key: cord-1049705-oo8p3dw4 authors: Lebohang Manoto, Sello; El-Hussein, Ahmed; Malabi, Rudzani; Thobakgale, Lebogang; Ombinda-Lemboumba, Saturnin; Attia, Yasser A.; Kasem, Mohamed.A.; Mthunzi-Kufa, Patience title: Exploring optical spectroscopic techniques and nanomaterials for virus detection date: 2020-08-27 journal: Saudi J Biol Sci DOI: 10.1016/j.sjbs.2020.08.034 sha: 32935a40b63f37564ba8e11791679e6f6a32d890 doc_id: 1049705 cord_uid: oo8p3dw4 Viral infections pose significant health challenges globally by affecting millions of people worldwide and consequently resulting in a negative impact on both socioeconomic development and health. Corona virus disease 2019 (COVID-19) is a clear example of how a virus can have a global impact in the society and has demonstrated the limitations of detection and diagnostic capabilities globally. Another virus which has posed serious threats to world health is the human immunodeficiency virus (HIV) which is a lentivirus of the retroviridae family responsible for causing acquired immunodeficiency syndrome (AIDS). Even though there has been a significant progress in the HIV biosensing over the past years, there is still a great need for the development of point of care (POC) biosensors that are affordable, robust, portable, easy to use and sensitive enough to provide accurate results to enable clinical decision making. The aim of this study was to present a proof of concept for detecting HIV-1 pseudoviruses by using anti-HIV1 gp41 antibodies as capturing antibodies. In our study, glass substrates were treated with a uniform layer of silane in order to immobilize HIV gp41 antibodies on their surfaces. Thereafter, the HIV pseudovirus was added to the treated substrates followed by addition of anti-HIV gp41 antibodies conjugated to selenium nanoparticle (SeNPs) and gold nanoclusters (AuNCs). The conjugation of SeNPs and AuNCs to anti-HIV gp41 antibodies was characterized using UV-vis spectroscopy, transmission electron microscopy (TEM) and zeta potential while the surface morphology was characterized by fluorescence microscopy, atomic force microscopy (AFM) and Raman spectroscopy. The UV-vis and zeta potential results showed that there was successful conjugation of SeNPs and AuNCs to anti-HIV gp41 antibodies and fluorescence microscopy showed that antibodies immobilized on glass substrates were able to capture intact HIV pseudoviruses. Furthermore, AFM also confirmed the capturing HIV pseudoviruses and we were able to differentiate between substrates with and without the HIV pseudoviruses. Raman spectroscopy confirmed the presence of biomolecules related to HIV and therefore this system has potential in HIV biosensing applications. Mankind has battled many types of viruses over the years, which have caused infectious diseases in humans, plants and animals (Zhao et al., 2020) . Viruses are the main source of various diseases ranging from mild to life threatening effects with masked or severe symptoms (Virgin, 2014) . Different types of viruses have shown to be responsible for the development of certain cancers (Hausen, 2009) had significantly reduced by more than 40% since the infection peak in 1997. Sub-Saharan Africa has the highest prevalence of HIV, accounting for more than 70% of all HIV infections . Despite, the deep effects and severe impacts of viral infections on the global health and economy, our capabilities for quick, efficient and rapid viral detection are still limited and incomplete. This may be due to many reasons, one of which is the selectivity characteristic of viral infection that could be to specific tissues and/or organs in part due to the differential ability of viruses to infect selected tissues and organ systems (Hausen 2009 ). The presence of reliable diagnostic tool is an essential for the delivery of proper health services that are needed to the patients and the surrounding communities. In case of the HIV, there is a large number of HIV diagnostics that have been developed. The currently used HIV diagnostics include; western blotting, enzyme linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). Albeit these techniques have proved to be very reliable and sensitive, identification of the virus and quantification methods are still laboratory based, time consuming and often expensive, this necessitates the need for accurate, rapid and sensitive virus biosensors (Lee et al., 2015) . Regardless of the type of biosensor and the detection of the analyte either at solidliquid interphase or in a solution involving the use of nanoparticles, the attachment of enzymes, antibodies, DNA