Applications of Graphene Field Effect Biosensors for Biological Sensing.

This chapter provides a comprehensive overview of the principles, applications, and advancements in graphene field-effect transistor (gFET) biosensors for biological sensing. The unique properties of graphene that make it ideal for biosensing, including its high conductivity, chemical stability, and ability to facilitate label-free detection, will be discussed. The chapter also explores various applications of gFET biosensors, from detecting pH and salinity changes to complex protein-protein interactions and DNA/RNA sensing. It also addresses the challenges and future directions in gFET biosensor technology, emphasizing the need for scalable manufacturing, sophisticated surface chemistry, and the integration of multiomics approaches to enhance biosensing capabilities.

CRISPR-Cas-Based Biomonitoring for Marine Environments: Toward CRISPR RNA Design Optimization Via Deep Learning.

Almost all of Earth's oceans are now impacted by multiple anthropogenic stressors, including the spread of nonindigenous species, harmful algal blooms, and pathogens. Early detection is critical to manage these stressors effectively and to protect marine systems and the ecosystem services they provide. Molecular tools have emerged as a promising solution for marine biomonitoring. One of the latest advancements involves utilizing CRISPR-Cas technology to build programmable, rapid, ultrasensitive, and specific diagnostics. CRISPR-based diagnostics (CRISPR-Dx) has the potential to allow robust, reliable, and cost-effective biomonitoring in near real time. However, several challenges must be overcome before CRISPR-Dx can be established as a mainstream tool for marine biomonitoring. A critical unmet challenge is the need to design, optimize, and experimentally validate CRISPR-Dx assays. Artificial intelligence has recently been presented as a potential approach to tackle this challenge. This perspective synthesizes recent advances in CRISPR-Dx and machine learning modeling approaches, showcasing CRISPR-Dx potential to progress as a rising molecular tool candidate for marine biomonitoring applications.

Development of Single Molecule Techniques for Sensing and Manipulation of CRISPR and Polymerase Enzymes.

Clustered regularly interspaced short palindromic repeats (CRISPR) and polymerases are powerful enzymes and their diverse applications in genomics, proteomics, and transcriptomics have revolutionized the biotechnology industry today. CRISPR has been widely adopted for genomic editing applications and Polymerases can efficiently amplify genomic transcripts via polymerase chain reaction (PCR). Further investigations into these enzymes can reveal specific details about their mechanisms that greatly expand their use. Single-molecule techniques are an effective way to probe enzymatic mechanisms because they may resolve intermediary conformations and states with greater detail than ensemble or bulk biosensing techniques. This review discusses various techniques for sensing and manipulation of single biomolecules that can help facilitate and expedite these discoveries. Each platform is categorized as optical, mechanical, or electronic. The methods, operating principles, outputs, and utility of each technique are briefly introduced, followed by a discussion of their applications to monitor and control CRISPR and Polymerases at the single molecule level, and closing with a brief overview of their limitations and future prospects.

Small-animal blood exchange is an emerging approach for systemic aging research.

We describe a small-animal blood exchange approach developed for aging research as an alternative to heterochronic parabiosis or plasma injections. In parabiosis, animals are surgically coupled, which has several disadvantages, including difficulty controlling experimental procedure, the effects of shared organs, environmental enrichment from jointly exploring the housing enclosure, involuntary exercise and an imprecise onset of blood sharing. Likewise, in plasma injections, the added volumes need to be small, and there is little flexibility in changing the relative contributions of ectopic to endogenous blood components. These factors complicate the conclusions and interpretations, including the identification of key mechanisms and molecular or cellular determinants. Our approach, where blood is exchanged between animals without them being surgically coupled, is less invasive than parabiosis. The percentage of exchanged blood or other exchanged fluids is known and precise. The age of plasma and cells can be mixed and matched at all desired relative contributions to the endogenous systemic milieu, and the onset of the effects can be accurately delineated. In this protocol, we describe the preparatory and animal surgery steps required for small-animal blood exchange in mice and compare this process with parabiosis and plasma injections. We also provide the design, hardware and software for the blood exchange device and compare automated and manual exchange methods. Lastly, we report mathematical modeling of the dilution of blood factors. The fluid exchange takes ~30 min when performed by a well-trained biomedical scientist; the entire process takes ~2 h.

