Exploring optical spectroscopic techniques and nanomaterials for virus detection (original) (raw)
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Quantitative Detection of HIV-1 Particles Using HIV-1 Neutralizing Antibody-Conjugated Beads
Analytical Chemistry, 2009
The human immunodeficiency virus (HIV) pandemic mainly affects developing countries, where the Joint United Nations Programme on HIV/AIDS (UNAIDS) estimates suggest that less than 1 in 10 people are aware of their HIV sero-status. In order to enhance epidemiological surveys, prevention programs, and therapeutic interventions, development of specific, rapid, and convenient diagnostic detection systems is still warranted. Here we report the direct detection of HIV particles using broadly HIV-1 neutralizing gp120 monoclonal antibody (gp120MAbs)-conjugated magnetic beads (MBs) and fluorescent nanosized polymeric beads (FNBs). The HIV-1 envelope glycoprotein gp120 is anchored to the viral surface through gp41 and mediates entry into target cells by interaction with the main cellular receptor (CD4) and coreceptors (e.g., CCR5 and CXCR4). FNBs conjugated to gp120MAbs (gp120MAbs-FNBs) were used to generate fluorescent signals, whereas MBs conjugated to gp120MAbs (gp120MAbs-MBs) were employed to isolate HIV-1 particles. In presence of HIV-1 particles, addition of gp120MAbs-FNBs and gp120MAbs-MBs leads to the formation of a MBs/HIV-1 particles/FNB complex, which can be easily isolated and concentrated by common magnet separation. We demonstrate the ability of detecting HIV-1 particles specifically and directly using MBs and FNBs with low sample volume (less than 100 µL) and rapidity (less than 1.5 h) without any pretreatment of test samples. The specific binding of FNBs with HIV-1 particles on the surface of MBs was confirmed by fluorescence microscopy and fluorescence-activated cell sorting (FACS). Imaging and FACS analysis revealed the specific and quantitative detection of HIV-1 particles. These results provide proof-of-principle that broadly HIV-1 neutralizing gp120 antibodies coupled to nanobeads can be employed for the direct detection of HIV-1 particles with potential implication for the development of specific, rapid, and convenient diagnostic systems.
Talanta, 2019
Human immunodeficiency virus (HIV) is a lentivirus that leads to acquired immunodeficiency syndrome (AIDS). With increasing awareness of AIDS emerging as a global public health threat, different HIV testing kits have been developed to detect antibodies (Ab) directed toward different parts of HIV. A great limitation of these tests is that they can not detect HIV antibodies during early virus infection. Therefore, to overcome this challenge, a wide range of biosensors have been developed for early diagnosis of HIV infection. A significant amount of these studies have been focused on the application of nanomaterials for improving the sensitivity and accuracy of the sensing methods. Following an introduction into this field, a first section of this review covers the synthesis and applicability of such nanomaterials as metal nanoparticles (NPs), quantum dots (QDs), carbon-based nanomaterials and metal nanoclusters (NCs). A second larger section covers the latest developments concerning nanomaterial-based biosensors for HIV diagnosis, with paying a special attention to the determination of CD4 + cells as a hall mark of HIV infection, HIV gene, HIV p24 core protein, HIV p17 peptide, HIV-1 virus-like particles (VLPs) and HIV related enzymes, particularly those that are passed on from the virus to the CD 4+ T lymphocytes and are necessary for viral reproduction within the host cell. These studies are described in detail along with their diverse principles/mechanisms (e.g. electrochemistry, fluorescence, electromagnetic-piezoelectric, surface plasmon resonance (SPR), surface enhanced Raman spectroscopy (SERS) and colorimetry). Despite the significant progress in HIV biosensing in the last years, there is a great need for the development of point-of-care (POC) technologies which are affordable, robust, easy to use, portable, and possessing sufficient quantitative accuracy to enable clinical decision making. In the final section, the focus is on the portable sensing devices as a new standard of POC and personalized diagnostics.
Development of nanobiosensors for human immunodeficiency virus detection—a mini review
Exploration of Medicine
Acquired immunodeficiency syndrome (AIDS) is a global disease caused by human immunodeficiency virus (HIV). About 50 million people have died worldwide due to HIV-1 infection alone. HIV is a primary sexually transmitted infection but can also spread via breastfeeding, blood transfer, organ transfer, etc. Early detection with the maintenance of the disease is the only way to reduce the spread and severity of the disease. There are many conventional techniques for the detection of the virus. Still, recently nano-based diagnostic method remains a little ahead of these techniques due to advancements in nanotechnology. Nanomaterial-based biosensors constitute a significant part of the discussion because of their high sensitivity and accuracy. Nanobiosensors like electronic nano biosensors, quantum dot (QD)-based biosensors, optical biosensors, electronic biosensors, electrochemiluminescence nanosensors, field-effect transistor (FET) biosensors, surface acoustic wave (SAW) biosensors, gra...
