Bohee Maenga,
Youngkyu Parkb and
Jungyul Park*ac
aDepartment of Mechanical Engineering, Sogang University, Sinsu-dong, Mapo-gu, Seoul, 121-742, Korea. E-mail: sortpark@sogang.ac.k; Fax: +82-2-712-0799; Tel: +82-2-705-8642
bAgency for Defense Development, Yuseong, P.O. Box 35, Daejeon, 305-600, Korea
cInterdisciplinary Program of Integrated Biotechnology, Sogang University, Sinsu-dong, Mapo-gu, Seoul, 121-742, Korea
First published on 5th January 2016
To date, there is no commercial point-of-care test (POCT) for intact virus detection. A POCT should show high sensitivity and have proper structure through simple and cost effective fabrication; moreover it should not require a complex and bulky instrument for measurement. In this study, a direct label-free biosensor based on 3D photonic crystal (PC) structures for Rotavirus has been demonstrated, which can be developed for POCT. The proposed sensor can detect the target with viral loading from 6.35 μg ml−1 to 1.27 mg ml−1 without any sample pretreatment. Sensitivity and selectivity performance are analyzed quantitatively by measuring peak wavelength value (PWV) and comparing the performance using an ELISA assay. In order to facilitate the target virus accessibility to inner structures in the sensor, the hydrogel based inverse opal structure is etched by O2 plasma and the connection window between nanopores becomes enlarged. This process is critical for enhancing virus sensing performance. Our PWV shift range is larger than of the shift in a 1D photonic crystal based virus sensor because the larger surface area in 3D inverse opal structures is realized. These results show that the proposed method is useful for developing a direct and easy-to-use virus detection kit in the form of a POCT in the near future.
PC sensors can be divided into two types: fluorescence-based detectors and label-free detectors.15 In fluorescence-based detection, PC play the role of a Bragg mirror to enhance the excitation signal, which matches the PWV; therefore, the sensitivity of sensors can be improved dramatically. Label-free biosensors detect biomolecules through changes of the PWV when biological molecules, such as proteins, viruses or cells, bind to the PC surface.3,15 This label-free method is more attractive than fluorescence labeling for many reasons: (1) portable point-of-care testing (POCT) is possible without the time-consuming and expensive fluorescence labeling step for the targets and the heavy and expensive fluorescent equipment required. (2) Quantitative measurement of molecule interaction is allowable. (3) Finally, this PC biosensor can be used as a colorimetric biosensor that can identify the quantity of biomolecule binding levels.8
Rotavirus is a viral agent of gastroenteritis in animals and humans and is distributed worldwide.16,17 It is one of the common enteric agents and mortality associated with its infection can be very significant for infants and children under 5 year, especially in developing countries.3 For detection of the virus, in earlier days, electron microscopy was used by confirming their morphology.18 Currently, there are many techniques and publications for virus diagnosis. ELISA and RT-PCR are the usual and classic diagnostic methods,16,18,19 and surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) are also studied for detection of intact virus particles.3,16 Although SPR and QCM offer a label-free biosensing with high sensitivity, their expensive and complex instrument is a bottleneck for development as a POCT. Virus detection through POCT has been technically challenging and no POCT platform for viral load has been available commercially.20
In this study, we present a direct label-free detection method for Rotavirus using a 3D PC biosensor, as shown in Fig. 1. The 3D inverse opal sensor that we proposed in this study for detection of the virus is essentially made up of a hydrogel backbone. Because these hydrogels are known to show high capacity for biomolecule immobilization, greater probability of interacting with the target molecules and high LOD, S/N, and sensitivity, they have been commercialized by some companies in microarrays.21–24 Previously, highly sensitive biosensing for biomolecular screening has been investigated using 3D nanoporous hydrogel by several research groups, including us.21,25,26 Another advantage is that a 3D inverse opal structure has larger voids that can accommodate more intensive variation in their refractive index compared to an opal structure.15,27 In addition, the presence of nanopores can not only provide greater surface area and more interaction sites, but also offer easier access for the analytes to the recognition sites.15,27 Especially, in order to facilitate the permeability of target analytes into nanoporous structures in the sensor, the hydrogel backbones in the inverse opal structure were treated with reactive ion etching (RIE). This resulted in enlarging the area of the interconnection windows between nanopores, and the analytes could access deeply into the sensor through the windows, and it critically affects the detection performance. For pathogen specificity, the surface of inverse opal structures is modified with protein A and specific monoclonal Rotavirus antibody. The proposed system can quantitatively detect a target virus load from 6.35 μg ml−1 to 1.27 mg ml−1 by measuring the PWV shift without any pretreatment. The specific capturing on the functionalized surfaces is confirmed by fluorescence and is visualized with gold nanoparticle labelling using scanning electron microscopy (SEM). Finally, the virus detection limit is compared with a commercial ELISA kit.