Mechanisms and Minimization of False Discovery of Metabolic Bioorthogonal Noncanonical Amino Acid Proteomics.

Metabolic proteomics has been widely used to characterize dynamic protein networks in many areas of biomedicine, including in the arena of tissue aging and rejuvenation. Bioorthogonal noncanonical amino acid tagging (BONCAT) is based on mutant methionine-tRNA synthases (MetRS) that incorporates metabolic tags, for example, azidonorleucine [ANL], into newly synthesized proteins. BONCAT revolutionizes metabolic proteomics, because mutant MetRS transgene allows one to identify cell type-specific proteomes in mixed biological environments. This is not possible with other methods, such as stable isotope labeling with amino acids in cell culture, isobaric tags for relative and absolute quantitation and tandem mass tags. At the same time, an inherent weakness of BONCAT is that after click chemistry-based enrichment, all identified proteins are assumed to have been metabolically tagged, but there is no confirmation in mass spectrometry data that only tagged proteins are detected. As we show here, such assumption is incorrect and accurate negative controls uncover a surprisingly high degree of false positives in BONCAT proteomics. We show not only how to reveal the false discovery and thus improve the accuracy of the analyses and conclusions but also approaches for avoiding it through minimizing nonspecific detection of biotin, biotin-independent direct detection of metabolic tags, and improvement of signal to noise ratio through machine learning algorithms.

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.

Diversification of the CRISPR Toolbox: Applications of CRISPR-Cas Systems Beyond Genome Editing.

The discovery of CRISPR has revolutionized the field of genome engineering, but the potential of this technology is far from reaching its limits. In this review, we explore the broad range of applications of CRISPR technology to highlight the rapid expansion of the field beyond gene editing alone. It has been demonstrated that CRISPR technology can control gene expression, spatiotemporally image the genome in vivo, and detect specific nucleic acid sequences for diagnostics. In addition, new technologies are under development to improve CRISPR quality controls for gene editing, thereby improving the reliability of these technologies for therapeutics and beyond. These are just some of the many CRISPR tools that have been developed in recent years, and the toolbox continues to diversify.

Rapid and Electronic Identification and Quantification of Age-Specific Circulating Exosomes via Biologically Activated Graphene Transistors.

Increasing access to modern clinical practices concomitantly extends lifespan, ironically revealing new classes of degenerative and inflammatory diseases of later years. Here, an electronic graphene field-effect transistor (gFET) is reported, termed EV-chip, for label-free, rapid identification and quantification of exosomes (EV) associated with aging through specific surface markers, CD63 and CD151. Studies suggest that blood-derived exosomes carry specific biomolecules that can be used toward diagnostic applications of age and health. However, to observe improvements in patient outcomes, earlier detection at the point-of-care (POC) is required. Unfortunately, conventional techniques and other electronic-based platforms for exosome sensing are burdensome and inept for the POC distinction of aged blood factors. It is shown that EV-chip can quantitatively detect purified exosomes from plasma, with a limit of detection (LOD) of 2 × 104 particles mL-1 and a limit of quantification (LOQ) of 6 × 104 particles mL-1 . The sensitivity and compact electronics of the EV-chip improves upon previously published electronic biosensors, making it ideal for a physician's office or a simple biological laboratory. The sensitivity, selectivity, and portability of the EV-chip demonstrate the potential of the biosensor as a powerful point-of-care diagnostic and prognostic tool for age-related diseases.

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.

The Microfluidic Toolbox for Analyzing Exosome Biomarkers of Aging.

