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A Supported Lipid Bilayer-Based Lab-on-a-Chip Biosensor for the Rapid Electrical Screening of Coronavirus Drugs
Abstract
With the rapid spread and multigeneration variation of coronavirus, rapid drug development has become imperative. A major obstacle to addressing this issue is adequately constructing the cell membrane at the molecular level, which enables in vitro observation of the cell response to virus and drug molecules quantitatively, shortening the drug experiment cycle. Herein, we propose a rapid and label-free supported lipid bilayer-based lab-on-a-chip biosensor for the screening of effective inhibition drugs. An extended gate electrode was prepared and functionalized by an angiotensin-converting enzyme II (ACE2) receptor-incorporated supported lipid bilayer (SLB). Such an integrated system can convert the interactions of targets and membrane receptors into real-time charge signals. The platform can simulate the cell membrane microenvironment in vitro and accurately capture the interaction signal between the target and the cell membrane with minimized interference, thus observing the drug action pathway quantitatively and realizing drug screening effectively. Due to these label-free, low-cost, convenient, and integrated advantages, it is a suitable candidate method for the rapid drug screening for the early treatment and prevention of worldwide spread of coronavirus.
... For example, SLBs have been successfully formed on bioelectronic devices, such as electrodes or transistors, and sometimes use conducting polymers as the underlying substrates. These SLB-based biosensing devices have been applied across a broad spectrum of applications, ranging from virus detection and screening [20,21] to the identification of toxins and pathogens in food [22,23]. ...
... The modification of transistor surfaces with SLBs is not only advantageous for detecting clinically significant biomarkers but also serves as a preliminary platform for characterizing a drug's effectiveness. In a recent study [20], an SLB-based lab-on-a-chip biosensor was developed for the rapid screening of drugs targeting coronavirus infections (Fig. 7a). The authors fabricated an extended-gate electrode functionalized with an angiotensin-converting enzyme II (ACE2) receptor-incorporated SLB to mimic the cell membrane environment for detecting viral interactions. ...
... The device also analyzed the effects of pore-forming peptides, such as gramicidin A, highlighting its capacity to detect fine ionic Fig. 7 a Schematic illustration of the spike protein/drug binding and detection process (upper) and the response of the drug inhibition efficiency of the interaction between coronavirus and ACE2 receptors in the presence of different drugs, weak inhibition by the hexapeptide, and strong inhibition by the HD5 peptide (lower). Reproduced with permission [20]. Copyright 2022, American Chemical Society. ...
Transistor-based platforms offer several advantages for chemical and biological sensing application over conventional electrochemical systems, including enhanced sensitivity, portability, cost-effectiveness, and biocompatibility. However, these devices often require functionalization with specific recognition units, introducing challenges related to the chemical stability of conjugated units, their conformation, and Debye length effects. Lipid-based biomembranes, particularly supported lipid bilayers (SLBs), can mimic the native architecture of cell membranes, acting as biointerfaces that facilitate signal transduction between extra- and intracellular environments. They also provide selective permeability to ions, specificity to biochemicals, as well as ease of integration with diverse materials. Over the past two decades, researchers have focused on integrating biomembranes with transistor platforms to advance bioelectronic sensing technologies and enhance the understanding and monitoring of biological processes. This review explores integrating various lipid-based biomembrane types with transistor-based devices. We review fundamental techniques for producing and characterizing biomembranes, the advantages and limitations of different transistor types, and their working principles in biomembrane-based systems. Additionally, we highlight recent developments in biomembrane-integrated sensing platforms, including their incorporation into transistor architectures, further functionalization with biorecognition units, and applications in detecting analytes.
... The detection results showed that the presence of two different drugs had an effect on the interaction between coronavirus and the ACE2 receptor, with weak inhibition by hexapeptide and strong inhibition by HD5 peptide. The integrated system could translate the interaction between biological target analytes and receptors into real-time charge signal, so as to realize effective screening of therapeutic drugs [89]. Hajian et al. prepared CRISPR-Chip by modifying graphene surface with CRISPR-Cas9 complex. ...
... the ACE2 receptor, with weak inhibition by hexapeptide and strong inhibition by HD5 peptide. The integrated system could translate the interaction between biological target analytes and receptors into real−time charge signal, so as to realize effective screening of therapeutic drugs [89]. Hajian et al. prepared CRISPR-Chip by modifying graphene surface with CRISPR−Cas9 complex. ...
... (a) Schematic of biosensor chip modified by SLB (top) and the inhibitory response of two different drugs to the interaction between coronavirus and ACE2 receptor (bottom). Reproduced with permission from[89]. Copyright 2022, American Chemical Society. ...
In comparison with traditional clinical diagnosis methods, field−effect transistor (FET)−based biosensors have the advantages of fast response, easy miniaturization and integration for high−throughput screening, which demonstrates their great technical potential in the biomarker detection platform. This mini review mainly summarizes recent advances in FET biosensors. Firstly, the review gives an overview of the design strategies of biosensors for sensitive assay, including the structures of devices, functionalization methods and semiconductor materials used. Having established this background, the review then focuses on the following aspects: immunoassay based on a single biosensor for disease diagnosis; the efficient integration of FET biosensors into a large−area array, where multiplexing provides valuable insights for high−throughput testing options; and the integration of FET biosensors into microfluidics, which contributes to the rapid development of lab−on−chip (LOC) sensing platforms and the integration of biosensors with other types of sensors for multifunctional applications. Finally, we summarize the long−term prospects for the commercialization of FET sensing systems.
... The design of the EG should optimally respond to the demands of the assay to be performed. At the same time, it is essential to consider the stability of the EG layers under continuous exposure to the analyte solution with potentially variable properties such as temperature, pH, ionic strength, etc. Gold (Au) is a universal material for making EG conductive layers due to its chemical resistance and well-known processing protocols [23,[30][31][32][33][34][35][36]. Other popular materials include graphene [20,37,38] and metal oxides, such as indium tin oxide (ITO) [21,38], InZnO [39], MgZnO [40], and Ga 2 O 3 [41] due to their good electrical and/or optical properties (high optical transmittance). ...
... This option is known for its high reproducibility and cost-effectiveness. EG-FET platforms employing commercially available FETs are approaching real-world applications, including the screening of coronavirus drugs [32], studying of Alzheimer's disease [31], and monitoring of wastewater [60]. Although off-the-shelf FETs have found extensive application in diverse fields, their sensitivity remains limited. ...
... (b) Adapted with permission from [102]), (c) Adapted with permission from [103]. (d) (Adapted with permission from [104]. domains in a highly sensitive and selective manner [87]. ...
... Following that, The SLB was paved using the vesicle fusion method on the plasma-processed gold surface layer. The suggested FET approach has a low detection limit of 2.5 pM (Fig. 3D) [104]. To detect MTB, a multichannel microfluidic device with an incredibly sensitive silicon nanowire field effect transistor (SiNW-FET) biosensor for Mycobacterium tuberculosis (MTB Ag85B protein) was constructed by Ma et al. in 2022. ...
Lab-on-a-chip (LOC) biosensors have recently piqued the interest of the research community as a result of its potential utility in personal healthcare and disease diagnostics. LOCs devices have been consistently developed over the last decades as merging microfluidics onto a single chip for performing many lab studies at the same time, such as biochemical and biomolecular detection. Molding, microcontact printing, micromachining, and other techniques were used in the preliminary advancement of miniature sensing platforms known as micro total analysis systems (μTAS). These time-consuming and multi-step processes were utilized to create structures on a micrometer size. As time passes, new approaches and modifications for replacing rigid substrates with flexible substrates were developed. Over the years, paper and plastic substrates have shown unique properties such as durability, flexibility, mobility, cost-effective, and simple manufacturing procedure owing to their compatibility with a wide range of printing equipment. This review discusses the different types of fabrication methods and techniques such as photolithography, soft lithography, screen printing, inkjet printing, laser micromachining, nanoimprinting for designing LOC sensing devices extensively. The types of transduction systems which includes electrochemical, optical and mechanical that play an important role in sensing devices have also been extensively described in this manuscript. Additionally, detection of numerous analytes categorized into small molecules, macromolecules and cell bodies have been comprehensively reviewed in this manuscript with illustrations and tabulated form.
... [2][3][4][5] Additionally, integration of SLBs with surface-based sensors can be relatively straightforward, depending on the lipid composition and surface characteristics. SLBs can be incorporated into a broad range of sensor types, including optical 6-8 and electrical [9][10][11] sensors, and chemical imaging techniques [12][13][14] can be used to investigate SLBs. Therefore, they have found utility as receptor layers for sensors that probe peptide-lipid, 15 lipid-lipid, 16 nanoparticle-lipid, 17 ion-lipid, 18 and small molecule-lipid 19 interactions. ...
