Liquid gated ZnO nanorod FET sensor for ultrasensitive detection of Hepatitis B surface antigen with vertical electrode configuration
Introduction
Detection of viral infections like Hepatitis-B, Hepatitis-C, HIV and others are of utmost priority, not only for human health but also to ensure security against bioterrorism. Recurrent onset of Hep-B virus infection may lead to hepatocellular carcinoma and cirrhosis (Lee et al., 2012). Out of two billion Hep-B infected people, 350 million are chronically infected worldwide. Hep-B surface antigen (HBsAg) is a part of the Hep-B virus and is used as a biomarker for Hep-B infection (Bonino et al., 2010, Trépo et al., 1993). When the body gets exposed to Hep-B, HBsAg appears in the serum, which is the first serologic marker. One of the requisite for early detection of HBsAg is ensuring an ultra low level of detection, such that even for trace quantities in presymptomatic condition, indication of antigen concentration is obtained.
HBsAg can be diagnosed using rapid diagnostic tests (RDT) in lateral flow or simple agglutination assay formats. Laboratory-based immunoassays for the detection of HBsAg include conventional methods like radioimmunoassay (RIA) and enzyme immunoassays (EIA), as well as latest technologies such as electrochemiluminescence immunoassays (ECLIA), chemiluminescent microparticle immunoassays (CMIA) and microparticle enzyme immunoassays (MEIA) (Amini et al., 2017). Virus detection techniques based on enzyme linked immunosorbent assay (ELISA) exist commercially which suffer from poor sensitivity, resulting in diagnosis at higher viral loads, typically of the order of nanomolars (Rutstein et al., 2014). This in turn enhances the treatment expenses. Thus, lowering the level of detection towards sub-femtomolar levels in physiological analyte by amplification of transduced signal is worthy investigating. One of the extensively used approaches for amplified signal transduction is the deployment of nanostructured materials in various configurations. Recent report on plasmonic gold nanoparticles (Hakimian et al., 2018) resulted in level of detection (LOD) in the aM range. An electronic adaptation of ELISA has been interfaced with indium oxide nanoribbons for virus detection down to sub-femtomolar regime (Aroonyadet et al., 2015). However, these technologies are not label free and require extensive biochemical processing. For point-of care diagnostic applications, label free nanostructured biosensors based on impedance, conductance and amperometric measurements have been extensively investigated. The widely fabricated nanostructures for this purpose include nanoribbons (Chen et al., 2013), nanowires (Li et al., 2015), ordered and random nanopores (Ghosh and Roychaudhuri, 2013), nanorods (Xue et al., 2015), nanotubes (Hidaka et al., 2016), nanogrids (Basu and Chaudhuri, 2016) and nanoparticles (Aydoğdu et al., 2013) on substrates like silicon and its oxide, carbon, graphene, indium oxide, zinc oxide, anodized alumina and various polymers. Amongst the different sensing mechanisms, FET based transconductance measurements on 1D silicon nanowires, carbon nanotubes and zinc oxide nanowires have been explored because of the intrinsic amplification effect (Lee et al., 2015, Marques et al., 2017). But their LODs are limited mostly in the pM range. This is attributed to the scarcity of available binding sites on the 1D nanoscale materials, especially in the fM or sub-fM range. We have reported FET based sensing of HBsAg in the aM range using graphene nanogrids (Basu and Chaudhuri, 2016) in buffer. However, in physiological samples, the detection limits are supposed to be further deteriorated with high salt concentration due to Debye screening effect, whereby sensing in aM regime is indeed a challenge. This condition becomes severe for most of the antibody-antigen conjugates since their dimensions typically exceed the Debye screening length of 1 nm in physiological analytes like serum or urine. For HBsAg also, the condition will be severe since they are large molecules greater than 10 nm (Gilbert et al., 2005). In this aspect, we have previously reported nanostructured silicon oxide based impedance biosensor which shows an interesting peak frequency phenomenon, not affected by the Debye screening effect and the shift in the peak frequency can detect down to 0.1 fM HBsAg in serum (Chakraborty et al., 2017). However, their performance is largely affected by surface roughness of the pores and suffers from reliability issues (Das and RoyChaudhuri, 2015).
