Recent advances in micro/nanotechnologies for global control of hepatitis B infection

Abstract

The control of hepatitis B virus (HBV) infection is a challenging task, specifically in developing countries there is limited access to diagnostics and antiviral treatment mainly due to high costs and insufficient healthcare infrastructure. Although the current diagnostic technologies can reliably detect HBV, they are relatively laborious, impractical and require expensive resources that are not suitable for resource-limited settings. Advances in micro/nanotechnology are pioneering the development of new generation methodologies in diagnosis and screening of HBV. Owing to combination of nanomaterials (metal/inorganic nanoparticles, carbon nanotubes, etc.) with microfabrication technologies, utilization of miniaturized sensors detecting HBV and other viruses from ultra-low volume of blood, serum and plasma is realized. The state-of-the-art microfluidic devices with integrated nanotechnologies potentially allow for inexpensive HBV screening at low cost. This review aims to highlight recent advances in nanotechnology and microfabrication processes that are employed for developing point-of-care (POC) HBV assays.

Keywords: Diagnosis; Graphene; Hepatitis B; Microfluidic; Nanobiotechnology; Nanotechnology; Plasmonics; Point-of-care; Screening.

Fig 1

Global distribution of HBV infection according to the WHO

Fig 2

Serum biomarkers for acute HBV infection. During acute HBV infection, HBsAg is the first antigen that appears. It clears out prior to the emergence of anti-HBs antibody. The period in between is called “window period”, in which anti-HBc IgM may be the only serological indicator of HBV infection. Therefore, HBsAg and the total anti-HBc antibodies (including IgM and IgG) are employed for HBV diagnosis.

Fig 3

HBV Infection, Diagnosis and Treatment Loop. Upper panel illustrates the viral genetic material, and hepatits B virus. Serum level of HBV DNA and ALT activity in acute and chronic phase. Middle panel shows the conventional methods: serologic assays and nucleic acid assays, advanced technologies: electrochemical sensor (interdigitated electrodes made of graphene, carbon nanotubes), mechanical sensors (quartz crystal microbalance and microcantilevers) and optical sensors (plasmonic platforms made of gold nanoparticles and photonic crystal based platforms). Lower panel shows chemical structure of immune modulator PEG-IFN and nucleos(t)ide analogs: LAM, ADV, ETV, LdT, TDF. (Reproduced from: (Arlett et al., 2011, Mannoor, Tao, 2012, Michael R. Kierny, 2012, Shafiee, 2014, Wynne et al., 1999))

Fig 4

An electrochemical immunosensor array enabling simultaneous detection of hepatitis virus antigens (hepatitis A, B, C, D, E) were utilized to develop a facile assay methodology. Five-type HBV virus antibodies functionalized self-made electrochemical sensor that were made of gold nanoparticles and protein A. The prototype immunosensor array used for a single-step antigen capturing. The detection relies on the potential change between priori and post antigen-antibody interaction. a) Illustration shows the immunosensor array integrated into fluidic system. Graphs (b) shows comparison of the results obtained by using the developed immunosensor array and the standard ELISA for HBV samples from 43 serum specimens. (Tang, Tang, 2010). c) DNA biosensors proposed by Wang et al. (2013a), FE-SEM images of aligned SWCNTs-gold nanoparticle hybrid sensors for the detection of hepatitis B DNAs. d) electrochemical response of the proposed sensor that compares 1-base mismatched and complementary DNAs. e) The use of individual single-walled carbon nanotubes as nanoelectrodes for electrochemistry (Heller et al., 2005), Micro-fabricated device schematic. CNTs are grown on Si wafers with 500 nm thermal SiO2 and contacted by Ti leads. A layer of SiOx and PMMA is used as insulating layer, in which windows are opened to selectively expose the SWNTs. (f) AFM-amplitude image of an exposed section of a CNT crossing the bottom of the pit through the PMMA and SiOx layers. (g) Low current measurement setup. The SWNT is exposed to a solution containing a redox-active species. (h) Sampled current voltammograms measured from two metallic SWNT devices (1 μm and 2 μm exposed). i) CNT network field-effect transistors (NTNFETs) that function as selective detectors of DNA immobilization and hybridization (Star et al., 2005). Scanning electron microscopy image of the random network NTNFET device. The distance between source (S) and drain (D) interdigitated metal electrodes is 10 μm. j) the response of the micro-fabricated device before and after incubation with 12-meroligonucleotide capture probes (5-CCT AAT AAC AAT-3), as well as after incubation with the complementary DNA targets.

Fig 5

The illustration of the FET-type micropatterned graphene nano-biohybrid immunosensor (GMNS) relies on GMs with close-packed carboxylated polypyrrole nanoparticle (CPPyNPs) on the flexible substrate. (photolithography, PR: photoresist, RIE: reactive-ion etching, GM: graphene micropattern). Images (at middle left) shows flexi-FET coupled with microfluidic channel and illustration (low left) of the GMNS integrated microchannels. The utilized system was tested against leakage that exhibits no leakage. Graph at lower right shows I_ds responses of the device for different HIV-2gp 36 Ab concentrations (1 to10 pM, and 1 nM) at V_ds = −10 mV (V_g = 0 V), a gradual increase in current was recorded upon Ab injection into the microchannel. The greater Ab concentrations the higher current values and saturation occurs rapidly (Kwon, Lee, 2013).

Fig 6

a) Schematic illustration of SPR microfluidic device that are used for the development of SPR based HBV detection technology. The described assay is designed to detect recombinant HBV surface antigens (HBsAg) by immobilizing anti-HBsAg polyclonal antibodies to a dextran layer of N-hydroxysuccinimide activated CM5 sensor chips. b) typical angular shift upon binding event c) real time monitoring of binding of HBV on to modified sensor surface (Xiaoqing Liu, Fuan Wang, 2013) d) SPR-LAMP platform. Cartridge is constructed by integrating a polymethyl methacrylate (PMMA) with two micro-wells and a 50 nm Au film coated polycarbonate (PC) prism. e) block diagram of SPR-LAMP microfludic device. f) Top view of the fluidic compartment. g) Data collected by the SPR-LAMP set up. The LAMP reaction is performed in the micro-wells, and the changes in the refractive index of reaction mixture is recorded in real-time (Tsung-Liang Chuanga, Shih-Chung Weib, 2012) h) Nanoplasmonic viral load detection platform, HIV was captured on the antibody immobilized biosensing surface. i) Schematic representation of surface modification strategy to capture HIV on the biosensing surface. j) viral load ranging 1.3 ± 0.7 log₁₀ copies/mL to 4.3 ± 1.2 log₁₀ copies/mL (Inci, Tokel, 2013).