Surface Plasmon Resonance Assay for Label-Free and Selective Detection of HIV-1 p24 Protein
Biosensors, 2021
The early detection of the human immunodeficiency virus (HIV) is of paramount importance to achieve efficient therapeutic treatment and limit the disease spreading. In this perspective, the assessment of biosensing assay for the HIV-1 p24 capsid protein plays a pivotal role in the timely and selective detection of HIV infections. In this study, multi-parameter-SPR has been used to develop a reliable and label-free detection method for HIV-1 p24 protein. Remarkably, both physical and chemical immobilization of mouse monoclonal antibodies against HIV-1 p24 on the SPR gold detecting surface have been characterized for the first time. The two immobilization techniques returned a capturing antibody surface coverage as high as (7.5 ± 0.3) × 1011 molecule/cm2 and (2.4 ± 0.6) × 1011 molecule/cm2, respectively. However, the covalent binding of the capturing antibodies through a mixed self-assembled monolayer (SAM) of alkanethiols led to a doubling of the p24 binding signal. Moreover, from th...
Multi-scale silica structures for improved HIV-1 Capsid (p24) antigen detection
The Analyst, 2016
Silica (SiO2) micro- and nanostructures fabricated with pre-stressed thermoplastic shrink wrap film have been shown to yield far-field fluorescence signal enhancements over their planar or wrinkled counterparts. The SiO2 structures have previously been used for improved detection of fluorescently labelled proteins and DNA. In this work, we probe the mechanism responsible for the dramatic increases in fluorescence signal intensity. Optical characterization studies attribute the fluorescence signal enhancements to increased surface density and light scattering from the rough SiO2 structures. Using this information, we come up with a theoretical approximation for the enhancement factor based off the scattering effects alone. We show that increased deposition thickness of SiO2 yields improved fluorescence signal enhancements, with an optimal SiO2 thin layer achieved at 20 nm. Finally, we show that the SiO2 substrates serve as a suitable platform for disease diagnostics, and show improve...
Nano Letters, 2004
Arrays of antibodies with well-defined feature size and spacing are necessary for developing highly sensitive and selective immunoassays to detect macromolecules in complex solutions. Here we report the application of nanometer-scale antibody array-based analysis to determine the presence of the human immunodeficiency virus type 1 (HIV-1) in blood samples. Dip-pen nanolithography (DPN) was used to generate nanoscale patterns of antibodies against the HIV-1 p24 antigen on a gold surface. Feature sizes were less than 100-nanometers, and the activity of the antibody was preserved. HIV-1 p24 antigen in plasma obtained directly from HIV-1-infected patients was hybridized to the antibody array in situ, and the bound protein was hybridized to a gold antibody-functionalized nanoparticle probe for signal enhancement. The nanoarray features in the three-component sandwich assay were confirmed by atomic force microscopy (AFM). Demonstration of measurable amounts of HIV-1 p24 antigen in plasma obtained from men with less than 50 copies of RNA per ml of plasma (corresponding to 0.025 pg per ml) illustrates that the nanoarray-based assay can exceed the limit of detection of conventional enzyme-linked immunosorbent assay (ELISA)based immunoassays (5 pg per ml of plasma) by more than 1000-fold.
Advancements in Biosensing Technologies for the Detection of Human Immunodeficiency Virus
Advances in Preventive Medicine and Health Care, 2021
HIV (Human Immunodeficiency Virus) is one of the major health concerns and continues to be an epidemic causing threat to a large population of the world. Despite great efforts and strategies being employed for containing its spread, it still is hard to tackle in resource-limited settings. Conventional molecular detection methods are laborious and require trained staff and well-equipped laboratory facilities. In this review we have discussed biosensors for rapid, portable and highly sensitive point-of-care detection providing timely diagnosis and monitoring of infectious agents like HIV. Different types of biosensors for the detection of HIV have been developed over the last twenty years including Electrochemical, Optical and Piezoelectric biosensors. Although biosensors provide various advantages over standard diagnostic methods still there are technical limitations that need to be addressed for their commercial implementation. Advances in Preventive Medicine and Health Care S Rafiq...
