On-chip Analysis Research Articles

Rapid and simple single-chamber nucleic acid detection system prepared through nature-inspired surface engineering.

Background: Nucleic acid (NA)-based diagnostics enable a rapid response to various diseases, but current techniques often require multiple labor-intensive steps, which is a major obstacle to successful translation to a clinical setting.Methods: We report on a surface-engineered single-chamber device for NA extraction and in situ amplification without sample transfer. Our system has two reaction sites: a NA extraction chamber whose surface is patterned with micropillars and a reaction chamber filled with reagents for in situ polymerase-based NA amplification. These two sites are integrated in a single microfluidic device; we applied plastic injection molding for cost-effective, mass-production of the designed device. The micropillars were chemically activated via a nature-inspired silica coating to possess a specific affinity to NA.Results: As a proof-of-concept, a colorimetric pH indicator was coupled to the on-chip analysis of NA for the rapid and convenient detection of pathogens. The NA enrichment efficiency was dependent on the lysate incubation time, as diffusion controls the NA contact with the engineered surface. We could detect down to 1×103 CFU by the naked eye within one hour of the total assay time.Conclusion: We anticipate that the surface engineering technique for NA enrichment could be easily integrated as a part of various types of microfluidic chips for rapid and convenient nucleic acid-based diagnostics.

Fill your Boots: Enhanced Embedded Bootloader Exploits via Fault Injection and Binary Analysis

The bootloader of an embedded microcontroller is responsible for guarding the device’s internal (flash) memory, enforcing read/write protection mechanisms. Fault injection techniques such as voltage or clock glitching have been proven successful in bypassing such protection for specific microcontrollers, but this often requires expensive equipment and/or exhaustive search of the fault parameters. When multiple glitches are required (e.g., when countermeasures are in place) this search becomes of exponential complexity and thus infeasible. Another challenge which makes embedded bootloaders notoriously hard to analyse is their lack of debugging capabilities.This paper proposes a grey-box approach that leverages binary analysis and advanced software exploitation techniques combined with voltage glitching to develop a powerful attack methodology against embedded bootloaders. We showcase our techniques with three real-world microcontrollers as case studies: 1) we combine static and on-chip dynamic analysis to enable a Return-Oriented Programming exploit on the bootloader of the NXP LPC microcontrollers; 2) we leverage on-chip dynamic analysis on the bootloader of the popular STM8 microcontrollers to constrain the glitch parameter search, achieving the first fully-documented multi-glitch attack on a real-world target; 3) we apply symbolic execution to precisely aim voltage glitches at target instructions based on the execution path in the bootloader of the Renesas 78K0 automotive microcontroller. For each case study, we show that using inexpensive, open-design equipment, we are able to efficiently breach the security of these microcontrollers and get full control of the protected memory, even when multiple glitches are required. Finally, we identify and elaborate on several vulnerable design patterns that should be avoided when implementing embedded bootloaders.

Fully integrated optofluidic SERS platform for real-time and continuous characterization of airborne microorganisms

An optofluidic, surface-enhanced Raman spectroscopy (SERS) platform was developed to detect airborne microorganisms, continuously and in real time. The platform consists of an on-chip analysis system integrated with an aerosol sampler and Raman spectrometer. A stratified two-phase flow, consisting of the sampled air stream and a stream of collection medium, is formed in the curved channel. The inertia of collected particles, such as bacterial cells, carries them across the phase boundary in the curved channel such that they impact the liquid stream directly. The collection efficiency of the microchannel was evaluated using different-sized standard polystyrene-latex particles. A collection efficiency of 99.6% was attained for particles with an average aerodynamic diameter of 1 μm, a typical size for bacterial aerosols, by optimizing the flow rates of the sample air and liquid medium. A silver colloid in the collection medium was used as the SERS adsorbent. After passing through a serpentine mixing channel, bacterial particles were detected by SERS in real time using the custom Raman spectroscopy system. The detection system was evaluated with five test bacteria: S. epidermidis, M. luteus, E. hirae, B. subtilis, and E. coli. The concentration of airborne S. epidermidis corresponded to a Raman peak at 732.5 cm−1. The limit of detection was approximately 102 CFU/mL and the total bacterial aerosol concentration was determined in real time based on the ratio of sampling air to SERS colloid.