As the fields of aging and neurological disease expand to liquid biopsies, there is a need to identify informative biomarkers for the diagnosis of neurodegeneration and other age-related disorders such as cancers. A means of high-throughput screening of biomolecules relevant to aging can facilitate this discovery in complex biofluids, such as blood. Exosomes, the smallest of extracellular vesicles, are found in many biofluids and, in recent years, have been found to be excellent candidates as liquid biopsy biomarkers due to their participation in intercellular communication and various pathologies such as cancer metastasis. Recently, exosomes have emerged as novel biomarkers for age-related diseases. Hence, the study of exosomes, their protein and genetic cargo can serve as early biomarkers for age-associated pathologies, especially neurodegenerative diseases. However, a disadvantage of exosome studies includes a lack in standardization of isolating, detecting, and profiling exosomes for downstream analysis. In this review, we will address current techniques for high-throughput isolation and detection of exosomes through various microfluidic and biosensing strategies and how they may be adapted for the detection of biomarkers of age-associated disorders.

Structural determination of Enzyme-Graphene Nanocomposite Sensor Material.

State-of-the-art ultra-sensitive blood glucose-monitoring biosensors, based on glucose oxidase (GOx) covalently linked to a single layer graphene (SLG), will be a valuable next generation diagnostic tool for personal glycemic level management. We report here our observations of sensor matrix structure obtained using a multi-physics approach towards analysis of small-angle neutron scattering (SANS) on graphene-based biosensor functionalized with GOx under different pH conditions for various hierarchical GOx assemblies within SLG. We developed a methodology to separately extract the average shape of GOx molecules within the hierarchical assemblies. The modeling is able to resolve differences in the average GOx dimer structure and shows that treatment under different pH conditions lead to differences within the GOx at the dimer contact region with SLG. The coupling of different analysis methods and modeling approaches we developed in this study provides a universal approach to obtain detailed structural quantifications, for establishing robust structure-property relationships. This is an essential step to obtain an insight into the structure and function of the GOx-SLG interface for optimizing sensor performance.

Detection of unamplified target genes via CRISPR-Cas9 immobilized on a graphene field-effect transistor.

Most methods for the detection of nucleic acids require many reagents and expensive and bulky instrumentation. Here, we report the development and testing of a graphene-based field-effect transistor that uses clustered regularly interspaced short palindromic repeats (CRISPR) technology to enable the digital detection of a target sequence within intact genomic material. Termed CRISPR-Chip, the biosensor uses the gene-targeting capacity of catalytically deactivated CRISPR-associated protein 9 (Cas9) complexed with a specific single-guide RNA and immobilized on the transistor to yield a label-free nucleic-acid-testing device whose output signal can be measured with a simple handheld reader. We used CRISPR-Chip to analyse DNA samples collected from HEK293T cell lines expressing blue fluorescent protein, and clinical samples of DNA with two distinct mutations at exons commonly deleted in individuals with Duchenne muscular dystrophy. In the presence of genomic DNA containing the target gene, CRISPR-Chip generates, within 15 min, with a sensitivity of 1.7 fM and without the need for amplification, a significant enhancement in output signal relative to samples lacking the target sequence. CRISPR-Chip expands the applications of CRISPR-Cas9 technology to the on-chip electrical detection of nucleic acids.

Graphene-based biosensor for on-chip detection of bio-orthogonally labeled proteins to identify the circulating biomarkers of aging during heterochronic parabiosis.

