Retraction: A new polymer lab-on-a-chip (LOC) based on a microfluidic capillary flow assay (MCFA) for detecting unbound cortisol in saliva.

Retraction of 'A new polymer lab-on-a-chip (LOC) based on a microfluidic capillary flow assay (MCFA) for detecting unbound cortisol in saliva' by Vinitha T. U. et al., Lab Chip, 2020, 20, 1961-1974, DOI: https://doi.org/10.1039/D0LC00071J.

A new polymer lab-on-a-chip (LOC) based on a microfluidic capillary flow assay (MCFA) for detecting unbound cortisol in saliva.

Unbound cortisol in saliva, which can be detected with non-invasive sampling, is now considered as one of the most effective biomarkers for the biochemical evaluation of common mental disorders. In this work, a new polymer lab-on-a-chip (LOC) based on a microfluidic capillary flow assay (MCFA) with on-chip dried reagents was newly developed and fully characterized for the detection of unbound cortisol in saliva. The new MCFA device consisted of serially connected microchannels for sample loading, dried detection antibodies, time delay for incubation time control, a spiral reaction chamber for testing, positive and negative controls, and a capillary pump for waste fluid collection. In addition, a portable fluorescence analyzer was also developed for the rapid quantitative measurement of salivary cortisol with high accuracy. A linear dynamic range of 7.0 pg mL-1-16.0 ng mL-1 was achieved from spiked artificial saliva samples with an inter-chip CV of around 4.0% using the developed LOC and fluorescence analyzer. The achieved results support the effective biochemical analysis of common mental disorders such as chronic stress, depression, anxiety and post traumatic stress disorder (PTSD). The new LOC based on a microfluidic capillary flow assay (MCFA) developed in this work can be one of the most promising LOC platforms for high-sensitivity and quantitative POCT with saliva and blood plasma/serum samples.

A new microchannel capillary flow assay (MCFA) platform with lyophilized chemiluminescence reagents for a smartphone-based POCT detecting malaria.

There has been a considerable development in microfluidic based immunodiagnostics over the past few years which has greatly favored the growth of novel point-of-care-testing (POCT). However, the realization of an inexpensive, low-power POCT needs cheap and disposable microfluidic devices that can perform autonomously with minimum user intervention. This work, for the first time, reports the development of a new microchannel capillary flow assay (MCFA) platform that can perform chemiluminescence based ELISA with lyophilized chemiluminescent reagents. This new MCFA platform exploits the ultra-high sensitivity of chemiluminescent detection while eliminating the shortcomings associated with liquid reagent handling, control of assay sequence and user intervention. The functionally designed microchannels along with adequate hydrophilicity produce a sequential flow of assay reagents and autonomously performs the ultra-high sensitive chemiluminescence based ELISA for the detection of malaria biomarker such as PfHRP2. The MCFA platform with no external flow control and simple chemiluminescence detection can easily communicate with smartphone via USB-OTG port using a custom-designed optical detector. The use of the smartphone for display, data transfer, storage and analysis, as well as the source of power allows the development of a smartphone based POCT analyzer for disease diagnostics. This paper reports a limit of detection (LOD) of 8 ng/mL by the smartphone analyzer which is sensitive enough to detect active malarial infection. The MCFA platform developed with the smartphone analyzer can be easily customized for different biomarkers, so a hand-held POCT for various infectious diseases can be envisaged with full networking capability at low cost.

Highly Sensitive Lab on a Chip (LOC) Immunoassay for Early Diagnosis of Respiratory Disease Caused by Respirable Crystalline Silica (RCS).

Respirable crystalline silica (RCS) produced in mining and construction industries can cause life-threatening diseases such as silicosis, lung cancer, and chronic obstructive pulmonary disease (COPD). These diseases could be more effectively treated and prevented if RCS-related biomarkers were identified and measured at an early stage of disease progression, which makes development of a point of care test (POCT) platform extremely desirable for early diagnosis. In this work, a new, highly sensitive lab on a chip (LOC) immunoassay has been designed, developed, and characterized for tumor necrosis factor α (TNF-α), a protein biomarker that causes lung inflammation due to RCS exposure. The designed LOC device is composed of four reservoirs for sample, enzyme conjugated detection antibody, wash buffer, and chemiluminescence substrate in liquid form, along with three spiral reaction chambers for test, positive control, and negative control. All reservoirs and spiral microchannels were connected in series and designed to perform sequential delivery of immunoassay reagents with minimal user intervention. The developed LOC measured TNF-α concentrations as low as 16 pg/mL in plasma from RCS-exposed rats and also had a limit of detection (LOD) of 0.5 pg/mL in spiked artificial serum. In addition, the analysis time was drastically reduced to about 30 min, as opposed to hours in conventional methods. Successful implementation of a highly sensitive, chemiluminescence-based immunoassay on a preloaded LOC with proper quality control, as reported in this work, can pave the way toward developing a new rapid POCT platform for in-field clinical diagnosis.