In order to verify that the proposed surface modification works properly for virus detection, after the reaction of the virus samples on the sensor surface, Rotavirus polyclonal goat antibody (Fitzgerald, USA) and FITC labeled anti-goat IgG (Fitzgerald, USA) were incubated. Each step was reacted for 2 h at 37 °C. Detected viruses on the sensor pores were identified by a fluorescent signal using an inverted fluorescence microscope (Olympus IX71). For checking the successful attachment of Rotavirus on the sensor surface, scanning electron microscopy (SEM, SIGMA, Carl Zeiss) was used and gold nanoparticles-tagged antibody was conjugated on the virus for easy observation.28,29 For immobilizing gold nanoparticles, 18 nm gold nanoparticle-labeled immunoglobulin antibody was reacted (abcam, UK). In order to prepare samples for SEM, the 3D PC sensors that finally reacted with virus or gold nanoparticles-tagged virus were rinsed with PBS and fixed with 1% glutaraldehyde (Sigma Aldrich) in PBS at 4 °C for 1 h. Washing with PBS was followed by 1% osmium tetroxide (Sigma Aldrich) in PBS at 4 °C for 15 min. The structure was then washed with DI water three times. Samples were dehydrated using a graded ethanol series (25%, 50%, 75%, 80%, 90%, and 100% diluted ethanol in DI water) and a drying step was conducted.
λ = 2d(neff2 − cos2![]() |
The effective refractive index of the constituent PC is expressed as neff, d is the distance of diffracting plane spacing, and θ is the Bragg angle of incidence of the light falling on the nanostructures.1
The surface modification reaction for Rotavirus detection on the fabricated sensors was analyzed by PWV shift with respect to each modification step, as shown Fig. 3. Each PWV shift represents the difference in PWV between the current and the previous surface modification step. After functionalization with protein A, a PWV shift of 5.19 ± 0.82 nm was measured. Then, after the captured antibodies were reacted sequentially, the difference in PWV from the previous step was measured to be 7.47 ± 0.66 nm. Although the blocking step is important for preventing non-specific binding, it was known that this step did not cause a statistically significant PWV shift compared to the antibody capture step.20 The PWV shift after the blocking step with 1% BSA was 1.05 ± 0.45 nm.
The performance of the virus sensor for detection of Rotavirus was also evaluated using the PWV shift. Various concentrations from the diluted virus solutions were tested to determine the sensitivity of the sensor. As a result, the PWV shift, according to virus concentration, showed a statistically significant difference compared to control samples (just PBS solution). The PWV shift at the highest concentration 1.27 mg ml−1 was almost 3.25 ± 0.31 nm. Shifts of 2.36 ± 0.32 nm and 1.48 ± 0.24 nm were measured after the reaction with 10 (127 μg ml−1) and 100 times (12.7 μg ml−1) diluted virus solution, respectively (Fig. 4, n = 5, p < 0.05). We also found that the PWV shift in the 200 times diluted solution (6.35 μg ml−1) was 0.8 ± 0.31 nm (n = 5, p < 0.05). It could be clearly distinguishable compared to the control group. However, as described in Fig. 4, there was no clear difference in PWV shift between the 2.54 μg ml−1 viral sample and the control group (n = 5, p > 0.05). From these data, it was verified that the proposed sensor was able to detect virus quantitatively, according to the change of the virus concentration, and its minimum resolution was 6.35 μg ml−1. Pineda et al. previously reported a 1D photonic crystal biosensor for detecting Rotavirus that has active viral infection.3 However, in this study, commercially available inactivated virus was used for safety issue, so it is difficult to make a focus-forming assay (FFA). This means it is not possible to convert our virus loading unit into FFU or copies per ml for direct comparison of our result with another group's results, but the function of inactivated virus is not different from real active samples except for the inactive treatment for safety. They have the same size, shape and specific antibody reactions as real samples, which are commonly used for validation of biosensors. Previously, Nakano et al. used commercially available inactivated Rotavirus for sensor detection31 and inactivated Influenza A virus also used for sensor study.32 Many literatures mentioned that our Rotavirus (strain SA11, Simian group A Rotavirus) is the same strain as the Human serotypes of group A Rotavirus, which is a major cause of gastroenteritis in young children throughout the world.16,19,33 When considering the PWV shift range, our PWV shift range (nanometer change) was much larger than the shift range in previous work (picometer range) because the larger surface area in 3D inverse opal structures was realized. This means more sensitive virus detection is possible using our system.