Studies of heterochronic parabiosis, where two animals of different ages are joined surgically, provided proof-of-principle results that systemic proteins have broad age-specific effects on tissue health and repair. In an effort to identify these systemic proteins, we previously developed a method to selectively label the proteome of only one animal joined in parabiosis utilizing bio-orthogonal non-canonical amino acid tagging (BONCAT), which can metabolically label proteins during their de novo synthesis by incorporating a methionine substitute, azido-nor-leucine (ANL), in cells expressing a mutant methionyl-tRNA synthetase (MetRSL274G). Once labeled, we can selectively identify the proteins produced by the MetRSL274G transgenic mouse in the setting of heterochronic parabiosis. This approach enabled the detection of several rejuvenating protein candidates from the young parabiont, which were transferred to the old mammalian tissue through their shared circulation. Although BONCAT is a very powerful technology, the challenges associated with its complexity including large starting material requirements and cost of ANL-labeled protein detection, such as modified antibody arrays and mass spectrometry, limit its application. Herein, we propose a lab-on-a-chip technology, termed Click-A+Chip for facile and rapid digital detection of ANL-labeled proteomes present in minute amount of sample, to replace conventional assays. Click-A+Chip is a graphene-based field effect biosensor (gFEB) which utilizes novel on-chip click-chemistry to specifically bind to ANL-labeled biomolecules. In this study, Click-A+Chip is utilized for the capture of ANL-labeled proteins transferred from young to old parabiotic mouse partners. Moreover, we were able to identify the young-derived ANL-labeled Lif-1 and leptin in parabiotic systemic milieu, confirming previous data as well as providing novel findings on the relative levels of these factors in young versus old parabionts. Summarily, our results demonstrate that Click-A+Chip can be used for rapid detection and identification of ANL-labeled proteins, significantly reducing the sample size, complexity, cost and time associated with BONCAT analysis.

An oral microjet vaccination system elicits antibody production in rabbits.

Noninvasive immunization technologies have the potential to revolutionize global health by providing easy-to-administer vaccines at low cost, enabling mass immunizations during pandemics. Existing technologies such as transdermal microneedles are costly, deliver drugs slowly, and cannot generate mucosal immunity, which is important for optimal immunity against pathogens. We present a needle-free microjet immunization device termed MucoJet, which is a three-dimensional microelectromechanical systems-based drug delivery technology. MucoJet is administered orally, placed adjacent to the buccal tissue within the oral cavity, and uses a self-contained gas-generating chemical reaction within its two-compartment plastic housing to produce a high-pressure liquid jet of vaccine. We show that the vaccine jet ejected from the MucoJet device is capable of penetrating the buccal mucosal layer in silico, in porcine buccal tissue ex vivo, and in rabbits in vivo. Rabbits treated with ovalbumin by MucoJet delivery have antibody titers of anti-ovalbumin immunoglobulins G and A in blood serum and buccal tissue, respectively, that are three orders of magnitude higher than rabbits receiving free ovalbumin delivered topically by a dropper in the buccal region. MucoJet has the potential to accelerate the development of noninvasive oral vaccines, given its ability to elicit antibody production that is detectable locally in the buccal tissue and systemically via the circulation.

In vivo delivery of transcription factors with multifunctional oligonucleotides.

Therapeutics based on transcription factors have the potential to revolutionize medicine but have had limited clinical success as a consequence of delivery problems. The delivery of transcription factors is challenging because it requires the development of a delivery vehicle that can complex transcription factors, target cells and stimulate endosomal disruption, with minimal toxicity. Here, we present a multifunctional oligonucleotide, termed DARTs (DNA assembled recombinant transcription factors), which can deliver transcription factors with high efficiency in vivo. DARTs are composed of an oligonucleotide that contains a transcription-factor-binding sequence and hydrophobic membrane-disruptive chains that are masked by acid-cleavable galactose residues. DARTs have a unique molecular architecture, which allows them to bind transcription factors, trigger endocytosis in hepatocytes, and stimulate endosomal disruption. The DARTs have enhanced uptake in hepatocytes as a result of their galactose residues and can disrupt endosomes efficiently with minimal toxicity, because unmasking of their hydrophobic domains selectively occurs in the acidic environment of the endosome. We show that DARTs can deliver the transcription factor nuclear erythroid 2-related factor 2 (Nrf2) to the liver, catalyse the transcription of Nrf2 downstream genes, and rescue mice from acetaminophen-induced liver injury.

