One-step immunoassay without washing steps for influenza A virus detection using ISFET
Introduction
Immunoassays for the detection of target analytes are based on specific interactions between antibodies and antigens (Lei et al., 2016; Muneoka et al., 2018). In general, antibodies (or antigens) are immobilized on solid supports (microplate and bead) such that they are recognized by target analytes through affinity interactions (Bong et al., 2019; Quesada-González et al., 2019; Yuzon et al., 2019). Such antigen-antibody interactions are then detected using secondary antibodies labeled with enzymes that produce color products, electrochemical responses, fluorescence, or luminescence or other types of reporters such as gold nanoparticles (An et al., 2016; Heidari et al., 2019; Lee et al., 2019; Park et al., 2019b). The major advantage of immunoassays is their high specificity toward target analytes even in a complex mixture. However, immunoassays are time-consuming and involve cumbersome washing steps between reactions to remove any unbound reagents and analytes. The sensitivity of immunoassays is influenced by washing steps because incomplete washing may result in false positive signals due to non-specific interactions (Banala et al., 2013).
The development of one-step immunoassay methods has gained momentum to allow completion of the entire procedure via the simple addition of a sample solution. A lateral flow immunoassay uses an antibody-printed strip and dried gold nanoparticle-labeled antibodies (Park et al., 2015). In this method, the result is analyzed based on the formation of a sandwich complex on the assay strip that is observed as a color change without the need for any additional instruments. This immunoassay could be effectively used for the qualitative determination of a target analyte in samples, e.g., a pregnancy test for the detection of human gonadotropin (hCG) in urine samples. For the quantitative analysis of any target analyte using the lateral flow immunoassay, many types of optical reader devices are available (Kim et al., 2019; Park, 2019).
In the present study, a new type of one-step immunoassay without washing steps was developed using two types of beads of different diameters and a filter within the lid of a reaction vessel. As shown in Fig. 1(a), detection (small diameter) and capture (large diameter) beads were mixed with test samples to produce a sandwich complex with target analytes. Samples negative for the test analyte would not produce any sandwich complex and the unbound detection bead would pass through the controlled pore size filter into the reaction vessel. The detection bead is coupled to an antibody against the target analyte and an enzyme urease, which would react with the substrate urea present in the reaction vessel and induce a pH change (Hu et al., 2016; Sun et al., 2019). The pH change is directly proportional to the concentration of the analyte and can be detected using indicators or other pH-sensitive devices. We compared the sensitivity of this assay using two different methods, phenol red and ion-selective field-effect transistor (ISFET), for the detection of pH change (Jang et al., 2017; Lee et al., 2016; Roh et al., 2019). The developed assay was used for the detection of influenza A virus in real patient samples and negative samples from healthy volunteers. Recently many reports were published on advantages and disadvantages of immunoassays. A self-powered α-fetoprotein (AFP) sensor was reported to quantify AFP concentration via thermoelectric changes based on enzyme product (Huang et al., 2020). This method showed a linear range of 0.5–60 ng/mL and LOD of 0.39 ng/mL. As the module could be reusable and inexpensive, this method was considered to have enormous potential for commercialization. However, the analysis required over 17 h including 12 h of incubation times for capture antibody. A novel DNA-based hybridization chain reaction (HCR) was reported to detect IgG using sandwich-type immunoassay (Zhang et al., 2012). Through the hybridization of hairpin oligomers, the labeled ferrocenes could be bound to AuNPs complex and it resulted in the amplification of immunoassay to reach the LOD of 0.1 fg/mL within the analysis time of 2.5 h. A near-infrared (near-IR) light activated photoelectrochemical immunoassay was also reported (Luo et al., 2019; Shu and Tang, 2017). This method used the quenching effect of copper ion for the detection of AFP and it required of 2.5–3 h to reach an excellent LOD of 1.2 pg/mL for AFP.
Section snippets
Materials
Urease, urea, and other chemicals were purchased from Sigma-Aldrich Korea (Seoul, Korea). Polystyrene microbeads of 3 and 15 μm diameters were procured from EasyChem (Chungcheongbuk-Do, Korea) and Sigma Aldrich Korea (Seoul, Korea), respectively. Reaction vessels (Dropper tube with filter cap) were obtained from Noble Biosciences (Kyunggido, Korea), The detection and the capture antibodies (monoclonal mouse antibodies) were supplied from Kyungsang National University (Prof. Won-Bo Shim). The
Principle of one-step immunoassay without washing steps
A one-step immunoassay for the detection of influenza A virus was developed using two different microbead types and a filter-inserted bottle. As shown in Fig. 1(a), the two beads of different diameters (15 μm for capture bead and 3 μm for detection bead) were prepared for the specific detection of influenza A virus. The surface of the detection bead (diameter of 3 μm) was coupled with anti-influenza A antibody and urease, whereas that of the capture bead (diameter of 15 μm) was labeled with
Conclusions
A one-step immunoassay for the detection of influenza A virus was developed using two different microbead types and a filter-inserted bottle. The two beads with different diameters (15 μm for capture bead and 3 μm for detection bead) were prepared for the specific detection of influenza A virus. Samples positive for influenza A virus produced a sandwich complex with the capture and detection beads. This complex could not pass through the filter, which have a controlled pore size. The negative
Funding
This work was supported by the National Research Foundation of Korea [grant number: NRF-2020R1A2B5B01002187] and Korea Institute of Science and Technology program [grant number: 2E30480].
CRediT authorship contribution statement
Ji-Hong Bong: Data curation, Writing - original draft. Hong-Rae Kim: Data curation, Writing - original draft. Jae-Woo Yoo: Data curation. Min-Jung Kang: Supervision. Myung-Geun Shin: Supervision, Resources. Jung-Su Lee: Supervision, Resources. Won-Bo Shim: Supervision, Resources. Sang-Dae Lee: Funding acquisition. Jae-Chul Pyun: Supervision, Funding acquisition, Writing - original draft, Writing - review & editing.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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These two authors contributed equally to this work.