HIV Virions as Nanoscopic Test Tubes for Probing Oligomerization of the Integrase Enzyme
ACS Nano, 2014
Employing viruses as nanoscopic lipid-enveloped test tubes allows the miniaturization of protein-protein interaction (PPI) assays while preserving the physiological environment necessary for particular biological processes. Applied to the study of the human immunodeficiency virus type 1 (HIV-1), viral biology and pathology can also be investigated in novel ways, both in vitro as well as in infected cells. In this work we report on an experimental strategy that makes use of engineered HIV-1 viral particles, to allow for probing PPIs of the HIV-1 integrase (IN) inside viruses with single-molecule Förster resonance energy transfer (FRET) using fluorescent proteins (FP). We show that infectious fluorescently labeled viruses can be obtained and that the quantity of labels can be accurately measured and controlled inside individual viral particles. We demonstrate, with proper control experiments, the formation of IN oligomers in single viral particles and inside viral complexes in infected cells. Finally, we show a clear effect on IN oligomerization, of small molecule inhibitors of interactions of IN with its natural human co-factor LEDGF/p75, corroborating that IN oligomer enhancing drugs are active already at the level of the virus and strongly suggesting the presence of a dynamic, enhanceable equilibrium between the IN dimer and tetramer in viral particles. Although applied to the HIV-1 IN enzyme, our methodology for utilizing HIV virions as nanoscopic test tubes for probing PPIs is generic, i.e. other PPIs targeted into the HIV-1, or PPIs targeted into other viruses, can potentially be studied with a similar strategy. KEYWORDS single-molecule fluorescence microscopy, HIV-1 integrase, stoichiometry, Förster resonance energy transfer, protein-protein interactions, nanoscopy AlphaScreen (PerkinElmer, Zaventem, Belgium) is a proximity based chemiluminescence assay that allows studying bimolecular interactions in vitro. The assay was performed largely as described before. 81 In brief, glutathione S transferase (GST) tagged HIV-1 IN was purified as described previously. 81 Wild-type IN-His 6 and the point mutant IN W108G -His 6 , were purified as described previously. 26 The assay buffer was 25 mM Tris/HCl pH 7.5, 150 mM NaCl, 1 mM dithiothreitol, 1 mM MgCl 2 0.1% (w/V) BSA, 0.1% (V/V) Tween-20. Per well in a 384-well microtiter plate (OptiPlate, PerkinElmer), 5 µL buffer, 5 µL of 5× working solutions of GST-IN and IN-His 6 or IN W108G -His 6 were added. The plate was sealed and left to incubate for 3 hours at 4 °C. Next, 10 µL of a mix of glutathione donor beads and Ni 2+ -chelate acceptor beads was added, bringing the final volume to 25 µL and establishing final concentrations of 20 µg/mL for the beads, 15 nM for GST-IN and 0-150 nM for IN-His 6 or IN W108G -His 6 . The plate was sealed again and incubated for 2 more hours at room temperature. The AlphaScreen signal (520-620 nm) was read out with a plate reader (EnVision Multilabel, PerkinElmer) and data were analyzed using Prism 5.0 (GraphPad Software, La Jolla, USA). JHendrix and ZD conceived and designed the research, DB performed experiments, DB and
ACS Nano, 2011
Virus life stages often constitute a complex chain of events, difficult to track in-vivo and in realtime. Challenges are associated with spatial and time limitations of current probes: most viruses are smaller than the diffraction limit of optical microscopes while the entire timescale of virus dynamics spans over 8 orders of magnitude. Thus, virus processes such as entry, disassembly, and egress have generally remained poorly understood. Here we discuss photothermal heterodyne imaging (PHI) as a possible alternative to fluorescence microscopy in the study of single virus-like nanoparticle (VNP) dynamics, with relevance in particular to virus uncoating. Being based on optical absorption rather than emission, PHI could potentially surpass some of the current limitations associated with fluorescent labels. As proof-of-principle, single VNPs self-assembled from 60 nm DNA-functionalized gold nanoparticles (DNA-Au NPs) encapsulated in a Gag protein shell of the Human immunodeficiency virus (HIV-1) were imaged and their photothermal response compared with DNA-Au NPs. For the first-time, the protein stoichiometry of a single virus-like particle was estimated by a method other than electron microscopy.
Rapid HIV-1 capsid interaction screening using fluorescence fluctuation spectroscopy
bioRxiv (Cold Spring Harbor Laboratory), 2020
The HIV capsid is a multifunctional protein capsule for delivery of the viral genetic material into the nucleus of the target cell. Host cell proteins bind to a number of repeating binding sites on the capsid to regulate steps in the replication cycle. Here we develop a fluorescence fluctuation spectroscopy method using self-assembled capsid particles as the bait to screen for fluorescence-labelled capsid-binding analytes ('prey' molecules) in solution. The assay capitalizes on the property of the HIV capsid as a multivalent interaction platform, facilitating high sensitivity detection of multiple prey molecules that have accumulated onto capsids as spikes in fluorescence intensity traces. By using a scanning stage, we reduced the measurement time to 10 s without compromising on sensitivity, providing a rapid binding assay for screening libraries of potential capsid interactors. The assay can also identify interfaces for host molecule binding by using capsids with defects in known interaction interfaces. Two-color coincidence detection using fluorescent capsid as bait further allows quantification of binding levels and determination of binding affinities. Overall, the assay provides new tools for discovery and characterization of molecules used by HIV capsid to orchestrate infection. The measurement principle can be extended for the development of sensitive interaction assays utilizing natural or synthetic multivalent scaffolds as analyte-binding platforms. .