Plasma Biomarker for Post-concussive Syndrome: A Pilot Study Using an Alternating Current Electro-Kinetic Platform

Background: Technology platforms that afford biomarker discovery in patients suffering from traumatic brain injury (TBI) remain an unmet medical need. Here, we describe an observational pilot study to explore the utility of an alternating current electrokinetic (ACE) microchip device in this context.Methods: Blood samples were collected from participating subjects with and without minor TBI. Plasma levels of glial fibrillary acidic protein (GFAP), Tau, ubiquitin C-terminal hydrolase L1 (UCH-L1), and cell-free DNA (cfDNA) were determined in subjects with and without minor TBI using ACE microchip device followed by on-chip immunofluorescent analysis. Post-concussive symptoms were assessed using the Rivermead Post Concussion Symptoms Questionnaire (RPCSQ) at one-month follow-up.Results: Highest levels of GFAP, UCH-L1, and Tau were seen in two minor TBI subjects with abnormality on head computed tomography (CT). In patients without abnormal head CT, Tau and GFAP levels discriminated between plasma from minor-TBI and non-TBI patients, with sensitivity and specificity of 64–72 and 50%, respectively. Plasma GFAP, UCH-L1, and Tau strongly correlated with the cumulative RPCSQ score. Plasma UCH-L1 and GFAP exhibited highest correlation to sensitivity to noise and light (r = 0.96 and 0.91, respectively, p < 0.001). Plasma UCH-L1 and Tau showed highest correlation with headache (r = 0.74 and 0.78, respectively, p < 0.001), sleep disturbance (r = 0.69 and 0.84, respectively, p < 0.001), and cognitive symptoms, including forgetfulness (r = 0.76 and 0.74, respectively, p < 0.001), poor concentration (r = 0.68 and 0.76, respectively, p < 0.001), and time required for information processing (r = 0.77 and 0.81, respectively, p < 0.001). cfDNA exhibited a strong correlation with depression (r = 0.79, p < 0.01) and dizziness (r = 0.69, p < 0.01). While cfDNA demonstrated positive correlation with dizziness and depression (r = 0.69 and 0.79, respectively, p < 0.001), no significant correlation was observed between cumulative RPCSQ and cfDNA (r = 0.07, p = 0.81).Conclusion: We provide proof-of-principle results supporting the utility of ACE microchip for plasma biomarker analysis in patients with minor TBI.

On-chip analysis of magnetically labeled cells with integrated cell sorting and counting techniques

In studies on cell therapies, cells often need to be magnetically labeled and then tracked using magnetic resonance imaging (MRI) techniques. To achieve good imaging performance on infused cells, the analysis of the sorted, labeled cells before infusion is necessary. Herein, we developed a microfluidic chip to quantitatively analyze magnetically labeled cells. The chip was equipped with a magnetophoresis-based cell sorting function and an impedance-based cell counting function. Using RAW264.7 macrophages, we confirmed the two functions of the chip, obtained the number and the magnetic loading distribution of the sorted, labeled cells, and ultimately demonstrated the broad applications of the chip in rapidly selecting a proper flow rate for the buffer solution in the cell sorting process, determining the total average magnetic loading of the labeled cells for the cell labeling process, and offering a necessary reference for the processing of the sorted cells for high performance in vivo imaging. This work provides an integrated lab-on-a-chip design for quantitatively analyzing magnetically labeled cells and thus can promote MRI-based cell-tracking studies.

Reconfigurable Integrated Optofluidic Droplet Laser Arrays.

Optofluidic lasers are an emerging technology for the development of miniaturized light sources and biological and chemical sensors. However, most optofluidic lasers demonstrated to date are operated at the single optical cavity level, which limits their applications in high-throughput biochemical sensing, high-speed wavelength switching, and on-chip spectroscopic analysis. Here, we demonstrated an optofluidic droplet laser array on a silicon chip with integrated microfluidics, in which four individual droplet optical cavities are generated and controlled by a 2 × 2 nozzle array. Arrays of droplets with a diameter ranging from 115 to 475 μm can be generated, removed, and regenerated on demand. The lasing threshold of the droplet laser array is in the range of 0.63-2.02 μJ/mm2. An image-based lasing threshold analysis method is developed, which enables simultaneous lasing threshold measurement for all laser units within the laser array using a low-cost camera. Compared to the conventional spectrum-based threshold analysis method, the lasing threshold obtained from the image-based method showed consistent results. Our droplet laser array is a promising technology in the development of cost-effective and integrated coherent light source on a chip for point-of-care applications.