Supported lipid bilayers (SLBs) are useful structures for mimicking cellular membranes, and they can be integrated with a variety of sensors. While there are a variety of methods for forming SLBs, many of these methods come with limitations in terms of the lipid compositions that can be employed and the substrates upon which the SLBs can be deposited. Here we demonstrate the use of an all-aqueous chaotropic agent exchange process that can be used to form SLBs on two different substrate materials: SiO2, which is compatible with traditional SLB formation by vesicle fusion and Al2O3, which is not compatible with vesicle fusion. When examined with quartz crystal microbalance with dissipation monitoring, the SLBs generated by chaotropic agent exchange (CASLBs) have similar frequency and dissipation shifts to SLBs formed by the vesicle fusion technique. The CASLBs block nonspecific protein adsorption on the substrate and can be used to sense protein-lipid interactions. Fluorescence microscopy was used to examine the CASLBs, and we observed long-range lateral diffusion of fluorescent probes, which confirmed the CASLBs were composed of a continuous, planar lipid bilayer. Our CASLB method provides another option for forming planar lipid bilayers on a variety of surfaces, including those that are not amenable to the widely used vesicle fusion method.
... Biosensors, on the other hand, have been shown to differentiate between a virus protein and a whole virus particle. They have been successfully used as detection platforms for coronaviruses by exploiting the specificity of antigens for their respective receptors [14][15][16][17] . However, to comprehensively understand the unique properties of emerging mutants and their potential for infection beyond mere binding interactions, a functional assessment of infectivity potential is imperative. ...
Viral mutations frequently outpace technologies used to detect harmful variants. Given the continual emergence of SARS-CoV-2 variants, platforms that can identify the presence of a virus and its propensity for infection are needed. Our electronic biomembrane sensing platform recreates distinct SARS-CoV-2 host cell entry pathways and reports the progression of entry as electrical signals. We focus on two necessary entry processes mediated by the viral Spike protein: virus binding and membrane fusion, which can be distinguished electrically. We find that closely related variants of concern exhibit distinct fusion signatures that correlate with trends in cell-based infectivity assays, allowing us to report quantitative differences in their fusion characteristics and hence their infectivity potentials. We use SARS-CoV-2 as our prototype, but we anticipate that this platform can extend to other enveloped viruses and cell lines to quantifiably assess virus entry.
... For example, with the desalination and filtration of serum using micro hemodialysis, the dilution of the ionic strength of the solution effectively increases the Debye length, but it will have a certain impact on the activity of the sample [32,33]; the shortening of the distance of the target from the sensing surface occurs by pruning the antibody, but it is more complicated to prepare [34]; there are also the methods that entail replacing the functionalized antibody on the silicon nanowire with an aptamer [35]. Also, the incorporation of biomolecule-permeable polymers into semiconducting materials in order to change the dielectric constant can be effective in overcoming a certain degree of Debye shielding effect [36]. However, with the exception of Hwang et al., who reported the use of pleated graphene to overcome the effect of Debye shielding effect, few studies have been conducted on overcoming the Debye shielding effect from the point of view of optimizing devices [37]. ...
Silicon nanowire field effect (SiNW-FET) biosensors have been successfully used in the detection of nucleic acids, proteins and other molecules owing to their advantages of ultra-high sensitivity, high specificity, and label-free and immediate response. However, the presence of the Debye shielding effect in semiconductor devices severely reduces their detection sensitivity. In this paper, a three-dimensional stacked silicon nanosheet FET (3D-SiNS-FET) biosensor was studied for the high-sensitivity detection of nucleic acids. Based on the mainstream Gate-All-Around (GAA) fenestration process, a three-dimensional stacked structure with an 8 nm cavity spacing was designed and prepared, allowing modification of probe molecules within the stacked cavities. Furthermore, the advantage of the three-dimensional space can realize the upper and lower complementary detection, which can overcome the Debye shielding effect and realize high-sensitivity Point of Care Testing (POCT) at high ionic strength. The experimental results show that the minimum detection limit for 12-base DNA (4 nM) at 1 × PBS is less than 10 zM, and at a high concentration of 1 µM DNA, the sensitivity of the 3D-SiNS-FET is approximately 10 times higher than that of the planar devices. This indicates that our device provides distinct advantages for detection, showing promise for future biosensor applications in clinical settings.
... A modified gate electrode was created and tailored using a supported lipid bilayer (SLB) incorporating angiotensin-converting enzyme II (ACE2) receptors. This integrated system effectively translated interactions between targets and membrane receptors into immediate electrical signals in real time [73]. ...
... However, the major obstacle to addressing this issue is adequately constructing the cell membrane at the molecular level. 6 Recently, reports on the applications of this kind of biosensor have been reported for the detection of COVID-19 7 (SARS-CoV-2), 8 Avian Influenza A (H5N1), 9 Mycobacterium tuberculosis DNA, 10 and the H1N1 influenza virus. 11 For the development of this kind of biosensor, it is essential to characterize the physical and thermodynamic properties of the films to understand their heterogeneity (structure) and fluctuations (dynamics), such as stress, Young's modulus, and thickness. ...
This paper presents an alternative optical characterization of biosensors based on supported lipid monolayers (SLMs). Developing these biosensors requires precise thickness characterization of the films to understand their structure and dynamics. This paper proposes an optical technique to measure the thickness, optical properties, and location of a dipalmitoyl-phosphatidylcholine (DPPC) SLM on top of a metallic thin film. DPPC SLMs are of interest for biosensing applications, such as detecting pulmonary-related infections like SARS-CoV-2, Avian Influenza, and the H1N1 influenza virus. The monolayer was fabricated using the Langmuir–Blodgett technique, and the experimental characterization consisted of measuring the surface-plasmon resonance angle in the Kretschmann configuration. This technique provides an alternative option for real-time visual inspection and determination of the location and shape of DPPC monolayers in large areas. Therefore, it offers a useful tool for further developing SLM-based biosensors.
... This nanochip [6][7][8] is used as a drug reservoir, carrier, and release to control the drug load and delivery [9]. This nanochip is usually made of conductive polymers, endogenous metals, and ligands [10,11]. The natural conductive polymers consist of hyaluronic acid, alginate, agarose, chondroitin sulphate, carbomer, and xanthan gum [12,13]. ...
This paper is to discuss the potential of using CuZn in an electrical biosensor drug carrier for drug delivery systems. CuZn is the main semiconductor ingredient that has great promise as an electrochemical detector to trigger releases of active pharmaceutical ingredients (API). This CuZn biosensor is produced with a green metal of frameworks, which is an anion node in conductive polymers linked by bioactive ligands using metal–polymerisation technology. The studies of Cu, Zn, and their oxides are highlighted by their electrochemical performance as electrical biosensors to electrically trigger API. The three main problems, which are glucose oxidisation, binding affinity, and toxicity, are highlighted, and their solutions are given. Moreover, their biocompatibilities, therapeutic efficacies, and drug delivery efficiencies are discussed with details given. Our three previous investigations of CuZn found results similar to those of other authors’ in terms of multiphases, polymerisation, and structure. This affirms that our research is on the right track, especially that related to green synthesis using plant extract, CuZn as a nanochip electric biosensor, and bioactive ligands to bind API, which are limited to the innermost circle of the non-enzymatic glucose sensor category.
Nanoparticles have become a widely used material in various fields, yet their mechanism of action at the cellular level after entering the human body remains unclear. Accurate observing the effect...
Supported lipid bilayers (SLBs) are useful structures for mimicking cellular membranes, and they can be integrated with a variety of sensors. Although there are a variety of methods for forming SLBs, many of these methods come with limitations in terms of the lipid compositions that can be employed and the substrates upon which the SLBs can be deposited. Here we demonstrate the use of an all-aqueous chaotropic agent exchange process that can be used to form SLBs on two different substrate materials: SiO2, which is compatible with traditional SLB formation by vesicle fusion, and Al2O3, which is not compatible with vesicle fusion. When examined with a quartz crystal microbalance with dissipation monitoring, the SLBs generated by chaotropic agent exchange (CASLBs) have similar frequency and dissipation shifts to SLBs formed by the vesicle fusion technique. The CASLBs block nonspecific protein adsorption on the substrate and can be used to sense protein–lipid interactions. Fluorescence microscopy was used to examine the CASLBs, and we observed long-range lateral diffusion of fluorescent probes, which confirmed that the CASLBs were composed of a continuous, planar lipid bilayer. Our CASLB method provides another option for forming planar lipid bilayers on a variety of surfaces, including those that are not amenable to the widely used vesicle fusion method.
