrite spiked into EBC, and clinical EBC samples. The sensor demonstrated a sensitivity of 0.21 µA µM-1 cm-2 in the range of 20-100 µM and of 0.1 µA µM-1 cm-2 in the range of 100-1000 µM nitrite concentration and exhibited a low detection limit of 830 nM in the EBC matrix. To benchmark our platform, we tested our sensors using seven pre-characterized clinical EBC samples with concentrations ranging between 0.14 and 6.5 µM. This enzyme-free and label-free method of detecting biomarkers in EBC can pave the way for the development of portable breath analyzers for diagnosing and managing changes in respiratory inflammation and disease.

Permeation of nickel nanodots on carbon nanotubes: synthesis of 3D CNT-based nanomaterials.

In this paper, we report the fabrication of three-dimensional (3D) hybrid carbon nanotubes (CNT)-based nanostructures. Secondary carbon nanotubes are grown on the hydrogenated and unzipped horizontal carbon nanotubes without any further catalyst deposition. Hydrogenation of horizontal CNTs leads to out-diffusion of Ni nanoparticles that were trapped within the walls of nanotubes during the original growth process. This out-diffusion effect, as permeation, leads to the formation of nickel dots at the surfaces of carbon nanotubes which acts as the catalyst for the growth of secondary nanotubes. By controlling the secondary growth condition, a variety of 3D structures could be achieved. The permeation effect and the evolution of secondary nanostructures are studied extensively by means of scanning electron microscopy, transmission electron microscopy, atomic force microscopy, X-ray photoelectron spectroscopy, and X-ray diffraction analysis.

Fabrication of sensitive glutamate biosensor based on vertically aligned CNT nanoelectrode array and investigating the effect of CNTs density on the electrode performance.

In this report, the fabrication of vertically aligned carbon nanotube nanoelectrode array (VACNT-NEA) by photolithography method is presented. Electrochemical impedance spectroscopy as well as cyclic voltammetry was performed to characterize the arrays with respect to different diffusion regimes. The fabricated array illustrated sigmoidal cyclic voltammogram with steady state current dominated by radial diffusion. The fabricated VACNT-NEA and high density VACNTs were employed as electrochemical glutamate biosensors. Glutamate dehydrogenase is covalently attached to the tip of CNTs. The voltammetric biosensor, based on high density VACNTs, exhibits a sensitivity of 0.976 mA mM(-1) cm(-2) in the range of 0.1-20 µM and 0.182 mA mM(-1) cm(-2) in the range of 20-300 µM glutamate with a low detection limit of 57 nM. Using the fabricated VACNT-NEA, the sensitivity increases approximately to a value of 2.2 Am M(-1) cm(-2) in the range of 0.01 to 20 µM and to 0.1 A mM(-1) cm(-2) in the range of 20-300 µM glutamate. Using this electrode, a record of low detection limit of 10 nM was achieved for glutamate. The results prove the efficacy of the fabricated NEA for low cost and highly sensitive enzymatic biosensor with high sensitivity well suited for voltammetric detection of a wide range of clinically important biomarkers.

Mediator-less highly sensitive voltammetric detection of glutamate using glutamate dehydrogenase/vertically aligned CNTs grown on silicon substrate.

A sensitive glutamate biosensor is prepared based on glutamate dehydrogenase/vertically aligned carbon nanotubes (GLDH, VACNTs). Vertically aligned carbon nanotubes were grown on a silicon substrate by direct current plasma enhanced chemical vapor deposition (DC-PECVD) method. The electrochemical behavior of the synthesized VACNTs was investigated by cyclic voltammetry and electrochemical impedance spectroscopic methods. Glutamate dehydrogenase covalently attached on tip of VACNTs. The electrochemical performance of the electrode for detection of glutamate was investigated by cyclic and differential pulse voltammetry. Differential pulse voltammetric determinations of glutamate are performed in mediator-less condition and also, in the presence of 1 and 5 µM thionine as electron mediator. The linear calibration curve of the concentration of glutamate versus peak current is investigated in a wide range of 0.1-500 µM. The mediator-less biosensor has a low detection limit of 57 nM and two linear ranges of 0.1-20 µM with a sensitivity of 0.976 mA mM(-1) cm(-2) and 20-300 µM with a sensitivity of 0.182 mA mM(-1) cm(-2). In the presence of 1 µM thionine as an electron mediator, the prepared biosensor shows a low detection limit of 68 nM and two linear ranges of 0.1-20 with a calibration sensitivity of 1.17 mA mM(-1) cm(-2) and 20-500 µM with a sensitivity of 0.153 mA mM(-1) cm(-2). The effects of the other biological compounds on the voltammetric behavior of the prepared biosensor and its response stability are investigated. The results are demonstrated that the GLDH/VACNTs electrode even without electron mediator is a suitable basic electrode for detection of glutamate.