Lyophilization of chemiluminescent substrate reagents for high-sensitive microchannel-based lateral flow assay (MLFA) in point-of-care (POC) diagnostic system.

Over the last few years, lateral flow assay (LFA) devices have grown to be the most common point-of-care test (POCT) platform facilitating disease diagnostics in low-resource environments. However, the lack of consistency and the limited sensitivity of these devices often lead to misdiagnosis and generates the need for an alternate approach. A chemiluminescence based microchannel-based lateral flow assay (MLFA) in a POCT platform can result in a much higher sensitivity but involves multiple additional steps of liquid reagents for the sequential execution of the signal amplification protocol. One of the best ways to develop a sample-to-answer system with minimum user intervention is to dry reagents on a chip prior to sample addition and to control the flow of the biological fluid through the drying chambers resulting in the reconstitution of the reagents. This work reports the methods for the successful lyophilization of the chemiluminescent substrate and its reconstitution in artificial serum without any significant loss of functionality. The lyophilized reagents were reconstituted and incorporated into the reaction chambers of a designed polymer lab-on-a-chip to implement a sandwich assay for the detection of malarial biomarkers. The results report a limit of detection (LOD) of 5.75 ng mL-1 which is sensitive enough to detect active malarial infection. Successful lyophilization and reconstitution of the chemiluminescent substrate, as reported here, can pave the way towards developing an autonomous POCT system implementing chemiluminescence based sandwich ELISA for enhanced sensitivity, portability, and ease-of-use in resource limited settings.

Pathogen-specific DNA sensing with engineered zinc finger proteins immobilized on a polymer chip.

A specific double-stranded DNA sensing system is of great interest for diagnostic and other biomedical applications. Zinc finger domains, which recognize double-stranded DNA, can be engineered to form custom DNA-binding proteins for the recognition of specific DNA sequences. As a proof of concept, a sequence-enabled reassembly of a TEM-1 ß-lactamase system (SEER-LAC) was previously demonstrated to develop zinc finger protein (ZFP) arrays for the detection of a double-stranded bacterial DNA sequence. Here, we implemented the SEER-LAC system to demonstrate the direct detection of pathogen-specific DNA sequences present in E. coli O157:H7 on a lab-on-a-chip. ZFPs custom-designed to detect Shiga toxin in E. coli O157:H7 were immobilized on a cyclic olefin copolymer (COC) chip, which can function as a non-PCR based molecular diagnostic device. Pathogen-specific double-stranded DNA was directly detected by using engineered ZFPs immobilized on the COC chip with high specificity, providing a detection limit of 10 fmol of target DNA in a colorimetric assay. Therefore, in this study, we demonstrated the great potential of ZFP arrays on the COC chip for further development of a simple and novel lab-on-a-chip technology for the detection of pathogens.

Spiral-based microfluidic device for long-term time course imaging of Neurospora crassa with single nucleus resolution.

Real-time imaging of fluorescent reporters plays a critical role in elucidating fundamental molecular mechanisms including circadian rhythms in the model filamentous fungus, Neurospora crassa. However, monitoring N. crassa for an extended period of time with single nucleus resolution is a technically challenging task due to hyphal growth that rapidly moves beyond a region of interest during microscopy experiments. In this report, we have proposed a two-dimensional spiral-based microfluidic platform and applied for monitoring the single-nucleus dynamics in N. crassa for long-term time course experiments.

Development and application of a microfabricated multimodal neural catheter for neuroscience.

We present a microfabricated neural catheter for real-time continuous monitoring of multiple physiological, biochemical and electrophysiological variables that are critical to the diagnosis and treatment of evolving brain injury. The first generation neural catheter was realized by polyimide-based micromachining and a spiral rolling packaging method. The mechanical design and electrical operation of the microsensors were optimized and tailored for multimodal monitoring in rat brain such that the potential thermal, chemical and electrical crosstalk among the microsensors as well as errors from micro-environmental fluctuations are minimized. In vitro cytotoxicity analyses suggest that the developed neural catheters are minimally toxic to rat cortical neuronal cultures. In addition, in vivo histopathology results showed neither acute nor chronic inflammation for 7 days post implantation. The performance of the neural catheter was assessed in an in vivo needle prick model as a translational replica of a "mini" traumatic brain injury. It successfully monitored the expected transient brain oxygen, temperature, regional cerebral blood flow, and DC potential changes during the passage of spreading depolarization waves. We envisage that the developed multimodal neural catheter can be used to decipher the causes and consequences of secondary brain injury processes with high spatial and temporal resolution while reducing the potential for iatrogenic injury inherent to current use of multiple invasive probes.