The global miniature devices market is poised to surpass a valuation of 12–15 billion USD by the year 2030. Lab-on-a-chip (LOC) devices are a vital component of this market.
Comprising a network of microchannels, electrical circuits, sensors, and electrodes, LOC is a miniaturized integrated device platform used to streamline day-to-day laboratory functions, run cost-effective clinical analyses and curb the need for centralized instrumentation facilities in remote areas. Compact design, portability, ease of operation, low sample volume, short reaction time, and parallel investigation stand as the pivotal factors driving the widespread acceptance of LOC within the biomedical community.
In this book, the Editors meticulously explore LOC through three key ‘Ts’:
This comprehensive text not only provides readers with a thorough understanding of the current advancements in the LOC domain but also offers valuable insights to support the utilization of miniaturized devices for enhanced healthcare practices. Aimed at career researchers looking for instruction in the topic and newcomers to the area, the book is also useful for undergraduate and postgraduate students embarking on new studies or for those interested in reading about the LOC platform.
Nature has inspired the development of biomimetic membrane sensors in which the functionalities of biological molecules, such as proteins and lipids, are harnessed for sensing applications. This review provides an overview of the recent developments for biomembrane sensors compatible with either bulk or planar sensing applications, namely using lipid vesicles or supported lipid bilayers, respectively. We first describe the individual components required for these sensing platforms and the design principles that are considered when constructing them, and we segue into recent applications being implemented across multiple fields. Our goal for this review is to illustrate the versatility of nature's biomembrane toolbox and simultaneously highlight how biosensor platforms can be enhanced by harnessing it.
Transmembrane transport analysis is essential for understanding cell physiological processes. Based on an artificial simulation of internal and external cellular environment, this paper introduces an innovative approach to investigate the microscopic behavior of small molecules through porin protein under mechanical curvature of lipid membrane. A flexible device system is developed, enabling quantitative electronic transmembrane analysis. The key transistor comprises a flexible, microporous electrode covered with the support lipid bilayers (SLBs) to mimic artificial cellular membrane, serving as an extended gate of the field‐effect transistor (FET). The transmembrane behaviors of charged ions and small molecules can be effectively monitored in real time by this FET‐based flexible device system. The flexibility of the electrode allows analyzing the transmembrane behavior under different mechanical bends. In this study, the developed flexible device is employed for the first time to simulate the mechanical bending of cellular membrane embedded with channel proteins and to monitor the transmembrane behavior of small molecules, thus providing a more authentic representation of membrane protein at a curved state. This approach holds the potential to contribute as a platform‐based technological advancement, supporting research into toxicological mechanisms and facilitating drug screening endeavors.
Field‐effect transistor‐based biosensors (FET biosensors) have gained significant attention in the context of the global SARS‐CoV‐2 pandemic owing to their label free and highly sensitive detection capabilities. In this comprehensive review, recent advances in the use of electrochemical‐transistor‐based biosensors for the label‐free detection of SARS‐CoV‐2 are analyzed. A general introduction to the electrochemical‐transistor‐based biosensor system is initiated, highlighting its critical technical requirements. Various structural designs and working principles of transistor‐based transducers are described, and the essential aspects of interface engineering for improved sensing performance are summarized. The applications of transistor‐based biosensors for the detection of SARS‐CoV‐2 are discussed in detail. Finally, perspectives for the future development of transistor‐based sensing systems are provided. This review aims to provide valuable insights and guidance for the design and optimization of biochemical sensor systems with the potential to impact health monitoring, disease diagnosis, and biosafety in the field of in vitro diagnostic products.
Viral mutation rates frequently outpace the development of technologies used to detect and identify harmful variants; for SARS Coronavirus-2 (SARS-CoV-2), these are called variants of concern (VOC). Given the continual emergence of VOC, there is a critical need to develop platforms that can identify the presence of a virus and readily identify its propensity for infection. We present an electronic biomembrane sensing platform that recreates the multifaceted and sequential biological cues that give rise to distinct SARS-CoV-2 virus host cell entry pathways and reports the progression of entry steps of these pathways as electrical signals. Within these electrical signals, two necessary entry processes mediated by the viral Spike protein, virus binding and membrane fusion, can be distinguished. Remarkably, we find that closely related VOC exhibit distinct fusion signatures that correlate with trends reported in cell-based infectivity assays, allowing us to report quantitative differences in fusion characteristics among them that inform their infectivity potentials. This cell-free biomimetic infection platform also has a virus-free option that equally reports infectivity potential of the Spike proteins. We used SARS-CoV-2 as our prototype, but we anticipate that this platform will extend to other enveloped viruses and cell lines to quantifiably explore virus/host interactions. This advance should aid in faster determination of entry characteristics and fusogenicities of future VOC, necessary for rapid response.
The assembly of biomimetic, planar supported lipid bilayers (SLBs) by the popular vesicle fusion method, which relies on the spontaneous adsorption and rupture of small unilamellar vesicles from aqueous solution on a solid surface, typically works with a limited range of support materials and lipid systems. We previously reported a conceptual advance in the formation of SLBs from vesicles in the gel or fluid phase using the interfacial ion-pairing association of charged phospholipid headgroups with electrochemically generated cationic ferroceniums bound to a self-assembled monolayer (SAM) chemisorbed to gold. This redox-driven approach lays down a single bilayer membrane on the SAM-modified gold surface at room temperature within minutes and is compatible with both anionic and zwitterionic phospholipids. The present work explores the effects of the surface ferrocene concentration and hydrophobicity/hydrophilicity on the formation of continuous SLBs of dialkyl phosphatidylserine, dialkyl phosphatidylglycerol, and dialkyl phosphatidylcholine using binary SAMs of ferrocenylundecanethiolate (FcC11S) and dodecanethiolate (CH3C11S) or hydroxylundecanethiolate (HOC11S) comprising different surface mole fractions of ferrocene (χFcsurf). An increase in the surface hydrophilicity and surface free energy of the FcC11S/HOC11S SAM mitigates the decrease in the attractive ion-pairing interactions resulting from a reduced χFcsurf. SLBs of ≳80% area coverage form on the FcC11S/HOC11S SAM for all the phospholipid types down to χFcsurf of at least 0.2, composition yielding a water contact angle (θW) of 44 ± 4°. By contrast, a greater number of ion-pairing interactions is required on the hydrophobic FcC11S/CH3C11S surface to drive the vesicle fusion process; bilayers or bilayer patches form at χFcsurf ≳ 0.6 (θW = 97 ± 3°). These findings will aid in tailoring the surface chemistry of redox-active modified surfaces to widen the conditions that yield supported lipid membranes.
The COVID-19 pandemic has posed significant challenges to existing healthcare systems around the world. The urgent need for the development of diagnostic and therapeutic strategies for COVID-19 has boomed the demand for new technologies that can improve current healthcare approaches, moving towards more advanced, digitalized, personalized, and patient-oriented systems. Microfluidic-based technologies involve the miniaturization of large-scale devices and laboratory-based procedures, enabling complex chemical and biological operations that are conventionally performed at the macro-scale to be carried out on the microscale or less. The advantages microfluidic systems offer such as rapid, low-cost, accurate, and on-site solutions make these tools extremely useful and effective in the fight against COVID-19. In particular, microfluidic-assisted systems are of great interest in different COVID-19-related domains, varying from direct and indirect detection of COVID-19 infections to drug and vaccine discovery and their targeted delivery. Here, we review recent advances in the use of microfluidic platforms to diagnose, treat or prevent COVID-19. We start by summarizing recent microfluidic-based diagnostic solutions applicable to COVID-19. We then highlight the key roles microfluidics play in developing COVID-19 vaccines and testing how vaccine candidates perform, with a focus on RNA-delivery technologies and nano-carriers. Next, microfluidic-based efforts devoted to assessing the efficacy of potential COVID-19 drugs, either repurposed or new, and their targeted delivery to infected sites are summarized. We conclude by providing future perspectives and research directions that are critical to effectively prevent or respond to future pandemics.
Graphical Abstract
A novel SARS-like coronavirus (SARS-CoV-2) recently emerged and is rapidly spreading in humans1,2. A key to tackling this epidemic is to understand the virus’s receptor recognition mechanism, which regulates its infectivity, pathogenesis and host range. SARS-CoV-2 and SARS-CoV recognize the same receptor - human ACE2 (hACE2)3,4. Here we determined the crystal structure of the SARS-CoV-2 receptor-binding domain (RBD) (engineered to facilitate crystallization) in complex with hACE2. Compared with the SARS-CoV RBD, a hACE2-binding ridge in SARS-CoV-2 RBD takes a more compact conformation; moreover, several residue changes in SARS-CoV-2 RBD stabilize two virus-binding hotspots at the RBD/hACE2 interface. These structural features of SARS-CoV-2 RBD enhance its hACE2-binding affinity. Additionally, we show that RaTG13, a bat coronavirus closely related to SARS-CoV-2, also uses hACE2 as its receptor. The differences among SARS-CoV-2, SARS-CoV and RaTG13 in hACE2 recognition shed light on potential animal-to-human transmission of SARS-CoV-2. This study provides guidance for intervention strategies targeting receptor recognition by SARS-CoV-2.