Integrated Digital Microfluidic Platform for Colorimetric Sensing of Nitrite.

In this paper, a palm-size digital microfluidic (DMF) platform integrated with colorimetric analysis was developed for quantifying the concentration of nitrite. To realize the on-chip repeatable colorimetric analysis, a novel printed circuit board (PCB)-based DMF chip was designed with an embedded aperture on the actuator electrode, forming a vertical light path for online measurement of the droplets. The capabilities of the DMF platform enable automatic manipulation of microliter-level droplets to implement Griess assay without the use of external systems such as syringe, pump, or valve, which provides the benefits including high flexibility, portability, miniature size, and low cost. Results indicated the characteristics of good linearity (R2 = 0.9974), the ignorable crosstalk for reusability, and the limit of detection (LOD) of nitrite as low as 5 μg/L. Furthermore, the presented platform was successfully applied to determine nitrite levels in food products with reliable results and satisfactory recoveries. This integrated DMF platform can be a promising new tool for a wide range of applications involving step-by-step solution mixing and optical detection in environmental monitoring, food safety analysis, and point-of-care testing.

Diagnosis and prognosis of myocardial infarction on a plasmonic chip

Cardiovascular diseases lead to 31.5% of deaths globally, and particularly myocardial infarction (MI) results in 7.4 million deaths per year. Diagnosis of MI and monitoring for prognostic use are critical for clinical management and biomedical research, which require advanced tools with accuracy and speed. Herein, we developed a plasmonic gold nano-island (pGold) chip assay for diagnosis and monitoring of MI. On-chip microarray analysis of serum biomarkers (e.g., cardiac troponin I) afforded up to 130-fold enhancement of near-infrared fluorescence for ultra-sensitive and quantitative detection within controlled periods, using 10 μL of serum only. The pGold chip assay achieved MI diagnostic sensitivity of 100% and specificity of 95.54%, superior to the standard chemiluminescence immunoassay in cardiovascular clinics. Further, we monitored biomarker concentrations regarding percutaneous coronary intervention for prognostic purpose. Our work demonstrated a designed approach using plasmonic materials for enhanced diagnosis and monitoring for prognostic use towards point-of-care testing.

Investigation of Environmental Pollutant-Induced Lung Inflammation and Injury in a 3D Coculture-Based Microfluidic Pulmonary Alveolus System.

The health impact of environmental pollution involving an increase in human diseases has been subject to extensive study in recent decades. The methodology in biomimetic investigation of these pathophysiologic events is still in progress to uncover the gaps in knowledge associated with pollution and its influences on health. Herein, we describe a comprehensive evaluation of environmental pollutant-caused lung inflammation and injury using a microfluidic pulmonary alveolus platform with alveolar-capillary interfaces. We performed a microfluidic three-dimensional coculture with physiological microenvironment simulation at microscale control and demonstrated a reliable reconstruction of tissue layers including alveolar epithelium and microvascular endothelium with typical mechanical, structural, and junctional integrity, as well as viability. On-chip detection and analysis of pulmonary alveolus responses focusing on various inflammatory and injurious dynamics to the respective pollutant stimulations were achieved in the coculture-based microfluidic pulmonary alveolus model, in comparison with common on-chip monoculture and off-chip culture tools. We confirmed the synergistic effects of the epithelial and endothelial interfaces on the stimuli resistance and verified the importance of creating complex tissue microenvironments in vitro to explore pollution-involved human pathology. We believe the microfluidic approach presents great promise in environmental monitoring, drug discovery, and tissue engineering.

Dominant rule of community effect in synchronized beating behavior of cardiomyocyte networks.