Single probe for real-time simultaneous monitoring of neurochemistry and direct-current electrocorticography.

We report a novel single neural probe for real-time simultaneous monitoring of multiple neurochemicals and direct-current electrocorticography (DC-ECoG). A major advance of this probe is the inclusion of two iridium oxide reference electrodes to improve sensor accuracy. The ECoG reference electrode is identical to the ECoG recording electrodes to significantly improve DC stability, while the reference for electrochemical sensors has 10-fold lower polarization rate to minimize the small current-induced drift in the reference electrode potential. In vitro, the single probe selectively measured oxygen (r(2)=0.985 ± 0.01, concentration range=0-60 mmHg, limit of detection=0.4 ± 0.07 mmHg) and glucose (r(2)=0.989 ± 0.009, concentration range=0-4mM, limit of detection=31 ± 8 µM) in a linear fashion. The performance of the single probe was assessed in an in vivo needle prick model to mimic sequelae of traumatic brain injury. It successfully monitored the theoretically expected transient brain oxygen, glucose, and DC potential changes during the passage of spreading depolarization (SD) waves. We envision that the developed probe can be used to decipher the cause-effect relationships between multiple variables of brain pathophysiology with the high temporal and spatial resolutions that it provides.

Evaluation of microelectrode materials for direct-current electrocorticography.

OBJECTIVE: Direct-current electrocorticography (DC-ECoG) allows a more complete characterization of brain states and pathologies than traditional alternating-current recordings (AC-ECoG). However, reliable recording of DC signals is challenging because of electrode polarization-induced potential drift, particularly at low frequencies and for more conducting materials. Further challenges arise as electrode size decreases, since impedance is increased and the potential drift is augmented. While microelectrodes have been investigated for AC-ECoG recordings, little work has addressed microelectrode properties for DC-signal recording. In this paper, we investigated several common microelectrode materials used in biomedical application for DC-ECoG. APPROACH: Five of the most common materials including gold (Au), silver/silver chloride (Ag/AgCl), platinum (Pt), Iridium oxide (IrOx), and platinum-iridium oxide (Pt/IrOx) were investigated for electrode diameters of 300 µm. The critical characteristics such as polarization impedance, AC current-induced polarization, long-term stability and low-frequency noise were studied in vitro (0.9% saline). The two most promising materials, Pt and Pt/lrOx were further investigated in vivo by recording waves of spreading depolarization, one of the most important applications for DC-ECoG in clinical and basic science research. MAIN RESULTS: Our experimental results indicate that IrOx-based microelectrodes, particularly with composite layers of nanostructures, are excellent in all of the common evaluation characteristics both in vitro and in vivo and are most suitable for multimodal monitoring applications. Pt electrodes suffer high current-induced polarization, but have acceptable long-term stability suitable for DC-ECoG. Major significance. The results of this study provide quantitative data on the electrical properties of microelectrodes with commonly-used materials and will be valuable for development of neural recordings inclusive of low frequencies.

Highly accurate thermal flow microsensor for continuous and quantitative measurement of cerebral blood flow.

Cerebral blood flow (CBF) plays a critical role in the exchange of nutrients and metabolites at the capillary level and is tightly regulated to meet the metabolic demands of the brain. After major brain injuries, CBF normally decreases and supporting the injured brain with adequate CBF is a mainstay of therapy after traumatic brain injury. Quantitative and localized measurement of CBF is therefore critically important for evaluation of treatment efficacy and also for understanding of cerebral pathophysiology. We present here an improved thermal flow microsensor and its operation which provides higher accuracy compared to existing devices. The flow microsensor consists of three components, two stacked-up thin film resistive elements serving as composite heater/temperature sensor and one remote resistive element for environmental temperature compensation. It operates in constant-temperature mode (~2 °C above the medium temperature) providing 20 ms temporal resolution. Compared to previous thermal flow microsensor based on self-heating and self-sensing design, the sensor presented provides at least two-fold improvement in accuracy in the range from 0 to 200 ml/100 g/min. This is mainly achieved by using the stacked-up structure, where the heating and sensing are separated to improve the temperature measurement accuracy by minimization of errors introduced by self-heating.

A new smart fall-down detector for senior healthcare system using inertial microsensors.