Stimuli-responsive electrodes detect oxidative stress and liver injury.

A digital point-of-care biosensor for measuring reactive oxygen species is presented based on novel reactive oxygen species responsive polymer-based electrodes. The biosensor is able to detect a drug-induced liver injury by monitoring the oxidative stress in the blood.

Continuous monitoring of inflammation biomarkers during simulated cardiopulmonary bypass using a microfluidic immunoassay device - a pilot study.

This work demonstrates the use of a continuous online monitoring system for tracking systemic inflammation biomarkers during cardiopulmonary bypass (CPB) procedures. The ability to monitor inflammation biomarkers during CPB will allow surgical teams to actively treat inflammation and reduce harmful effects on postoperative morbidity and mortality, enabling improved patient outcomes. A microfluidic device has been designed which allows automation of the individual processing steps of a microbead immunoassay to allow continuous tracking of antigen concentrations. Preliminary experiments have demonstrated that the results produced by the microimmunoassay are comparable to results produced from a standard enzyme-linked immunosorbent assay (r = 0.98). Additionally, integration of the assay with a simulated CPB circuit has been demonstrated with temporal tracking of C3a concentrations within blood continuously sampled from the circuit. The presented work describes the motivation, design challenges, and preliminary experimental results of this project.

Microfiltration platform for continuous blood plasma protein extraction from whole blood during cardiac surgery.

This report describes the design, fabrication, and testing of a cross-flow filtration microdevice, for the continuous extraction of blood plasma from a circulating whole blood sample in a clinically relevant environment to assist in continuous monitoring of a patient's inflammatory response during cardiac surgeries involving cardiopulmonary bypass (CPB) procedures (about 400,000 adult and 20,000 pediatric patients in the United States per year). The microfiltration system consists of a two-compartment mass exchanger with two aligned sets of PDMS microchannels, separated by a porous polycarbonate (PCTE) membrane. Using this microdevice, blood plasma has been continuously separated from blood cells in a real-time manner with no evidence of bio-fouling or cell lysis. The technology is designed to continuously extract plasma containing diagnostic plasma proteins such as complements and cytokines using a significantly smaller blood volume as compared to traditional blood collection techniques. The microfiltration device has been tested using a simulated CPB circulation loop primed with donor human blood, in a manner identical to a clinical surgical setup, to collect plasma fractions in order to study the effects of CPB system components and circulation on immune activation during extracorporeal circulatory support. The microdevice, with 200 nm membrane pore size, was connected to a simulated CPB circuit, and was able to continuously extract ~15% pure plasma volume (100% cell-free) with high sampling frequencies which could be analyzed directly following collection with no need to further centrifuge or modify the fraction. Less than 2.5 ml total plasma volume was collected over a 4 h sampling period (less than one Vacutainer blood collection tube volume). The results tracked cytokine concentrations collected from both the reservoir and filtrate samples which were comparable to those from direct blood draws, indicating very high protein recovery of the microdevice. Additionally, the cytokine concentration increased significantly compared to baseline values over the circulation time for all cytokines analyzed. The high plasma protein recovery (over 80%), no indication of hemolysis and low level of biofouling on the membrane surface during the experimental period (over 4 h) were all indications of effective and reliable device performance for future clinical applications. The simple and robust design and operation of these devices allow operation over a wide range of experimental flow conditions and blood hematocrit levels to allow surgeons and clinicians autonomous usage in a clinical environment to better understand the mechanisms of injury resulting from cardiac surgery, and allow early interventions in patients with excessive postoperative complications to improve surgical outcomes. Ultimately, monolithic integration of this microfiltration device with a continuous microimmunoassay would create an integrated microanalysis system for tracking inflammation biomarkers concentrations in patients for point-of-care diagnostics, reducing blood analysis times, costs and volume of blood samples required for repeated assays.