or cells is inevitable (Sonawane and Nimse, 2016) . Assay sensitivity of biosensors depends on the conformation of the immobilised biomolecules such as antibodies and therefore performance of the system is greatly dependent on the surface chemistry used for immobilisation of biomolecules (Welch et al., 2017) . The introduction of nanotechnology in the biosensor field has increased the sensitivity and other analytical characteristics of the biosensor. Nanotechnology uses nanoparticles ranging between 1 and 1000 nm in size and these particles have unique properties such as surface chemistry, shape dependence and optical properties that enable a variety of application including bioimaging, biosensing, vaccine development and drug delivery (Jazayeri et al., 2016) . Various types of nanomaterials such as carbon nanotubes (CNTs), quantum dots, magnetic nanoparticles, selenium nanoparticles (SeNPs), silver (Ag) and gold nanoparticles (AuNPs) or gold nanoclusters (AuNCs) have been used in biosensors with gold particles being the most commonly used type of nanoparticles because of their ability to increase sensitivity and specificity (Zhang et al., 2009) . AuNCs have played a pivotal role in recent advances of fluorescent probe research because of their ability to fluoresce while SeNPs has attracted attention because of their large surface area, high surface activity, powerful adsorbing ability and low toxicity. AuNCs have been used in fields such as biomarkers detection, fluorescence detection and catalysis because of their good stability of fluorescence, low cytotoxicity and good biocompatibility. SeNPs also show favourable biocompatibility and can be easily conjugated with biomolecules without losing activity and preparing them is easy and cost effective (Wang et al., 2019) . Biomolecules such as antibodies can be immobilized on both AuNCs and SeNPs either passively through ionic or hydrophobic interactions or covalently through a chemical reaction with a surface group (Finetti et al., 2016) . The aim of the current study is to present as a proof of concept, the development of a biosensor system that would enable for rapid and robust detection of viral threats. The current research focused on HIV-1 pseudoviruses detection by using anti-HIV1 gp41 antibodies as capturing antibodies. A uniform and stable layer of silane was formed on glass substrates using (3-glycidoxypropyl)trimethoxysilane (GPTMS) for covalent attachment of the amine group of the HIV antibodies to the glass substrates. The HIV pseudovirus was added to the substrates, which then bound to the immobilised HIV antibodies. This was followed by the addition of AuNCs and SeNPs conjugated to HIV antibodies, which would bind to the HIV pseuodovirus. The surfaces and nanoparticles before and after conjugation were characterised by UVvis spectroscopy, transmission electron microscopy (TEM), atomic force microscopy (AFM), laser induced fluorescence (LIF) and Raman spectroscopy. Sodium selenite, ascorbic acid, hydrogen peroxide, sulfuric acid, polyvinylpyrrolidone (PVP), sodium borohydride (NaBH 4 , 95%), hydrogen tetrachloroauratetrihydrate A modified chemical reduction method was used, where 5 mM of sodium selenite solution was prepared. Ascorbic acid was used as a stock reducing agent at a concentration of 100 mM and the protecting agent, Polyvinylpyrrolidone (PVP) was made up at 0.1% (v/v) concentration. All solvents were dissolved in Milli-Q water at room temperature. Reactants were introduced in the following order: 2.5 ml of PVP was added to 15 ml of sodium selenite and followed by 2.5 ml of ascorbic acid. The solution was mixed under mild stirring rates for 1 -2 min, until a deep red clear colour was achieved, this colour demonstrates reaction progression. The prepared SeNPs were conjugated to anti-HIV1 gp41 antibody (Abcam, ab9065) by mixing the SeNPs and anti-HIV1 antibody at a ratio of 1:1 and sonicating the mixture for 40 minutes. The solution was centrifuged at 2500 rpm for 1 minute to remove unbound antibody and the supernatant was discarded. Conjugates were resuspended in 1X phosphate buffered saline (PBS) with a pH of 7.4. AuNCs were prepared by modifying Attia et al., 2016 method. In brief, 5 ml of 0.2 M cetyltrimethylammonium chloride (CTAC) solution was mixed with 5 ml of 5 mM hydrogen tetrachloroauratetrihydrate (HAuCl 4 ) solution. While stirring the solution, 60 ml of ice-cold freshly prepared 0.1 M sodium borohydride (NaBH 4 ) was added and this resulted in the formation of a solution with a brownish yellow colour. Vigorous stirring of the cluster