A new smart fall-down detector for senior healthcare system using inertial microsensors and Wi-Fi technology has been designed, prototyped and characterized in this work. The detector can reduce the risk of severe injury or death caused by falling down with minimum false alarm rate. The different patterns of motion are sensed by a set of inertial sensors composed of a tri-axial accelerometer and a tri-axial gyroscope. The signals of motion are sampled and processed by a microcontroller with integrated algorithms. The smart algorithm integrated with machine learning can be customized according to different habits of different seniors to reduce false alarms. The fall-down signal is transmitted through Wi-Fi to the client via Internet.

An innovative sample-to-answer polymer lab-on-a-chip with on-chip reservoirs for the POCT of thyroid stimulating hormone (TSH).

A new sample-to-answer polymer lab-on-a-chip, which can perform immunoassay with minimum user intervention through on-chip reservoirs for reagents and single-channel assay system, has been designed, developed and successfully characterized as a point-of-care testing (POCT) cartridge for the detection of thyroid stimulating hormone (TSH). Test results were obtained within 30 minutes after a sample was dropped into the POCT cartridge. The analyzed results of TSH showed a linear range of up to 55 µIU mL(-1) with the limit of detection (LOD) of 1.9 µIU mL(-1) at the signal-to-noise ratio (SNR) of 3. The reagents stored in the on-chip reservoirs maintained more than 97% of their initial volume for 120 days of storage time while the detection antibody retained its activity above 98% for 120 days. The sample-to-answer polymer lab-on-a-chip developed in this work using the mass-producible and low-cost polymer is well suited for the point-of-care testing of rapid in vitro diagnostics (IVD) of TSH.

Superhydrophilic multilayer silica nanoparticle networks on a polymer microchannel using a spray layer-by-layer nanoassembly method.

Nanoporous and superhydrophilic multilayer silica nanoparticle networks have been developed on a hydrophobic cyclic olefin copolymer (COC) microchannel using a spray layer-by-layer (LbL) electrostatic nanoassembly method. This powerful and promising LbL method provides a simple, cost-effective, and high-throughput nanoporous silica multilayer selectively onto the hydrophobic polymer surfaces. These newly developed multilayer networks have also been successfully characterized by contact angle measurement, environmental scanning electron microscopy (ESEM), energy-dispersive X-ray spectroscopy (EDS), and atomic force microscopy (AFM). The superhydrophilic effect, which was confirmed by the contact angle measurements, of the silica networks ensured the hydrophilic nature of the selectively constructed nanoporous silica nanoparticles onto the patterned hydrophobic COC microchannel. The capillary effect of the developed surface was characterized by measuring the length of a test liquid driven by the induced capillary forces in an on-chip capillary pumping platform with horizontal microchannels. The pumping capability achieved from the sprayed nanoporous surface for the on-chip micropump was mainly due to the strong capillary imbibition driven by the multicoated bilayers of hydrophilic silica nanoparticles. The developed networks with spray-assembled nanoparticles were also applied for an on-chip blood plasma separation platform with closed microchannels. The spray LbL method developed in this work can be a highly practical approach for the modification of various polymer microchannels because of several advantages such as an extremely simple process for the multilayer formation and flexibly controlled surface functionality at room temperature.

A new on-chip whole blood/plasma separator driven by asymmetric capillary forces.

A new on-chip whole blood/plasma separator driven by asymmetric capillary forces, which are produced through a microchannel with sprayed nanobead multilayers, has been designed, fabricated and fully characterized. The silica nanobead multilayers revealing as superhydrophilic surfaces have been fabricated using a spray layer-by-layer (LbL) nano-assembly method. This new on-chip blood plasma separator has been targeted for a sample-to-answer (S-to-A) microfluidic lab-on-a-chip (LOC) toward point-of-care clinical testing (POCT). Effective plasma separation from undiluted whole blood was achieved through the microchannel which was composed of asymmetric superhydrophilic surfaces with a 10 mm hydrophobic patch. Blood cells were continuously accumulated over the hydrophobic patch while the blood plasma was able to flow over the patch. Therefore, the blood plasma was successfully separated from the whole blood throughout the accumulated blood cells which worked as a so-called 'self-built-in blood cell microfilter'. The separated plasma was approximately 102 nL from a single drop of 3 µL whole blood within 10 min, which is very suitable for single-use disposable POCT devices.

A micro blood sampling system for catheterized neonates and pediatrics in intensive care unit.