In the present paper, Na3BiO4–Bi2O3 films have been tested for hydrogen ion sensing. Na3BiO4–Bi2O3 mixed oxide nanostructures were deposited on the Indium–Tin-Oxide (ITO) coated glass substrate using a low-cost electrodeposition technique at room temperature. The nanostructures have been characterized using FESEM and XPS to study their morphology and composition, respectively. Vertically aligned nanostructures (thickness~90 nm) with well-defined edges were seen in FESEM studies. XPS analysis indicates the presence of Na3BiO4 and Bi2O3. The crystallinity and the mixed phase of the film were further confirmed by X-ray diffraction study. These nanostructures were explored as a potential candidate for pH sensing using them as a sensing film for Extended-Gate Field-Effect Transistor (EGFET). A sensitivity of 49.63 mV/pH has been observed with good linearity. To the best of our knowledge, for the first time vertically aligned Na3BiO4–Bi2O3 mixed oxide nanostructures are demonstrated as an EGFET-based (Hydrogen ion) pH sensor.
Quantification of the multivalent interactions of influenza viruses binding at interfaces may provide ways to tackle key biological questions regarding influenza virulence and zoonoses. Yet, the deconvolution of the contributions of molecular and interfacial parameters, such as valency, interaction area and receptor density, to the binding of whole viruses is hindered by difficulties in the direct determination of these parameters. We report here a chemical platform technology to study the binding of multivalent recombinant hemagglutinin (rHA) nanoparticles at artificial sialoglycan cell receptor-presenting interfaces in which all these parameters can be derived, thus allowing the desired full and quantitative binding analysis. SiO2 substrates were functionalized with supported lipid bilayers containing a targeted and tunable fraction of a biotinylated lipid, followed by the adsorption of streptavidin and biotinylated polyvalent 2,3- or 2,6-sialyl lactosamine (SLN). rHA nanoparticles were used as a virus mimic to provide a good prediction of the number of interactions involved in binding. Low nanomolar affinities and selectivities for binding at the 2,6-SLN platforms were observed for rHA particles from three different virus variants. When fitting the data to a multivalency model, the nanomolar overall affinity appears to be achieved by 6-9 HA-sugar molecular interaction pairs, which individually present a rapid association/dissociation behavior. This dynamic behavior may be an essential biological attribute in the functioning of the influenza virus.
Prostaglandin E receptor EP4, a G-protein-coupled receptor, is involved in disorders such as cancer and autoimmune disease. Here, we report the crystal structure of human EP4 in complex with its antagonist ONO-AE3-208 and an inhibitory antibody at 3.2 Å resolution. The structure reveals that the extracellular surface is occluded by the extracellular loops and that the antagonist lies at the interface with the lipid bilayer, proximal to the highly conserved Arg316 residue in the seventh transmembrane domain. Functional and docking studies demonstrate that the natural agonist PGE2 binds in a similar manner. This structural information also provides insight into the ligand entry pathway from the membrane bilayer to the EP4 binding pocket. Furthermore, the structure reveals that the antibody allosterically affects the ligand binding of EP4. These results should facilitate the design of new therapeutic drugs targeting both orthosteric and allosteric sites in this receptor family.
A graphene field-effect transistor (G-FET) with the spacious planar graphene surface can provide a large-area interface with cell membranes to serve as a platform for the study of cell membrane-related protein interactions. In this study, a G-FET device paved with a supported lipid bilayer (referred to as SLB/G-FET) was first used to monitor the catalytic hydrolysis of the SLB by phospholipase D. With excellent detection sensitivity, this G-FET was also modified with a ganglioside GM1-enriched SLB (GM1-SLB/G-FET) to detect cholera toxin B. Finally, the GM1-SLB/G-FET was employed to monitor amyloid-beta 40 (Aβ40) aggregation. In the early nucleation stage of Aβ40 aggregation, while no fluorescence was detectable with traditional thioflavin T (ThT) assay, the prominent electrical signals probed by GM1-SLB/G-FET demonstrate that the G-FET detection is more sensitive than the ThT assay. The comprehensive kinetic information during the Aβ40 aggregation could be collected with a GM1-SLB/G-FET, especially covering the kinetics involved in the early stage of Aβ40 aggregation. These experimental results suggest that SLB/G-FETs hold great potential as a powerful biomimetic sensor for versatile investigations of membrane-related protein functions and interaction kinetics.
The pathogenesis of the Ebola virus which leads to a severe hemorrhagic fever in hosts is a very complex process which is not completely understood. Glycoproteins of the viral envelope are believed to play a crucial role in receptor binding and subsequently in fusion of the virus with the target cells of the host. As a result, the virus enters the cells and replicates. This process causes further cytopathic, and pathological reactions in the host's body. To gain further insights into the fusogenic interactions of the virus with cell membranes, we used well-controlled simple biomimetic systems, consisting of solid-supported phospholipid layers together with a small sequence of the viral glycoprotein (EBO17), which is believed to be the most important part responsible for viral pathogenesis. We monitor the real-time interaction of a EBO17 peptide sequence from the Ebola virus with dipalmitoylphosphatidylcholine (DMPC) phospholipid membranes using quartz crystal microbalance with dissipation monitoring (QCM-D) as a label-free method. In particular, we focus on the influence of the concentration of the peptide and the lipid layer geometry on the disrupting mechanism of the EBO17 peptide. Results indicate that for 2D supported lipid bilayers, low peptide concentrations induce a small, but detectable change in layer stability due to the presence of an α-helix configuration of the peptide. With large peptide concentrations, the peptide acquires a β-sheet configuration and no significant layer changes can be observed. A different mechanism is responsible for the interaction of the EBO17 peptides with the more complex 3D supported vesicle layers, for which a concentration-dependent trend can be observed leading to thicker lipid layers. Complementary analysis of the lipids' main phase transition evidences the differences induced in layer organization on the two layer geometries. These results confirm the importance of the interplay between lipid layer geometry and related peptide organization as an essential marker in peptide activity.
The seminal importance of detecting ions and molecules for point-of-care tests has driven the search for more sensitive, specific, and robust sensors. Electronic detection holds promise for future miniaturized in-situ applications and can be integrated into existing electronic manufacturing processes and technology. The resulting small devices will be inherently well suited for multiplexed and parallel detection. In this review, different field-effect transistor (FET) structures and detection principles are discussed, including label-free and indirect detection mechanisms. The fundamental detection principle governing every potentiometric sensor is introduced, and different state-of-the-art FET sensor structures are reviewed. This is followed by an analysis of electrolyte interfaces and their influence on sensor operation. Finally, the fundamentals of different detection mechanisms are reviewed and some detection schemes are discussed. In the conclusion, current commercial efforts are briefly considered.
Biolayer interferometry (BLI) is a well-established optical label-free technique to study biomolecular interactions. Here we describe for the first time a cell-based BLI (cBLI) application that allows label-free real-time monitoring of signal transduction in living cells. Human A431 epidermoid carcinoma cells were captured onto collagen-coated biosensors and serum-starved, followed by exposure to agonistic compounds targeting various receptors, while recording the cBLI signal. Stimulation of the epidermal growth factor receptor (EGFR) with EGF, the β2-adrenoceptor with dopamine, or the hepatocyte growth factor receptor (HGFR/c-MET) with an agonistic antibody resulted in distinct cBLI signal patterns. We show that the mechanism underlying the observed changes in cBLI signal is mediated by rearrangement of the actin cytoskeleton, a process referred to as dynamic mass redistribution (DMR). A panel of ligand-binding blocking and non-blocking anti-EGFR antibodies was used to demonstrate that this novel BLI application can be efficiently used as a label-free cellular assay for compound screening and characterization.
Field-effect transistors (FETs) based on large-area graphene and other 2D materials can potentially be used as low-cost and flexible potentiometric biological sensors. However, there have been few attempts to use these devices for quantifying molecular interactions and to compare their performance to established sensor technology. Here, gold-coated graphene FETs are used to measure the binding affinity of a specific protein-antibody interaction. Having a gold surface gives access to well-known thiol chemistry for the self-assembly of linker molecules. The results are compared with potentiometric silicon-based extended-gate sensors and a surface plasmon resonance system. The estimated dissociation constants are in excellent agreement for all sensor types as long as the active surfaces are the same (gold). The role of the graphene transducer is to simply amplify surface potential changes caused by adsorption of molecules on the gold surface.
