Reusable surface amplified nanobiosensor for the sub PFU/mL level detection of airborne virus

Abstract

We developed a reusable surface-amplified nanobiosensor for monitoring airborne viruses with a sub-PFU/mL level detection limit. Here, sandwich structures consisted of magnetic particles functionalized with antibodies, target viruses, and alkaline phosphatases (ALPs) were formed, and they were magnetically concentrated on Ni patterns near an electrochemical sensor transducer. Then, the electrical signals from electrochemical markers generated by ALPs were measured with the sensor transducer, enabling highly-sensitive virus detection. The sandwich structures in the used sensor chip could be removed by applying an external magnetic field, and we could reuse the sensor transducer chip. As a proof of concepts, the repeated detection of airborne influenza virus using a single sensor chip was demonstrated with a detection limit down to a sub-PFU/mL level. Using a single reusable sensor transducer chip, the hemagglutinin (HA) of influenza A (H1N1) virus with different concentrations were measured down to 10 aM level. Importantly, our sensor chip exhibited reliable sensing signals even after more than 18 times of the repeated HA sensing measurements. Furthermore, airborne influenza viruses collected from the air could be measured down to 0.01 PFU/mL level. Interestingly, the detailed quantitative analysis of the measurement results revealed the degradation of HA proteins on the viruses after the air exposure. Considering the ultrasensitivity and reusability of our sensors, it can provide a powerful tool to help preventing epidemics by airborne pathogens in the future.

Figure 1

Schematic diagram depicting the cyclic process for the repeated sensing measurements of surface-amplified sandwich immunoassay. (a) Preparation of sandwich structures in PBS solution including magnetic particles conjugated with 1st antibody, alkaline phosphatase (ALP) conjugated with 2nd antibody, and influenza viruses. (b) Fabrication of a reusable sensor transducer chip including an interdigitated gap sensor and ferromagnetic Ni patterns. (c) Trapping of sandwich structures on Ni patterns between Au electrodes via an external magnetic field. (d) Electrochemical sensing measurement of surface-amplified electrical signals. (e) Detrapping of sandwich structures via an external magnetic field with an opposite direction to that for trapping.

Figure 2

Basic trapping and detrapping operations of our reusable sensor chip. (a) Schematic diagram (i) showing our reusable sensor chip and the field emission scanning electron microscopy (SEM) image (ii) of a sensor chip. (b) Simulation results showing cross-sectional views of a magnetic field strength around a Ni pattern during trapping (i) and detrapping (ii) processes. (c) SEM images after trapping (i) and detrapping (ii) magnetic particles on Ni patterns.

Figure 3

Repeated sensing measurements to detect hemagglutinin (HA) proteins of influenza A (H1N1) virus via surface-amplified sandwich immunoassay. (a) Simulation result showing AP concentrations after AP diffusion in the surface-amplified sandwich immunoassay. (b) Real-time sensor responses to various concentrations of HA with and without the surface amplification of signals. (c) Dose-dependence response curves of the surface-amplified immunoassay (n = 3) and conventional ELISA measurements (n = 5) using the same 1st and 2nd antibodies (i). Each data point and error bar represents the average value and standard deviation of the experiments. Log–log scale plot showing linearity of our sensor response against HA concentrations (ii).

Figure 4

Repeated sensing measurements for the detection of airborne influenza A (H1N1) virus via the surface-amplified sandwich immunoassay. (a) Schematic diagram depicting the aerosolization and collection of influenza virus solution. (i) Aerosolization of influenza virus solution. (ii) Dilution of the aerosolized influenza virus with clean air. (iii) Collection of the aerosolized influenza virus. (b) Dose-dependence response curves of surface-amplified immunoassay to influenza viruses before and after the aerosolization and collection processes. Each data point and error bar represents the average value and standard deviation of the experiments (n = 3). (c) Comparison of the sensing measurement results obtained before and after the sampling processes.