A new micro blood sampling system has been designed, fabricated, and characterized to reduce iatrogenic blood loss from the catheterized neonates and pediatrics in intensive care unit by providing micro-volume of blood to analytical biomedical microdevices which can do point-of-care testing for their critical care. The system can not only save enormous iatrogenic blood loss through 1 to 10 µL of blood sampling and re-infusion of 1 to 5 mL of discard blood but also reduce the infection risk through the closed structure while satisfying the key criteria of the blood sampler. The sampled blood preserved its quality without rupturing of red blood cells verified by blood potassium concentrations of 3.86 ± 0.07 mM on the sampled blood which is similar to 3.81 ± 0.04 mM measured from the blood which did not go through the system. The sampling volume among the sampling channels showed consistency with the relative standard deviation of 1.41 %. In addition to the micro blood sampling capability, the sampling system showed negligible sample cross-contamination. The analyte-free samples collected after aspirating 7,500 times higher signal sample showed the same output signal as blank. The system was also demonstrated not to cause air-embolism by having no bubble generation during flushing procedure and the system was verified as leak-free since there was no fluid leakage under 30 times higher pressure than central venous pressure for 24 h.

Circadian rhythms in Neurospora crassa on a polydimethylsiloxane microfluidic device for real-time gas perturbations.

Racetubes, a conventional system employing hollow glass tubes, are typically used for monitoring circadian rhythms from the model filamentous fungus, Neurospora crassa. However, a major technical limitation in using a conventional system is that racetubes are not amenable for real-time gas perturbations. In this work, we demonstrate a simple microfluidic device combined with real-time gas perturbations for monitoring circadian rhythms in Neurospora crassa using bioluminescence assays. The developed platform is a useful toolbox for investigating molecular responses under various gas conditions for Neurospora and can also be applied to other microorganisms.

A novel microfluidic microplate as the next generation assay platform for enzyme linked immunoassays (ELISA).

In this work we introduce a novel microfluidic enzyme linked immunoassays (ELISA) microplate as the next generation assay platform for unparalleled assay performances. A combination of microfluidic technology with standard SBS-configured 96-well microplate architecture, in the form of microfluidic microplate technology, allows for the improvement of ELISA workflows, conservation of samples and reagents, improved reaction kinetics, and the ability to improve the sensitivity of the assay by multiple analyte loading. This paper presents the design and characterization of the microfluidic microplate, and its application in ELISA.

Formation of lipid bilayers inside microfluidic channel array for monitoring membrane-embedded nanopores of phi29 DNA packaging nanomotor.

An efficient method to form lipid bilayers inside an array of microfluidic channels has been developed and applied to monitor the membrane-embedded phi29 DNA packaging motor with an electrochemical characterization on a lab-on-a-chip (LOC) platform. A push-pull junction capturing approach was applied to confine a small amount of the lipid solution inside a microchannel. The selective permeability between solvents and water in PDMS was utilized to extract the solvent from the lipid solution, resulting in a self-formation of the lipid bilayer in the microchannel array. Each microchannel was independently connected to a silver/silver chloride (Ag/AgCl) electrode array, leading to a high-throughput monitoring of the nanopore insertion in the formed lipid bilayers. The formation of multiple lipid bilayers inside an array of microchannels and the simultaneous electrical and optical monitoring of multiple bilayer provides an efficient LOC platform for the further development of single phi29 motor pore sensing and high throughput single pore dsDNA sequencing.

Micromachined lab-on-a-tube sensors for simultaneous brain temperature and cerebral blood flow measurements.

This work describes the development of a micromachined lab-on-a-tube device for simultaneous measurement of brain temperature and regional cerebral blood flow. The device consists of two micromachined gold resistance temperature detectors with a 4-wire configuration. One is used as a temperature sensor and the other as a flow sensor. The temperature sensor operates with AC excitation current of 500 µA and updates its outputs at a rate of 5 Hz. The flow sensor employs a periodic heating and cooling technique under constant-temperature mode and updates its outputs at a rate of 0.1 Hz. The temperature sensor is also used to compensate for temperature changes during the heating period of the flow sensor to improve the accuracy of flow measurements. To prevent thermal and electronic crosstalk between the sensors, the temperature sensor is located outside the "thermal influence" region of the flow sensor and the sensors are separated into two different layers with a thin-film Copper shield. We evaluated the sensors for accuracy, crosstalk and long-term drift in human blood-stained cerebrospinal fluid. These in vitro experiments showed that simultaneous temperature and flow measurements with a single lab-on-a-tube device are accurate and reliable over the course of 5 days. It has a resolution of 0.013 °C and 0.18 ml/100 g/min; and achieves an accuracy of 0.1 °C and 5 ml/100 g/min for temperature and flow sensors respectively. The prototype device and techniques developed here establish a foundation for a multi-sensor lab-on-a-tube, enabling versatile multimodality monitoring applications.