For FET biosensor, the problem of reduced Debye screening at high ionic strengths, has been addressed by two methods- applying a high frequency signal between drain and source which initiates breakdown of the electric double layer near FET channel surface and coating the FET channel surface with polyethylene glycol which enhances the effective screening length (Chu et al., 2017, Gao et al., 2015, Kulkarni and Zhong, 2012). In the former, fast switching of the applied bias leads to deeper penetration of the electric potential of target protein but its application in carbon nanotubes has resulted in only pM detection limit, the reasons for which have already been indicated. The latter method has been utilized in graphene and carbon nanotube FETs for detection of numerous biomolecules like prostate specific antigen, Zika virus, lead ions and brain natriuretic peptide (Afsahi et al., 2018, Gao et al., 2016, Lei et al., 2017, Wang et al., 2016). But the detection limits vary from nM to a few hundreds of fMs primarily because the thick functionalization layer results in weakened capacitive coupling leading to a decrease in transconductance. At this stage, it is evident, that development of FET based biosensors on a high surface area to volume ratio substrate with a high intrinsic transconductance is the need of the time. In this aspect, ZnO nanorod is a desirable material compared to graphene in terms of sensing application due to its high on-off ratio (Ahn et al., 2018). Additionally ZnO nanorods lead to enhancement of surface area to volume ratio and have been experimented for protein, virus and enzyme detection using optical, amperometric, impedance and transconductance principles (Ahmad et al., 2017a, Ahmad et al., 2017b, Ahmad et al., 2017c, Ahmad et al., 2013, Ahmad et al., 2014, Ahmad et al., 2015, Ahn et al., 2018, Dang et al., 2015, Fathollahzadeh et al., 2017, Han et al., 2016, Ibupoto et al., 2011, Kumar et al., 2016, Zong et al., 2017). The detection limit obtained for virus is around few femtomolars in buffer (Han et al., 2016). ZnO nanorod integrated microfluidic biosensor has been reported (Yu et al., 2017) as a sensitive immunofluorescence platform for the detection of avian influenza virus (AIV). The high sensitivity has been attributed to the combined effect of faster carrier diffusion process of the biomolecules within the nanorod structures and enhanced capture of the surface probes. But, virus detection in physiological analyte using label free FET based ZnO nanorod structure has not yet been explored. The fM detection limit of virus in ZnO nanorod FETs in buffer can be extended to physiological analyte in the aM regime by adopting high frequency heterodyne mode of sensing and incorporating some signal amplification strategies. Here, we improve the electrical contact configurations for signal enhancement, which also does not impose any additional consumable burden. Presently, all the existing drain-source contacts are fabricated laterally below the ZnO seed layer and the biomolecules on the surface of the ZnO nanorods mostly affect the conduction of the seed layer indirectly. Conduction through ZnO nanorods is limited by the presence of available interconnections. On the contrary, if the contacts are fabricated vertically, one on the ZnO nanorod surface and the other at the bottom, there will be enhanced conduction through the nanorods and the direct impact of the surface potential alteration within the nanorods is expected to result in additional current change.
In this paper, electrical sensing of HBsAg as a target protein down to aM range in serum has been demonstrated on liquid gated ZnO nanorods. Firstly, implementing the high frequency heterodyne signal between the drain source contacts fabricated below the seed layer enable detection of HBsAg down to 1 fM in serum. But with the vertical drain-source contact configuration, the detection limit has been lowered to 20 aM through a sensitivity enhancement of more than 200%.
Section snippets
Fabrication of ZnO nanorod based FET
For the fabrication of ZnO nanorods FET biosensor, low cost glass substrate has been used. ZnO nanorods based FET has been fabricated with two drain-source electrode configurations- lateral and vertical as depicted in Fig. 1(a) and (b) respectively. For the former, on one side of the glass substrate, contacts have been fabricated by photolithography. Positive photoresist S1813 has been spin coated on the glass surface at 2500 rpm for 30 s and further it has been dried at 100 °C on a hot plate
Structural and optical characterization
The schematic illustration of the FET sensor with the different electrode configurations are shown in Fig. 1(a) and (b). Overall picture of the FET sensor has been displayed in Fig. 1(c). XRD pattern of the ZnO nanorods (post annealing) has been represented in Fig. S1(a). ZnO nanorods grown on glass surface exhibit diffraction peak in [0002] plane at 2θ ≈ 34.4°. The rapidly grown [0002] diffraction peak denotes hexagonal growth of ZnO crystal with its c-axis perpendicular to the glass
Conclusion
To summarize, in this paper, it has been demonstrated that FETs with ZnO nanorod array, operated in heterodyne mode at high frequency and immobilized with antibody probes can be applied for label free ultra-sensitive detection of HBsAg with a detection limit as low as 20 aM in physiological analyte using vertical electrode configuration. This has been possible due to a 200% sensitivity amplification compared to lateral electrode configuration, attributed to the enhanced penetration of the
Acknowledgement
This research has been supported by Visvesvaraya Ph.D. Scheme and Visvesvaraya Young Faculty Research Award funded by the Ministry of Electronics & Information Technology, Government of India. One of the authors, Mr. N. Das would like to acknowledge UGC-RGNF for providing his fellowship.
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