solution was continued and used after 5 min of preparation. The prepared AuNCs were conjugated to anti-HIV1 gp41 antibody as before. The absorption spectra were analysed using the NanoDrop ND 1000 spectrophotometer to determine the red shifts before and after functionalisation of nanoparticles with anti-HIV1 gp41 antibodies. Several UV-vis spectra (~5) were taken for the SeNPs and AuNCs before and after they were conjugated to anti-HIV1 gp41 antibodies. The zeta potential of SeNPs and AuNCs dispersed in water before and after conjugation with anti-HIV1 gp41 antibodies were analysed using the Malvern Zetasizer Nano ZS (Malvern Instruments Ltd., United Kingdom). Characterisation of SeNPs and AuNCs before and after they were conjugated to anti-HIV1 gp41 antibodies was done using TEM. The samples were prepared by drying a drop of solution containing nanoparticles on the TEM grid. The images of the samples on the grid were taken using the JEOL TEM (JEM 2100F). For LIF measurements, we used a continuous wave (CW) diode pumped solid state laser (DPSS) with 100 mW power and emission wavelength was measured at 405 nm. LIF was used to analyse SeNPs and AuNCs before and after conjugation with anti-HIV gp41 antibodies. The examined samples were in a quartz cuvette of 10 mm thickness and the laser light was delivered by means of an optical fibre. The emitted fluorescence was collected and delivered to a spectrometer (USB2000 FLG Ocean Optics, USA) via another optical fibre placed at 90° to the excitation light. Spectra Suit software (Ocean Optics, USA) was used for acquiring and collecting the data, while spectra analysis was done by using the Origin Lab. Penicillin-Streptomycin (Gibco, 15140122). The cells were incubated at 37 °C in 5% CO 2 and 85% humidity until they reached a confluency of 80% which was usually after 2 to 3 days. After reaching confluency, the cells were then co-transfected with components of HIV-1 in order to produce the HIV pseudoviruses. The HIV-1 pseudoviruses were produced by transfecting HEK293 T cells using the Glass substrates were activated using piranha solution (H 2 SO 4 :H 2 O 2 , 7:3 v/v) at 75 °C for 45 minutes in order to hydroxylate the surface of the glass substrates. After activation, glass substrates were thoroughly washed in ultrapure water and blow dried using nitrogen and placed in an oven to remove any water on the surface. Substrates were then exposed to (3-glycidoxypropyl)trimethoxysilane (GPTMS) at 80 °C in vacuum for 6 hours. After silanization, the glass substrates were sonicated in toluene, methanol, and ultrapure water and dried under a stream of nitrogen. Anti-HIV1 gp41 antibodies were immobilized on the silanized glass substrates by incubating the glass substrates with HIV antibodies overnight at 4 °C. After overnight incubation, HIV-1 pseudoviruses at a concentration of 300 pg/ml were added to the surface for 1 hour at room temperature, followed by washing with ultra-pure water. Anti-HIV1 gp41 antibodies conjugated to SeNPs and AuNCs were the added to the surface of the substrates. The surface morphology of glass substrates coated with anti-HIV1 gp41 antibodies, HIV pseudoviruses, SeNPs and AuNCs conjugated to HIV antibodies were analysed using atomic force microscopy (AFM). The AFM images were captured using a Vecco AFM system (Digital Instruments, USA) using a silicon cantilever tip. The tip has a curvature radius of 10 nm and is n-doped silicon, with a resonance frequency of 204-497 kHz and a force constant of 10-130 N/m. The images acquired by the AFM instrument were analysed using the Nanoscope software. An illustration of the custom-built Raman optical setup that was used to perform Raman measurements on the surface of the substrates is shown in figure 1 . The Raman excitation source was a 527nm single mode diode laser (Evolution Nd:YLF, diode-pumped, Q-switched) of 1 KHz repetition rate with 5 µs pulse duration. The laser beam was expanded using two lenses telescope (L1 and L2) and delivered to the sample through a 100X microscope objective (NA = 1.25). A dichroic mirror was used to direct the laser beam to the back of the microscope objective lens and remove the Rayleigh scattering. The spot size of the beam at the sample was approximately 1 μm with a power of 10 mW. After passing through a notch filter (to further remove the Rayleigh scattering), the backscattered Raman signal was collected and guided to a spectrograph (Andor Shamrock spectrometer) and a deep cooling CCD camera (Newton, DU9-20P, Andor CCD camera) through an optical fiber. A grating with 1200 g/mm and 500 nm