Zinc oxide (ZnO) nanostructures are promising candidates as electronic components for biological and chemical applications. In this study, ZnO ultra-fine nanowire (NW) and nanoflake (NF) hybrid structures have been prepared by Au-assisted chemical vapor deposition (CVD) under ambient pressure. Their surface morphology, lattice structures, and crystal orientation were investigated by scanning electron microscopy (SEM), x-ray diffraction (XRD), and transmission electron microscopy (TEM). Two types of ZnO nanostructures were successfully integrated as gate electrodes in extended-gate field-effect transistors (EGFETs). Due to the amphoteric properties of ZnO, such devices function as pH sensors. We found that the ultra-fine NWs, which were more than 50 μm in length and less than 100 nm in diameter, performed better in the pH sensing process than NW–NF hybrid structures because of their higher surface-to-volume ratio, considering the Nernst equation and the Gouy–Chapman–Stern model. Furthermore, the surface coating of (3-Aminopropyl)triethoxysilane (APTES) protects ZnO nanostructures in both acidic and alkaline environments, thus enhancing the device stability and extending its pH sensing dynamic range.
Significance
Linear polyprenols are recurring molecular components in the biosynthetic pathways responsible for the assembly of essential glycoconjugates, including peptidoglycan and N-linked glycoproteins. Despite their highly conserved presence in all domains of life, the role of the extended linear polyprenyl groups in the dynamics of membrane-bound glycan assembly pathways remains a mystery. Here we apply the nanodisc model membrane platform to simultaneously assess the interactions and activities of the polyprenyl-linked substrates, enzymes, and lipid bilayer by investigating initial steps from the Campylobacter jejuni N-linked glycosylation pathway. This work represents a proof-of-concept demonstrating that nanodiscs can be used for the precise manipulation and study of polyprenol-dependent pathways.
Biosensors allowing for the rapid and sensitive detection of viral pathogens in environmental or clinical samples are urgently needed to prevent disease outbreaks and spreading. We present a bioanalytical assay for the detection of whole viral particles with single virus sensitivity. Specifically, we focus on the detection of human norovirus, a highly infectious virus causing gastroenteritis. In our assay configuration, virus-like particles are captured onto a supported lipid bilayer containing a virus-specific glycolipid and detected after recognition by a glycolipid-containing fluorescent vesicle. Read-out is performed after illumination of the vesicle labels by total internal reflection fluorescence microscopy. This allows for visualization of individual vesicles and for recording of their binding kinetics under equilibrium conditions (equilibrium fluctuation analysis), as demonstrated previously. In this work we extend the concept and demonstrate that this simple assay setup can be used as a bioanalytical assay for the detection of virus particles at a limit of detection of 16 fM. Furthermore, we demonstrate how the analysis of the single vesicle-virus-like particle interaction dynamics can contribute to increase the accuracy and sensitivity of the assay by discriminating specific from non-specific binding events. This method is suggested to be generally applicable, provided that these events display different interaction kinetics.
Human α-defensins are cationic peptides that self-associate into dimers and higher-order oligomers. They bind protein toxins,
such as anthrax lethal factor (LF), and kill bacteria, including Escherichia coli and Staphylococcus aureus, among other functions. There are six members of the human α-defensin family: four human neutrophil peptides, including HNP1,
and two enteric human defensins, including HD5. We subjected HD5 to comprehensive alanine scanning mutagenesis. We then assayed
LF binding by surface plasmon resonance, LF activity by enzyme kinetic inhibition, and antibacterial activity by the virtual
colony count assay. Most mutations could be tolerated, resulting in activity comparable with that of wild type HD5. However,
the L29A mutation decimated LF binding and bactericidal activity against Escherichia coli and Staphylococcus aureus. A series of unnatural aliphatic and aromatic substitutions at position 29, including aminobutyric acid (Abu) and norleucine
(Nle) correlated hydrophobicity with HD5 function. The crystal structure of L29Abu-HD5 depicted decreased hydrophobic contacts
at the dimer interface, whereas the Nle-29-HD5 crystal structure depicted a novel mode of dimerization with parallel β strands.
The effect of mutating Leu29 is similar to that of a C-terminal hydrophobic residue of HNP1, Trp26. In addition, in order to further clarify the role of dimerization in HD5 function, an obligate monomer was generated by
N-methylation of the Glu21 residue, decreasing LF binding and antibacterial activity against S. aureus. These results further characterize the dimer interface of the α-defensins, revealing a crucial role of hydrophobicity-mediated
dimerization.
Severe acute respiratory syndrome (SARS) is an acute infectious disease that spreads mainly via the respiratory route. A distinct coronavirus (SARS-CoV) has been identified as the aetiological agent of SARS. Recently, a metallopeptidase named angiotensin-converting enzyme 2 (ACE2) has been identified as the functional receptor for SARS-CoV. Although ACE2 mRNA is known to be present in virtually all organs, its protein expression is largely unknown. Since identifying the possible route of infection has major implications for understanding the pathogenesis and future treatment strategies for SARS, the present study investigated the localization of ACE2 protein in various human organs (oral and nasal mucosa, nasopharynx, lung, stomach, small intestine, colon, skin, lymph nodes, thymus, bone marrow, spleen, liver, kidney, and brain). The most remarkable finding was the surface expression of ACE2 protein on lung alveolar epithelial cells and enterocytes of the small intestine. Furthermore, ACE2 was present in arterial and venous endothelial cells and arterial smooth muscle cells in all organs studied. In conclusion, ACE2 is abundantly present in humans in the epithelia of the lung and small intestine, which might provide possible routes of entry for the SARS-CoV. This epithelial expression, together with the presence of ACE2 in vascular endothelium, also provides a first step in understanding the pathogenesis of the main SARS disease manifestations.
Background
Cardiac troponin is the gold standard biomarker used to rule in or out acute myocardial infarction (AMI) in patients. Highly sensitive and reliable detection mechanisms for this important biomarker are required for advancing the field of early detection in medical applications. Field effect transistor (FET) sensors, specifically functionalised with troponin antibodies, can perform specific detection of cardiac troponin I or T, with high sensitivity and low limit of detection (LoD), in fully scalable and low power operated intelligent sensing systems.
Purpose
To design and realize a new wearable blood or sweat on-chip sensor, able to perform a real-time reliable detection of the cardiac troponin level in a patient suffering of AMI symptoms or during the post-operative phase. The sensor exploits the voltammetric detection of biomarkers, and offers the potential for miniaturization and reduced hospitalization costs.
Methods
Electrical current modulation in field effect transistor-based sensors is used as transducing principle, as a result of the surface potential change following exposure to biological fluids containing different concentrations of antigens. The use of antibodies fragments, properly attached to the sensor surface, enhances the sensitivity. Proof of concept tests involve the detection of fluorescein.
Results
The specific antigen detection by means of an electrical measurement has been proved, with a detection limit of <1 nM (24ng/mL), linear range over one decade and a sensitivity around 70mV/decade. Fluorescein antibodies fragments, commercially purchased, were attached to the gate of an EGFET device; the binding events between fluorescein molecules and the correspondent antibodies modify the electrical conduction properties of the transducer, creating a current shift whose amplitude is univocally related to the antigen concentration. The exploitation of the antibodies fragments allows the antigens to bind closer to the sensing surface, creating a more noticeable electrical perturbation. The device is made of a commercial Metal Oxide Semiconductor FET (MOSFET), whose gate is extended by a platinum electrode for sensing. The metal is then functionalised by attaching covalently antibodies fragments that act as probes for the antigens in the tested biofluids. Laboratory samples were prepared in house, using fluorescein as antigen. Concentrations between 45pM and 1uM have been tested, and the absence of non-specific binding has been successfully proved with bovine serum albumin (BSA), for which any noticeable response has been recorded.
Conclusion
We report an important step towards a real-time and reliable detection of cardiac troponin by means of nano-ISFETs, demonstrating the fluorescein antigens-antibodies specific binding. Further steps consists in correctly achieve a surface functionalisation with cardiac troponin I antibodies, and further enhance the sensor LoD.