Exploiting the combination of latest microfabrication technologies and single cell measurement technologies, we can measure the interactions of single cells, and cell networks from "algebraic" and "geometric" perspectives under the full control of their environments and interactions. However, the experimental constructive single cell-based approach still remains the limitations regarding the quality and condition control of those cells. To overcome these limitations, mathematical modeling is one of the most powerful complementary approaches. In this review, we first explain our on-chip experimental methods for constructive approach, and we introduce the results of the "community effect" of beating cardiomyocyte networks as an example of this approach. On-chip analysis revealed that (1) synchronized interbeat intervals (IBIs) of cell networks were followed to the more stable beating cells even their IBIs were slower than the other cells, which is against the conventional faster firing regulation or "overdrive suppression," and (2) fluctuation of IBIs of cardiomyocyte networks decreased according to the increase of the number of connected cells regardless of their geometry. The mathematical simulation of this synchronous behavior of cardiomyocyte networks also fitted well with the experimental results after incorporating the fluctuation-dissipation theorem into the oscillating stochastic phase model, in which the concept of spatially arranged cardiomyocyte networks was involved. The constructive experiments and mathematical modeling indicated the dominant rule of synchronization behavior of beating cardiomyocyte networks is a kind of stability-oriented synchronization phenomenon as the "community effect" or a fluctuation-dissipation phenomenon. Finally, as a practical application of this approach, the predictive cardiotoxicity is introduced.

Selective Retrieval of Individual Cells from Microfluidic Arrays Combining Dielectrophoretic Force and Directed Hydrodynamic Flow.

Hydrodynamic-based microfluidic platforms enable single-cell arraying and analysis over time. Despite the advantages of established microfluidic systems, long-term analysis and proliferation of cells selected in such devices require off-chip recovery of cells as well as an investigation of on-chip analysis on cell phenotype, requirements still largely unmet. Here, we introduce a device for single-cell isolation, selective retrieval and off-chip recovery. To this end, singularly addressable three-dimensional electrodes are embedded within a microfluidic channel, allowing the selective release of single cells from their trapping site through application of a negative dielectrophoretic (DEP) force. Selective capture and release are carried out in standard culture medium and cells can be subsequently mitigated towards a recovery well using micro-engineered hybrid SU-8/PDMS pneumatic valves. Importantly, transcriptional analysis of recovered cells revealed only marginal alteration of their molecular profile upon DEP application, underscored by minor transcriptional changes induced upon injection into the microfluidic device. Therefore, the established microfluidic system combining targeted DEP manipulation with downstream hydrodynamic coordination of single cells provides a powerful means to handle and manipulate individual cells within one device.

Automated on-chip analysis of tuberculosis drug-resistance mutation with integrated DNA ligation and amplification.

Detection of a single base mutation in Mycobacterium tuberculosis DNA can provide fast and highly specific diagnosis of antibiotic-resistant tuberculosis. Mutation-specific ligation of padlock probes (PLPs) on the target followed by rolling circle amplification (RCA) is highly specific, but challenging to integrate in a simple microfluidic device due to the low temperature stability of the phi29 polymerase and the interference of phi29 with the PLP annealing and ligation. Here, we utilized the higher operation temperature and temperature stability of Equiphi29 polymerase to simplify the integration of the PLP ligation and RCA steps of an RCA assay in two different strategies performed at uniform temperature. In strategy I, PLP annealing took place off-chip and the PLP ligation and RCA were performed in one pot and the two reactions were clocked by a change of the temperature. For a total assay time of about 1.5h, we obtained a limit of detection of 2pM. In strategy II, the DNA ligation mixture and the RCA mixture were separated into two chambers on a microfluidic disc. After on-disc PLP annealing and ligation, the disc was spun to mix reagents and initiate RCA. For a total assay time of about 2h, we obtained a limit of detection of 5pM. Graphical abstract.

