Emerging Biosensing Technologies for the Diagnostics of Viral Infectious Diseases.

Several viral infectious diseases appear limitless since the beginning of the 21st century, expanding into pandemic lengths. Thus, there are extensive efforts to provide more efficient means of diagnosis, a better understanding of acquired immunity, and improved monitoring of inflammatory biomarkers, as these are all crucial for controlling the spread of infection while aiding in vaccine development and improving patient outcomes. In this regard, various biosensors have been developed recently to streamline pathogen and immune response detection by addressing the limitations of traditional methods, including isothermal amplification-based systems and lateral flow assays. This review explores state-of-the-art biosensors for detecting viral pathogens, serological assays, and inflammatory biomarkers from the material perspective, by discussing their advantages, limitations, and further potential regarding their analytical performance, clinical utility, and point-of-care adaptability. Additionally, next-generation biosensing technologies that offer better sensitivity and selectivity, and easy handling for end-users are highlighted. An emerging example of these next-generation biosensors are those powered by novel synthetic biology tools, such as clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated proteins (Cas), in combination with integrated point-of-care devices. Lastly, the current challenges are discussed and a roadmap for furthering these advanced biosensing technologies to manage future pandemics is provided.

The promise of graphene-based transistors for democratizing multiomics studies.

As biological research has synthesized genomics, proteomics, metabolomics, and transcriptomics into systems biology, a new multiomics approach to biological research has emerged. Today, multiomics studies are challenging and expensive. An experimental platform that could unify the multiple omics approaches to measurement could increase access to multiomics data by enabling more individual labs to successfully attempt multiomics studies. Field effect biosensing based on graphene transistors have gained significant attention as a potential unifying technology for such multiomics studies. This review article highlights the outstanding performance characteristics that makes graphene field effect transistor an attractive sensing platform for a wide variety of analytes important to system biology. In addition to many studies demonstrating the biosensing capabilities of graphene field effect transistors, they are uniquely suited to address the challenges of multiomics studies by providing an integrative multiplex platform for large scale manufacturing using the well-established processes of semiconductor industry. Furthermore, the resulting digital data is readily analyzable by machine learning to derive actionable biological insight to address the challenge of data compatibility for multiomics studies. A critical stage of systems biology will be democratizing multiomics study, and the graphene field effect transistor is uniquely positioned to serve as an accessible multiomics platform.

Erythrocytes, a New Contributor to Age-Associated Loss of Blood-Brain Barrier Integrity.

Blood exchanges between young and old partners demonstrate old blood has a detrimental effect on brain health of young animals. Previous studies primarily investigate soluble blood factors, such as transforming growth factor-beta, on the brain and the blood-brain barrier (BBB). However, the role of blood cellular components, particularly erythrocytes, has not been defined. Erythrocyte morphology and rigidity change as mammals age, altering their transport within the capillary bed. This impacts downstream biological events, such as the release of reactive oxygen species and hemoglobin, potentially compromising the BBB. Here, a micro electrical BBB (µE-BBB), with cocultured endothelial and astrocytic cells, and a built-in trans-endothelial electrical resistance (TEER) system is described to monitor the effect of capillary shear stress on erythrocytes derived from young and old mice and people and the subsequent effects of these cells on BBB integrity. This is monitored by the passage of fluorescein isothiocyanate-dextran and real-time profiling of TEER across the BBB after old and young erythrocyte exposure. Compared to young erythrocytes, old erythrocytes induce an increased permeability by 42% and diminished TEER by 2.9% of the µE-BBB. These results suggest that changes in circulating erythrocytes are a biomarker of aging in the context of BBB integrity.

Discrimination of single-point mutations in unamplified genomic DNA via Cas9 immobilized on a graphene field-effect transistor.

Simple and fast methods for the detection of target genes with single-nucleotide specificity could open up genetic research and diagnostics beyond laboratory settings. We recently reported a biosensor for the electronic detection of unamplified target genes using liquid-gated graphene field-effect transistors employing an RNA-guided catalytically deactivated CRISPR-associated protein 9 (Cas9) anchored to a graphene monolayer. Here, using unamplified genomic samples from patients and by measuring multiple types of electrical response, we show that the biosensors can discriminate within one hour between wild-type and homozygous mutant alleles differing by a single nucleotide. We also show that biosensors using a guide RNA-Cas9 orthologue complex targeting genes within the protospacer-adjacent motif discriminated between homozygous and heterozygous DNA samples from patients with sickle cell disease, and that the biosensors can also be used to rapidly screen for guide RNA-Cas9 complexes that maximize gene-targeting efficiency.

Integrated nucleic acid testing system to enable TB diagnosis in peripheral settings.