Antigens detection with EGFET
Funding Acknowledgement
Type of funding source: Public grant(s) – National budget only. Main funding source(s): Swiss National Science Foundation: ERA-NET, FLAG-ERA CONVERGENCE
The burgeoning epidemic caused by novel coronavirus 2019 (2019-nCoV) is currently a global concern. Angiotensin-converting enzyme-2 (ACE2) is a receptor of 2019-nCoV spike 1 protein (S1) and mediates viral entry into host cells. Despite the abundance of ACE2 in small intestine, few digestive symptoms are observed in patients infected by 2019-nCoV. Herein, we investigated the interactions between ACE2 and human defensins (HDs) specifically secreted by intestinal Paneth cells. The lectin-like HD5, rather than HD6, bound ACE2 with a high affinity of 39.3 nM and weakened the subsequent recruitment of 2019-nCoV S1. The cloak of HD5 on the ligand-binding domain of ACE2 was confirmed by molecular dynamic simulation. A remarkable dose-dependent preventive effect of HD5 on 2019-nCoV S1 binding to intestinal epithelial cells was further evidenced by in vitro experiments. Our findings unmasked the innate defense function of lectin-like intestinal defensin against 2019-nCoV, which may provide new insights into the prevention and treatment of 2019-nCoV infection.
Drug discovery is a complex, multistep process, in which many challenges need to be overcome at each stage, from the discovery of a biomolecular target to the ensuring of the efficacy and safety of a compound in humans. Today's analytical methods allow tens of thousands of drug candidates to be screened for their ability to inhibit specific enzymes and the miniaturization of these approaches is highly desirable, accelerating the drug discovery process and reducing the associated costs. Herein, it is reviewed the miniaturized techniques currently used to evaluate enzymatic activity and inhibition giving special attention to microplate formats, microarrays, nanoarrays, and microfluidic technologies. It is, also, highlighted some of the characteristics and their abilities for potential uses are compared and discussed. In addition, the challenges of their applications in diagnosis, analysis, and therapy, which should help to improve the quality of healthcare globally are also pointed out.
The emergence of SARS-CoV-2 has resulted in >90,000 infections and >3,000 deaths. Coronavirus spike (S) glycoproteins promote entry into cells and are the main target of antibodies. We show that SARS-CoV-2 S uses ACE2 to enter cells and that the receptor-binding domains of SARS-CoV-2 S and SARS-CoV S bind with similar affinities to human ACE2, correlating with the efficient spread of SARS-CoV-2 among humans. We found that the SARS-CoV-2 S glycoprotein harbors a furin cleavage site at the boundary between the S1/S2 subunits, which is processed during biogenesis and sets this virus apart from SARS-CoV and SARS-related CoVs. We determined cryo-EM structures of the SARS-CoV-2 S ectodomain trimer, providing a blueprint for the design of vaccines and inhibitors of viral entry. Finally, we demonstrate that SARS-CoV S murine polyclonal antibodies potently inhibited SARS-CoV-2 S mediated entry into cells, indicating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited upon vaccination.
Exploring new strategies for simple and on-demand methods of manipulating the sensing ability of sensor devices functionalized with artificial receptors embedded in a molecular assembly is important to realizing high-throughput on-site sensing systems based on integrated and miniaturized devices such as field-effect transistors (FETs). Although FET-based chemical sensors can be used for rapid, quantitative, and simultaneous determination of various desired analytes, detectable targets in conventional FET sensors are currently restricted owing to the complicated processes used to prepare sensing materials. In this study, we investigated the relationship between the sensing features of FETs and the nanostructures of mixed self-assembled monolayers (mSAMs) for the detection of biomolecules. The FET devices were systematically functionalized using mixtures of benzenethiol derivatives (4-mercaptobenzoic acid and benzenethiol), which changed the nanostructure of the SAMs formed on gold sensing electrodes. The obtained cross-reactivity in the FETs modified with the mSAMs was derived from the multidimensional variations of the SAM characteristics. Our successful demonstration of continuous control of the molecular recognition ability in the FETs by applying the mSAM system could lead to the development of next-generation versatile analyzers, including chemical sensor arrays for the determination of multiple analytes anytime, anywhere.
Membrane proteins (MPs) are playing important roles in several biological processes. Screening new candidate compounds targeting MPs is important for drug discovery. However, it remains challenging to characterize the interactions between MPs and small-molecule ligands in a label-free method. In this study, an SPR-based membrane protein-targeted active ingredients recognition strategy was constructed. This strategy contains two major modules: affinity detection module and ligand screening module. Through the combination of these two functional modules, it is feasible to screen small molecular ligands targeting MPs from herbal medicines. First, we have constructed high/low comparative C-X-C chemokine receptor type 4 (CXCR4)-expressed lentiviral particles (LVPs) models and characterized the expression levels. Then we immobilized LVPs on CM5 chips and detected the affinity between AMD3100 and CXCR4 by using affinity detection module. The KD of AMD3100 was 32.48 ± 3.17 nM. Furthermore, the suitability and robustness of the ligand screening module were validated by using AMD3100 as a positive compound. Subsequently, this module was applied in the screening of CXCR4 small molecular ligands from herbal medicine extracts. Senkyunolide I was screened out from Chuanxiong extract. The affinity constant between senkyunolide I and CXCR4 was 2.94 ± 0.36 μM. Boyden chamber assay revealed that senkyunolide I could inhibit cell migration process. In conclusion, an SPR-based small molecular ligand recognition strategy combined with virus-based membrane protein stabilization method was constructed. The SPR-based membrane protein-targeted active ingredients recognition strategy will be an effective tool to screen target components from complex systems acting on MPs.
Supported lipid bilayers (SLBs) have been widely used to provide native environments for membrane protein studies. In this study, we utilized graphene field-effect transistors (GFETs) coated with a fluid SLB to perform label-free detection of membrane-associated ligand-receptor interactions in their native lipid bilayer environment. It is known that the analyte-binding event needs to occur within the Debye length for it to be significantly sensed by an FET sensor. However, the thickness of a lipid bilayer is around 4-5nm thick, which is larger than the Debye length of a solution with physiologically relevant ionic strength. There is thus a question of whether an FET sensor can detect the binding event above the bilayer. In this study, we show how the existence of an SLB can influence the effective detection distance and the formation criterion of a fluid and continuous SLB on a graphene surface. We discovered that the water intercalation between the graphene and the underlying silica substrate hinders the SLB formation but is required for the stable electrical recording by a GFET. To verify the existence of a fluid SLB on graphene, which was previously complicated by the graphene fluorescence quenching-effect, we developed a modified fluorescence recovery after photobleaching method. In addition, our results showed that SLB coated GFETs can quantitatively detect ligand binding onto the receptors embedded in the SLBs. The comparison of our experimental data with a theoretical model shows that the contribution of the SLB acyl chain hydrophobic region to the screening effect can be negligible and, therefore, that the effective detection region can extend beyond the SLB.
Cryo-electron microscopy (cryo-EM) of non-crystalline single particles is a biophysical technique that can be used to determine the structure of biological macromolecules and assemblies. Historically, its potential for application in drug discovery has been heavily limited by two issues: the minimum size of the structures it can be used to study and the resolution of the images. However, recent technological advances — including the development of direct electron detectors and more effective computational image analysis techniques — are revolutionizing the utility of cryo-EM, leading to a burst of high-resolution structures of large macromolecular assemblies. These advances have raised hopes that single-particle cryo-EM might soon become an important tool for drug discovery, particularly if they could enable structural determination for 'intractable' targets that are still not accessible to X-ray crystallographic analysis. This article describes the recent advances in the field and critically assesses their relevance for drug discovery as well as discussing at what stages of the drug discovery pipeline cryo-EM can be useful today and what to expect in the near future.
A field-effect transistor-based cortisol sensor was demonstrated in physiological condition. Antibody-embedded polymer on the remote gate was proposed to overcome the Debye length issue (λ_D). The sensing membrane was made by linking polystyrene-co-methacrylic acid (PSMA) with anti-cortisol before coating the modified polymer on the remote gate. The embedded receptor in polymer showed sensitivity from 10 fg/ml to 10 ng/ml for cortisol and limit of detection (LOD) of 1 pg/ml in 1× PBS where λ_D is 0.2 nm. LOD of 1 ng/ml was shown in lightly buffered artificial sweat. Finally, a sandwich ELISA confirmed antibody binding activity of antibody-embedded PSMA.
To efficiently isolate maximal quantity of circulating tumor cells (CTCs) and circulating tumor cell microembolis (CTMs) from patient blood by antibody coated microfluidics, a multi-functional, pegylated polyamidoamine-dendrimers conjugated supported lipid bilayer surface construct was proposed to enhance accessibility of antibody molecules to the antigen molecules on target CTCs. The combination of a hydrated, stretchable dendrimer and a laterally-mobile supported lipid bilayer (SLB) provide attached antibody molecules with 2.5-dimensional chain movement, achieving multivalency between the surface antibody and cell antigen molecules. An over 170% enhancement is distinctive for Panc-1 cells that expresses low antigen level. Of seven pancreatic ductal adenocarcinoma patients, an average 440 single CTCs and 90 CTMs were collected in 2mL peripheral blood, which were 1.6 times and 2.3 times more, than those captured by the SLB-only microfluidics. In summary, we have demonstrated a material design to enhance multivalent antibody-antigen interaction, which is useful for rare cell enrichment and cancer detection.