Laser assisted microfluidic membrane evaporator for sample crystallization separation

Microfluidic devices for sample crystallization separation plays crucially important role in various on-chip analytical and testing scenes. Evaporation-based microfluidic crystallization technology is one of the most important methods for on-chip sample enrichment and crystallization separation. However, the solvent evaporation process in closed chip device is severely limited by poor vapor transportation, unstable interfacial behaviors and low evaporation rate. Aiming to address these issues, here we designed a microfluidic membrane evaporator for fast continuous flow sample crystallization separation. In this design, laser induced heating effect was employed to trigger the evaporation of the solvent, gas-permeable hydrophobic polytetrafluoroethylene membrane was used to stabilize the gas-liquid interface and provide vapor pathways during the evaporation process. Effects of laser power, solution flow rate and sample concentration on the crystallization separation performance was investigated. Results indicates the designed microfluidic membrane evaporator has good crystallization separation performance under different operation conditions, which can be further applied to different types of microfluidic systems and platforms for on-chip analysis and testing.

Microfluidic single-cell analysis-Toward integration and total on-chip analysis.

Various types of single-cell analyses are now extensively used to answer many biological questions, and with this growth in popularity, potential drawbacks to these methods are also becoming apparent. Depending on the specific application, workflows can be laborious, low throughput, and run the risk of contamination. Microfluidic designs, with their advantages of being high throughput, low in reaction volume, and compatible with bio-inert materials, have been widely used to improve single-cell workflows in all major stages of single-cell applications, from cell sorting to lysis, to sample processing and readout. Yet, designing an integrated microfluidic chip that encompasses the entire single-cell workflow from start to finish remains challenging. In this article, we review the current microfluidic approaches that cover different stages of processing in single-cell analysis and discuss the prospects and challenges of achieving a full integrated workflow to achieve total single-cell analysis in one device.

On chip optical tractor beam by surface plasmon polariton

Since the discovery, surface wave tractor beams have drawn less attention compared to free-propagating optical tractor beams. For Mie-range objects, this article stands for one of the very first proposals of on chip SPP (surface plasmon polariton) based optical tractor beam where two SPP waves are excited by using gratings made of grooves and meta-surfaces. Such beam can pull dielectric particles within certain size ranges when it is non-paraxial enough. Our SPP tractor beam set-up enables on chip object transportation and radius based optical sorting which could further facilitate the on-chip manipulation and analysis of nanoparticles and biomolecules.

Closed Bipolar Electrode Array for On-Chip Analysis of Cellular Respiration by Cell Aggregates

Cell aggregates have attracted much attention owing to their potential applications in tissue engineering and drug screening. To evaluate cellular respiration of individual cell aggregates in these applications, noninvasive and on-chip high-throughput analytical tools are necessary. Electrochemical methods for detecting oxygen concentrations are useful because of their noninvasiveness. However, these conventional methods may be unsuitable for high-throughput detection because it is difficult to prepare many electrodes on a small chip owing to the limitation of area for connecting electrodes. Alternatively, a bipolar electrode (BPE) system offers clear advantages. In this system, electrochemical reactions are induced at both ends of a BPE without complex wiring. In this study, we present a BPE array for detecting the respiratory activity of cell aggregates. Oxygen concentrations near cell aggregates at cathodic poles of BPEs were converted to electrochemiluminescence (ECL) signals of [Ru(bpy)3]2+/tripropylamine at anodic poles of BPEs. To separate ECL chemicals from cell aggregates, we fabricated a closed BPE device containing analytical and reporter chambers. As a proof of concept, 32 BPEs were controlled wirelessly using a pair of driving electrodes, and the respiratory activities of individual MCF-7 cell aggregates as a cancer model were successfully detected by monitoring ECL signals. Compared with conventional electrode arrays for cell analysis, the wiring of the current device was simple because the multiple BPEs functioned with only a single power supply. To the best of our knowledge, this is the first study of on-chip analysis of cellular activity using a BPE system.

Thyroid hormone activates Xenopus MBD3 gene via an intronic TRE in vivo.