To facilitate treatment and limit transmission of tuberculosis (TB), new methods are needed to enable rapid and affordable diagnosis of the disease in high-burden low-resource settings. We have developed a prototype integrated nucleic acid testing device to detect Mycobacterium tuberculosis (M.tb) in sputum. The device consists of a disposable cartridge and compact, inexpensive instrument that automates pathogen lysis, nucleic acid extraction, isothermal DNA amplification and lateral flow detection. A liquefied and disinfected sputum sample is manually injected into the cartridge, and all other steps are automated, with a result provided in <1.5 h. Cell disruption and DNA extraction is executed within a four-port active valve containing a miniature bead blender (based on PureLyse® technology, Claremont BioSolutions LLC). The DNA-containing eluate is combined with dry master-mix reagents and target DNA is isothermally amplified. Amplified master-mix is then pumped into a lateral flow strip chamber for detection. The entire process is performed in a single-use closed-system cartridge to prevent amplicon carryover. For testing of M.tb-spiked sputum the system provided a limit of detection of 5 × 103 colony forming units (CFU) per mL. None of the negative sputum-only controls yielded a false-positive result. Testing of 45 clinical sputum specimens from TB cases and controls relative to a validated manual qPCR-based comparator method revealed a preliminary sensitivity of 90% and specificity of 96%. With further development, the herein described integrated nucleic acid testing device can enable TB diagnosis and treatment initiation in the same clinical encounter in near-patient low-resource settings of high TB burden countries.

Use of the Kalman Filter for Aortic Pressure Waveform Noise Reduction.

Clinical applications that require extraction and interpretation of physiological signals or waveforms are susceptible to corruption by noise or artifacts. Real-time hemodynamic monitoring systems are important for clinicians to assess the hemodynamic stability of surgical or intensive care patients by interpreting hemodynamic parameters generated by an analysis of aortic blood pressure (ABP) waveform measurements. Since hemodynamic parameter estimation algorithms often detect events and features from measured ABP waveforms to generate hemodynamic parameters, noise and artifacts integrated into ABP waveforms can severely distort the interpretation of hemodynamic parameters by hemodynamic algorithms. In this article, we propose the use of the Kalman filter and the 4-element Windkessel model with static parameters, arterial compliance C, peripheral resistance R, aortic impedance r, and the inertia of blood L, to represent aortic circulation for generating accurate estimations of ABP waveforms through noise and artifact reduction. Results show the Kalman filter could very effectively eliminate noise and generate a good estimation from the noisy ABP waveform based on the past state history. The power spectrum of the measured ABP waveform and the synthesized ABP waveform shows two similar harmonic frequencies.

Simple system for isothermal DNA amplification coupled to lateral flow detection.

Infectious disease diagnosis in point-of-care settings can be greatly improved through integrated, automated nucleic acid testing devices. We have developed an early prototype for a low-cost system which executes isothermal DNA amplification coupled to nucleic acid lateral flow (NALF) detection in a mesofluidic cartridge attached to a portable instrument. Fluid handling inside the cartridge is facilitated through one-way passive valves, flexible pouches, and electrolysis-driven pumps, which promotes a compact and inexpensive instrument design. The closed-system disposable prevents workspace amplicon contamination. The cartridge design is based on standard scalable manufacturing techniques such as injection molding. Nucleic acid amplification occurs in a two-layer pouch that enables efficient heat transfer. We have demonstrated as proof of principle the amplification and detection of Mycobacterium tuberculosis (M.tb) genomic DNA in the cartridge, using either Loop Mediated Amplification (LAMP) or the Exponential Amplification Reaction (EXPAR), both coupled to NALF detection. We envision that a refined version of this cartridge, including upstream sample preparation coupled to amplification and detection, will enable fully-automated sample-in to answer-out infectious disease diagnosis in primary care settings of low-resource countries with high disease burden.

Disposable Miniature Check Valve Design Suitable for Scalable Manufacturing.

We present a passive, miniature check valve which can be manufactured using standard techniques ideal for low-cost, disposable systems used in medical devices and other applications. The body of the valve consists of a hollow cylindrical core, closed at one end, with a side port and a cylindrical elastomeric sleeve placed over the core body, covering the side port. The pressure required for initial opening of the valve, referred to as cracking pressure, can be adjusted, and depends predominantly on the valve core outer diameter, the sleeve inner diameter, the sleeve wall thickness, and the sleeve material's modulus of elasticity. These parameters can be controlled to tight tolerances, while the tolerances on other features can be relaxed, which simplifies valve manufacturing and assembly. Valve embodiments produced from different materials, and with varying critical dimensions, exhibited distinct and reproducible cracking pressures in the range of 2 to 20 PSI. The cracking pressure did not vary significantly as a function of flow rate. No back flow leakage was encountered up to 30 PSI, the pressure limit of the sensor used in this experiment. Most of the valves tested had small internal volumes of 3-4 µL. The internal volume can be further reduced by selecting a core of smaller inner diameter. In contrast to lithography-based microvalves that generally must be manufactured in-situ within the fluidic device, the herein presented valve can be manufactured independently of, and can be readily integrated into fluidic systems manufactured via a wide selection of fabrication methods.

A study of EWOD-driven droplets by PIV investigation.

Despite the recent interest in droplet-based microfluidics using electrowetting-on-dielectric (EWOD), fundamental understanding of the fluid dynamics remains limited to two-dimensional (2D) reduction of the Navier-Stokes equation. Experimental data are in dire need to verify the predictions and advance the field. We report an investigation of the flow inside droplets actuated by EWOD in air using micro particle image velocimetry (micro-PIV). Using the continuity equation, we reconstruct the 3D velocity field from the 2D PIV experimental data. We present some fundamental findings and build valuable insights that will help design sophisticated EWOD microfluidic devices. For example, the results confirm that efficient mixing in a droplet may be achieved by moving the droplet along an irreversible pattern that breaks the symmetry of the two circulating inner flows.