In this work, a new strategy for electrochemical analysis of enzyme has been proposed based on a self-assembled lipid bilayer on an electrode surface mediated by hydrazone chemistry. Taking aldolase as an example, the enzyme can catalyze the formation of products containing carbonyl groups. These groups can react with hydrazine groups of the functional lipid derivative, resulting in the self-assembly of lipid bilayer on a guanidinium modified electrode surface. The lipid bilayer will then prevent the movement of hydrophilic electrochemical probes. Consequently, the catalytic reaction of the enzyme may result in the change of the obtained electrochemical peak current. Experimental results reveal that aldolase activity can be analyzed over a widely linear detection range from 5 mU/L to 100 U/L with a low detection limit of 1 mU/L. Meanwhile, the method can exhibit good precision and reproducibility, and it can be applied for real sample analysis. What is more, since lipid bilayer is the universal basis for cell-membrane structure, while hydrazone chemistry is popular in nature, this work may also provide a new insight for the development of electrochemical analysis and electrochemical biosensors.
Artificial lipid bilayers in the form of planar supported or vesicular bilayers are commonly used as models for studying interaction of biological membranes with different substances such as proteins and small molecule pharmaceutical compounds. Lipid membranes are typically regarded as inert and passive scaffolds for membrane proteins, but both non-specific and specific interactions between biomolecules and lipid membranes are indeed ubiquitous; dynamic exchange of proteins from the environment at the membrane interface can strongly influence the function of biological membranes. Such exchanges would either be of a superficial (peripheral) or integrative (penetrating) nature. In the context of viral membranes (termed envelopes), this could contribute to the emergence of zoonotic infections as well as change the virulence and/or pathogenicity of viral diseases. In this study, we analyze adsorption/desorption patterns upon challenging tethered liposomes and enveloped virus particles with proteins - or protein mixtures - such as bovine serum albumin, glycosylphosphatidylinositol anchored proteins and serum, chosen for their different lipid-interaction capabilities. We employed quartz crystal microbalance and dual polarization interferometry measurements to measure protein/membrane interaction in real time. We identified differences in mass uptake between the challenges, as well as differences between variants of lipid bilayers. Tethered viral particles showed a similar adsorption/desorption behavior to liposomes, underlining their value as model system. We believe that this methodology may be developed into a new approach in virology and membrane research by enabling the combination of biophysical and biochemical information.
This paper reports a high-performance glucose biosensor using ZnO nanorod-based field effect transistor (FET) to continuously monitor glucose concentration. Sensing area reduction of the sensor for minimally-invasive glucose monitoring with high sensitivity and good stability is demonstrated. The biosensor is fabricated via a hydrothermal growth of semiconducting ZnO nanorods between source and drain micro-electrodes incorporating with alternating current (AC) electric-field control. ZnO nanorod structured FET acts as a frequency mixer to transduce glucose concentration into a current change at certain difference frequency, which is measured by a lock-in amplifier. Due to the large surface-to-volume ratio of ZnO nanorods and the advanced frequency mixing detection scheme, good anti-jamming capability and long-term stability are realized. The glucose sensor achieves a high sensitivity of 1.6mA/(μM-cm²), a glucose concentration detection limit of 1μM, and excellent long-term stability shown in 38-h continuous monitoring. In contrast to the state-of-the-art glucose sensors, the developed biosensor exhibits competitive advantages in sensitivity, long-term stability, tiny size and fabrication cost, which indicates its promising application potential in wearable continuous glucose monitoring for diabetics.
Rapid and simultaneous detection of multiple potential pathogens by portable devices can facilitate early diagnosis of infectious diseases, and allow for rapid and effective implementation of disease prevention and treatment measures. The development of a ZnO nanorod integrated microdevice as a multiplex immunofluorescence platform for highly sensitive and selective detection of avian influenza virus (AIV) is described. The 3D morphology and unique optical property of the ZnO nanorods boost the detection limit of the H5N2 AIV to as low as 3.6 × 10³ EID50 mL⁻¹ (EID50: 50% embryo infectious dose), which is ≈22 times more sensitive than conventional enzyme-linked immunosorbent assay. The entire virus capture and detection process could be completed within 1.5 h with excellent selectivity. Moreover, this microfluidic biosensor is capable of detecting multiple viruses simultaneously by spatial encoding of capture antibodies. One prominent feature of the device is that the captured H5N2 AIV can be released by simply dissolving ZnO nanorods under slightly acidic environment for subsequent off-chip analyses. As a whole, this platform provides a powerful tool for rapid detection of multiple pathogens, which may extent to the other fields for low-cost and convenient biomarker detection.
The study reports the use of extended gate field-effect transistors (FET) for the label-free and sensitive detection of prostate cancer (PCa) bio-markers in human plasma. The approach integrates for the first time hybrid synthetic receptors compris-ing of highly selective aptamer-lined pockets (apta-MIP) with FETs for sensitive detection of prostate specific antigen (PSA) at clinically relevant concen-trations. The hybrid synthetic receptors were con-structed by immobilising an aptamer-PSA complex on gold and subjecting it to 13 cycles of dopamine electropolymerisation. The polymerisation resulted in the creation of highly selective polymeric cavities that retained the ability to recognize PSA post removal of the protein. The hybrid synthetic receptors were subsequently used in an extended gate FET setup for electrochemical detection of PSA. The sensor was reported to have a limit of detection of 0.1 pg/ml with a linear detection range from 0.1 pg/ml to 1 ng/ml PSA. Detection of 1-10 pg/mL PSA was also achieved in diluted human plasma. The present apta-MIP sensor developed in conjunction with FET devices demonstrates the potential for clinical application of synthetic hybrid receptors for the detection of clinically relevant biomarkers in complex samples.
Field-effect transistor (FET) biosensor has attracted extensive attentions, due to its unique features in detecting various biomolecules with high sensitivity and selectivity. However, currently used FET biosensors obtaining from expensive and elaborate micro/nano-fabrication are always disposable because the analyte cannot be efficiently removed after detection. In this work, we established a photocatalysis-induced renewable graphene-FET (G-FET) biosensor for protein detection, by layer-to-layer assembling reduced graphene oxide (RGO) and RGO-encapsulated TiO2 composites to form a sandwiching RGO@TiO2 structure on a pre-fabricated FET sensor surface. After immobilization of anti-D-Dimer on the graphene surface, sensitive detection of D-Dimer was achieved with the detection limits of 10 pg/mL in PBS and 100 pg/mL in serum, respectively. Notably, renewal of the FET biosensor for recycling measurements was significantly realized by photocatalytically cleaning the substances on the graphene surface. This work demonstrates for the first time the development and application of photocatalytically renewable G-FET biosensor, paving a new way for G-FET sensor towards a plethora of diverse applications.
By means of the in situ electrokinetic assessment of aqueous particles in conjunction with the addition of anionic adsorbates, we develop and examine a new approach to the scalable characterization of the specific accessible surface area of particles in water. For alumina powders of differing morphology in mildly acidic aqueous suspensions, the effective surface charge was modified by carboxylate anion adsorption through the incremental addition of oxalic and citric acids. The observed zeta potential variation as a function of the proportional reagent additive was found to exhibit inverse hyperbolic sine-type behavior predicted to arise from monolayer adsorption following the Grahame-Langmuir model. Through parameter optimization by inverse problem solving, the zeta potential shift with relative adsorbate addition revealed a nearly linear correlation of a defined surface-area-dependent parameter with the conventionally measured surface area values of the powders, demonstrating that the proposed analytical framework is applicable for the in situ surface area characterization of aqueous particulate matter. The investigated methods have advantages over some conventional surface analysis techniques owing to their direct applicability in aqueous environments at ambient temperature and the ability to modify analysis scales by variation of the adsorption cross section.
The interaction of cell and organelle membranes (lipid bilayers) with nanoelectronics can enable technologies to sense and measure electrophysiology in qualitatively new ways. To date, a variety of sensing devices have been demonstrated to measure membrane currents through macroscopic numbers of ion channels. However, nanoelectronic based sensing of single ion channel currents has been a challenge. Here, we report graphene-based field-effect transistors combined with supported lipid bilayers as a platform for measuring, for the first time, individual ion channel activity. We show that the supported lipid bilayers uniformly coat the single layer graphene surface, acting as a biomimetic barrier that insulates both electrically and chemically the graphene from the electrolyte environment. Upon introduction of pore-forming membrane proteins such as alamethicin and gramicidin A, single ion current pulses charging the quantum capacitance of the graphene are observed through the lipid bilayers from the graphene to the electrolyte. This approach combines nanotechnology with electrophysiology to demonstrate a qualitatively new way of measuring ion channel currents.