as blazing wavelength was chosen to disperse the Raman signal. The nanoparticles conjugated to anti-HIV1 gp41 antibodies were characterised using UV-vis spectroscopy ( Figure 2) . The spectrophotometer is able to identify components in a solution based on the unique absorbance characteristic of the components in the solution. The SeNPS and AuNCs showed absorption at wavelengths of ~300 and ~250 nm respectively before conjugation with the anti-HIV1 gp41 antibodies. After conjugation with the antibody there was a reduction in the absorption intensity in both SeNPS and AuNCs. The decrease in the UV-vis spectrum intensity could indicate that successful conjugation of nanoparticles to antibodies was achieved. It is generally accepted that the intensity and absorption peak of the nanoparticles is dependent on the size, shape and agglomeration of the particles (Link and El-Sayed, 2003) . Both SeNPs and AuNCs maintained their characteristic band shape after conjugating to biomolecules showing that the nanoparticles did not experience aggregation after binding to biomolecules (Vahdati and Moghadam, 2020). Interestingly, no red shifts were recorded after conjugation to HIV antibodies for both SeNPS and AuNCs which was unexpected since most conjugation of nanoparticles to antibodies results in a red shift. To achieve conjugation of nanoparticles to antibodies, physical and chemical interaction can be used and in this study the physical interaction method was adopted. The physical interaction between the antibodies and nanoparticles depends on three phenomena namely; hydrophobic interaction between the metallic surface and the antibody, ionic attraction between the positively charged antibodies and the negatively charged The nanoparticles morphology was characterised using TEM. Figure 3 shows TEM images of SeNPs and AuNCs before and after they were conjugated to anti-HIV1 The fluorescence spectrum of AuNCs and SeNPs before and after conjugation with HIV antibodies when irradiated with laser light are shown in Figure 4 . Metallic nanoparticles such as gold and semi-metallic elements like selenium have intrinsic fluorescence and a characteristic spectrum. Fluorescence staining was done in order to determine whether the immobilized anti-HIV1 gp41 antibodies on the surface of the glass substrates were able to capture the HIV pseudoviruses. Figure AFM was used to analyse the surface of the substrates and each of the scan represent a 2 m X 2 m lateral area that was scanned. AFM was used to characterize surfaces that were coated with anti-HIV1 gp41 antibodies followed by the addition of HIV-1 pseudoviruses and no viruses were added to the other substrates as shown in the figure 6. Both SeNPs and AuNCs conjugated to HIV antibodies were then added to surfaces and AFM images were taken (Figure 7 ). In the absence of HIV-1 pseudoviruses there were fewer complexes that resembled SeNPs and AuNCs as shown in Figure 7B The HIV viral particle is spherical in shape and has a diameter of approximately 120 nm which is 60 times smaller than the size of a red blood cell (Otange et al., 2017; Freed, 1998; Mascarenhas and Musier-Forsyth, 2009; Farzin et al., 2020) . The virus is surrounded by a phospholipid enveloped containing surface proteins; glycoprotein gp120 and glycoprotein gp41. On the inside, the virus has a cone shaped HIV-1 p24 antigen which is located on its core which is surrounded by a matrix composed of viral protein p17 (Farzin et al., 2020) . It is expected that when the virus is excited, the Raman scattered radiation will consist of bands associated with the composition of the virus. This study represents a proof of concept by using biosensors and nanomaterial as a reliable and potential tool for viral threat identification. In summary, we synthesized 2) can be targeted. This will involve the immobilization of SARS-CoV-2 spike antibody on salinized glass substrates for capturing SARS-CoV-2 viruses. The sample to be tested can then be added to the substrates and the viruses will be captured by the immobilized antibody. Nanoparticles conjugated SARS-CoV-2 spike antibody can be added to the surface of the substrates for signal enhancement and improvement of the sensitivity of system. The same spectroscopic approach followed in the study can be adopted for the diagnosis of COVID-19. Future studies are required to validate this concept with COVID-19 and other viruses with the identification of the setup limit of detection, sensitivity and selectivity. 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The authors declare no conflict of interest.