Thyroid hormone (T3) is important for adult organ function and vertebrate development. Amphibian metamorphosis is totally dependent on T3 and can be easily manipulated, thus offering a unique opportunity for studying how T3 controls vertebrate development. T3 controls frog metamorphosis through T3 receptor (TR)-mediated regulation of T3 response genes. To identify direct T3 response genes, we previously carried out a ChIP (chromatin immunoprecipitation)-on-chip analysis with a polyclonal anti-TR antibody on the tadpole intestine and identified many putative TR target genes. Among them is the methyl-CpG binding domain protein 3 (MBD3) gene, which has been implicated to play a role in epigenetic regulation of cellular processes as a subunit of the Mi-2/NuRD(Nucleosome Remodeling Deacetylase)complex. We show here that MBD3 is upregulated in the intestine and tail by T3 and its expression peaks at stage 62, the climax of metamorphosis. We further show that a putative TRE within the first intron of the MBD3 gene binds to TR/RXR in vitro and in vivo, and mediates T3 regulation of the MBD3 promoter in vivo.

Direct loading of blood for plasma separation and diagnostic assays on a digital microfluidic device.

Finger-stick blood sampling is convenient for point of care diagnostics, but whole blood samples are problematic for many assays because of severe matrix effects associated with blood cells and cell debris. We introduce a new digital microfluidic (DMF) diagnostic platform with integrated porous membranes for blood-plasma separation from finger-stick blood volumes, capable of performing complex, multi-step, diagnostic assays. Importantly, the samples can be directly loaded onto the device by a finger "dab" for user-friendly operation. We characterize the platform by comparison to plasma generated via the "gold standard" centrifugation technique, and demonstrate a 21-step rubella virus (RV) IgG immunoassay yielding a detection limit of 1.9 IU mL-1, below the diagnostic cut-off. We propose that this work represents a critical next step in DMF based portable diagnostic assays-allowing the analysis of whole blood samples without pre-processing.

On-Chip DNA Analysis of Tuberculosis Based on Magnetic Nanoparticle Clustering Induced by Rolling Circle Amplification Products

We present on-chip implementation of an isothermal nucleic acid amplification assay with optomagnetic detection. Isothermal rolling circle amplification (RCA) was designed for the highly specific detection of a single point mutation in the subsequence of the katG catalase peroxidase gene responsible for resistance against the antibiotic isoniazid in Mycobacterium tuberculosis . The assay was integrated using streptavidin-coated magnetic microbeads (MMBs) as a movable substrate to transport the synthetic katG target and products between the sequential processing steps. RCA products were detected via optomagnetic measurements of the binding of functionalized magnetic nanoparticles (MNPs). Binding of MNPs to RCA products leads to an increase of their hydrodynamic size and consequently a shift of the optomagnetic signal to lower frequencies. We first analyzed the affinity of DNA probes used for target capture on MMBs and binding of MNPs to RCA products, respectively. This enabled us to choose conditions where amplification products were released from the MMBs and where their binding to MNPs was optimal. The optimized assay was transferred to a polymer chip with three connected chambers filled with reaction buffers for capture, RCA, and detection. The chip was operated in a setup integrating temperature control, transport of the DNA on magnetic carriers, and dose-dependent detection of MNP clustering. For a total assay time of about 2 h, we demonstrate specific detection of the antibiotic resistance in a synthetic tuberculosis DNA target with a detection limit of 4 pM.

On-Chip CMOS Self-Decoupling Battery Cell System for Security Protection

This article presents an effective on-chip power analysis attack countermeasure based on a new CMOS self-decoupling battery cell system that uses a self-decoupling circuit. The self-decoupling circuit dynamically controls an on-chip virtual power supply point, $V_{\mathrm {ddv}}$ , that can be used to power security-sensitive modules. The circuit automatically decouples an on-chip CMOS battery cell from powering a sensitive module when its voltage level reaches a designed minimum threshold level $V_{\mathrm {dd-min}}$ and connects it for a very short charging cycle to the chip’s main voltage supply, $V_{\mathrm {dd}}$ . The charging cycles for the experiments presented in this article are less than 10 ns and are designed to support the CMOS battery cell size and the minimum designed threshold voltage level $V_{\mathrm {dd-min}}$ . Simulation results of test designs implemented in the 45-nm CMOS technology process show that the proposed countermeasure is efficient when used with battery cell sizes that can power the protected cryptographic module for more than ten data operation cycles before recharging. In addition, using the on-chip self-decoupling battery cell system allows for power consumption savings within the protected module of up to 43 % due to the dynamic voltage scaling generated at the virtual power supply point.