The sensing membrane of extended-gate field-effect-transistor (EGFET) pH sensors, intrinsic zinc oxide (i-ZnO) thin-film and nanorod array, was fabricated using the vapor cooling condensation method together with anodic alumina membrane (AAM) template. Furthermore, the photoelectrochemical (PEC) method was employed to passivate the ZnO thin-film and the sidewall surface of ZnO nanorod array in order to suppress the influence of Femi level pinning. The resulting EGFET pH sensors with passivated i-ZnO thin-film and i-ZnO nanorod array exhibited significantly improved sensing performances owing to the lower surface state density and the larger sensing surface-to-volume ratio. The measured sensitivities of the pH sensors with unpassivated i-ZnO thin-film and unpassivated i-ZnO nanorod array are 38.46 mV/pH and 44.01 mV/pH, respectively, at the pH value ranging from 4 to 12. The higher sensitivity of 42.37 mV/pH and 49.35 mV/pH, respectively, were measured with the pH sensors with passivated i-ZnO thin-film and passivated i-ZnO nanorod array.
Interest in the identification and isolation of circulating tumor cells (CTCs) has been growing since the introduction of CTCs as an alternative to the tumor tissue biopsy, which can potentially be important indices for prognosis and cancer treatment. However, the contamination of non-specific binding of normal hematologic cells makes high purity CTCs detection problematic. Furthermore, preserving the viability of CTCs remains a challenge. In this study, we proposed to construct an anti-EpCAM functionalized supported lipid bilayer (SLB), a biomimetic and non-fouling membrane coating, for CTCs capturing, purification and maintaining the viability. Healthy human blood spiked with pre-stained colorectal cancer cell lines, HCT116 and colo205, were used to investigate interaction of cells with the anti-EpCAM functionalized SLB surfaces. Over 97% of HCT116, and 72% of colo205 were captured and adhered by the surface anti-EpCAM; conversely, the majority of blood cells were easily removed by gentle buffer exchange, with the overall purity of cancer cells exceeding 95%. The bound cancer cells were subsequently detached for cell culture. Both HCT116 and colo205 continued to proliferate over 2-week observation period, indicating that the anti-EpCAM functionalized SLB platform providing a simple strategy for capturing, purifying, and releasing viable targeted rare cells.
We report the formation of POPC lipid bilayers that span 130 nm pores in a freestanding silicon nitride film supported on a silicon substrate. These solvent-free lipid membranes self-assemble on organosilane-treated Si3N4 via the fusion of 200 nm unilamellar vesicles. Membrane fluidity is verified by fluorescence recovery after photobleaching (FRAP), and membrane resistance in excess of 1 GΩ is demonstrated using electrical impedance spectroscopy (EIS). An array of 40 000 membranes maintained high impedance over 72 h, followed by rupture of most of the membranes by 82 h. Membrane incorporation of gramicidin, a model ion channel, resulted in increased membrane conductance. This membrane conductance was diminished when the gramicidin channels were blocked with CaCl2, indicating that the change in membrane conductance results from gramicidin-mediated ion transport. These very stable, biologically functional pore-spanning membranes open many possibilities for silicon-based ion-channel devices for applications such as biosensors and high-throughput drug screening.
The excitation of surface plasmons in nanoslits in metal films is known to result in large electromagnetic field enhancements. The enhancement factor strongly depends on the exact geometrical parameters, such as widths and lengths. In this work, we numerically study field enhancement and transmission resonances inside short nanoslits (nanopores) with varying lengths using three-dimensional finite difference time domain (FDTD) simulations. The results indicate that the excitation of highly confined lateral Fabry–Pérot surface plasmon resonances results in enhancement factors greatly exceeding the two-dimensional case. (© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)
In vitro infection of Vero E6 cells by SARS coronavirus (SARS-CoV) is blocked by hexapeptide Tyr-Lys-Tyr-Arg-Tyr-Leu. The peptide also inhibits proliferation of coronavirus NL63. On human cells both viruses utilize angiotensin-converting enzyme 2 (ACE2) as entry receptor. Blocking the viral entry is specific as alpha virus Sindbis shows no reduction in infectivity. Peptide (438)YKYRYL(443) is part of the receptor-binding domain (RBD) of the spike protein of SARS-CoV. Peptide libraries were screened by surface plasmon resonance (SPR) to identify RBD binding epitopes. (438)YKYRYL(443) carries the dominant binding epitope and binds to ACE2 with K(D)=46 μM. The binding mode was further characterized by saturation transfer difference (STD) NMR spectroscopy and molecular dynamic simulations. Based on this information the peptide can be used as lead structure to design potential entry inhibitors against SARS-CoV and related viruses.
In this study, we have successfully demonstrated that a GaN nanowire (GaNNW) based extended-gate field-effect-transistor (EGFET) biosensor is capable of specific DNA sequence identification under label-free in situ conditions. Our approach shows excellent integration of the wide bandgap semiconducting nature of GaN, surface-sensitivity of the NW-structure, and high transducing performance of the EGFET-design. The simple sensor-architecture, by direct assembly of as-synthesized GaNNWs with a commercial FET device, can achieve an ultrahigh detection limit below attomolar level concentrations: about 3 orders of magnitude higher in resolution than that of other FET-based DNA-sensors. Comparative in situ studies on mismatches ("hotspot" mutations related to human p53 tumor-suppressor gene) and complementary targets reveal excellent selectivity and specificity of the sensor, even in the presence of noncomplementary DNA strands, suggesting the potential pragmatic application in complex clinical samples. In comparison with GaN thin film, NW-based EGFET exhibits excellent performance with about 2 orders higher sensitivity, over a wide detection range, 10(-19)-10(-6) M, reaching about a 6-orders lower detection limit. Investigations illustrate the unique and distinguished feature of nanomaterials. Detailed studies indicate a positive effect of energy band alignment at the biomaterials-semiconductor hybrid interface influencing the effective capacitance and carrier-mobility of the system.
The electronic properties of graphene can be modulated by charged lipid bilayer adsorbing on the surface. Biorecognition events which lead to changes in membrane integrity can be monitored electrically using an electrolyte-gated biomimetic membrane-graphene transistor. Here, we demonstrate that the bactericidal activity of antimicrobial peptides can be sensed electrically by graphene based on a complex interplay of biomolecular doping and ionic screening effect.
Membrane proteins, which are the target of most drugs, are implicated in many critical cellular functions such as signal transduction, bioelectricity, exocytosis and endocytosis. Therefore, developing techniques to investigate the functions of membrane proteins is obviously important. Here, we have developed a novel system by integrating artificial lipid bilayer (biomimetic membrane) with single-walled carbon nanotube networks (SWNT-net) based field-effect transistor (FET), and demonstrated that such hybrid nanoelectronic biosensors can specifically and electronically detect the presence and dynamic activities of ionophores (specifically, gramicidin and calcimycin) in their native lipid environment. This technique can potentially be used to examine other membrane proteins (e.g. ligand-gated ion channels, receptors, membrane insertion toxins, and antibacterial peptides) for the purposes of biosensing, fundamental studies, or high throughput drug screening.
Miniaturized technologies for high-throughput drug screening
Miniaturized technologies for high-throughput drug screening
enzymatic assays and diagnostics − A review. TrAC, Trends Anal
enzymatic assays and diagnostics − A review. TrAC, Trends Anal.
- L Chen
- D Lv
- S Wang
- D Wang
- X Chen
- Y Liu
- Z Hong
- Z Zhu
- Y Cao
- Y Chai
Chen, L.; Lv, D.; Wang, S.; Wang, D.; Chen, X.; Liu, Y.; Hong,
536 Z.; Zhu, Z.; Cao, Y.; Chai, Y. Surface Plasmon Resonance-Based
- W H Huang
- Z Zhang
- G J Zhang
Huang, W. H.; Zhang, Z.; Zhang, G. J. Photocatalysis-Induced
Renewable Field-Effect Transistor for Protein Detection
Renewable Field-Effect Transistor for Protein Detection. Anal. Chem.
549 2016, 88, 4048−4054.
Lipid-Modified Graphene-Transistor Biosensor for
- R Chen
R.; Chen, Y. T. Lipid-Modified Graphene-Transistor Biosensor for
Monitoring Amyloid-β Aggregation
Monitoring Amyloid-β Aggregation. ACS Appl. Mater. Interfaces 2018,
554 10, 12311−12316.
Specific and label-free immunosensing of
- B Akabayov
- G Shalev
Akabayov, B.; Shalev, G. Specific and label-free immunosensing of