Fig. 4 shows the comparison of sensitivity between the proposed PC sensor and ELISA. The PC sensor shows a similar limit of detection compared with the ELISA assay. In the previous work,3 the sensing performance using 1D photonic crystal was also comparable with the commercially available ELISA kit. Using the ELISA assay, it was difficult to detect less than 6.35 μg ml−1 concentration virus samples. This value was similar to the minimum detection level of the PC sensor, as described above. The ELISA assay results showed that the absorbance dropped off remarkably below 127 μg ml−1 level of virus concentration. However, in the PC sensor, the PWV shift has shown linear variation according to the virus concentration decrease; i.e., the PWV shift range went down steadily with respect to decreased virus concentration. Therefore, it will be a more useful tool to analyze virus concentration quantitatively. We demonstrated that the PC sensor had sufficient sensitivity, which was comparable or superior to commercially available ELISA.
Each 3D PC structure was functionalized with the same procedure that we established. The measured PWV shifts of each virus, including the control which was reacted with PBS buffer, are demonstrated in Fig. 5. In case of Echo and Influenza A virus, the PWV shifts were not distinguishable from the control group, which was reacted with no virus agents, just PBS solution, although the two viruses were exposed to the PC sensor with an almost twice higher concentration than Rotavirus. It was confirmed that the 3D PC sensor specifically detected Rotavirus, as we designed.
In order to check the specific binding performance, the proposed biomolecule binding reaction was carried out on a glass, a bare PEG-DA flat surface and an inverse opal PC structure. First, the fluorescent signal from the sample that allows non-specific binding (has no capture antibody) was compared to the reference sample that was not treated at all. There was no difference in fluorescent signals between the two groups. However, a definite intensity change was observed when the sample for specific binding was compared with the sample for non-specific binding. Especially, there was a much higher signal from the 3D inverse opal structure than the signal from the 2D flat surface, such as the glass or PEG-DA substrate, as shown in Fig. 6(d). This supported that the 3D inverse opal structure could have virus detection ability superior to the 2D flat glass and the flat PEG-DA substrate, because the 3D inverse opal structure could allow enough interconnection sites for binding the virus sample. Moreover, with the flat PEG-DA and glass surface, higher intensity was observed in the flat PEG-DA, and it was proven that intrinsically more biomolecule immobilization and higher probability of interacting with the target can be realized on hydrogel than on glass.
Metal nanoparticles tagged on viruses were used for clearer observation by SEM.28,29 Fig. 7 shows the surface of sensors after incubation with virus solution and tagging with 18 nm size gold nanoparticles conjugated with monoclonal antibody for 2 h. By using this method, it is possible to easily observe the detected virus particles on the inverse opal structure by finding the attached gold nanoparticles. It was well known that the wheel-like round shaped structure is the characteristic feature of the Rotavirus and its regular size is 50–80 nm in diameter.18 Herein, from the SEM image, we were able to identify the virus of about 50–70 nm size with round shape on the sensor surface (Fig. 7). Moreover, we found that the shiny gold particles tagged on the virus were scattered inside the pores from photonic crystal structures.
Finally, we compared the sensor performance upon RIE treatment that induces deeper penetration of the virus into internal structures. When the inverse opal structure was fabricated using colloidal particles, the connection windows between nanopores were narrow. It was not wide enough for viruses to pass through freely. To facilitate the target virus's access into inner structures, O2 plasma etching by RIE was implemented, and the size of the interconnection windows was evidently increased more than 100 nm, as shown in Fig. 7(c) and (d). The enhanced virus detection ability was verified definitely by comparing the SEM images between non-RIE treated and RIE treated samples. In the case of RIE treated sample (Fig. 7(c) and (d)), there were many gold particle-tagged viruses inside nanopores, but it was difficult to find the virus inside nanpores in the case of non-RIE treated sample (Fig. 7(b)). To determine the optimal etching condition, we compared the morphology in the nanopore structures depending on the reaction time. More detail is available in the ESI (Fig. S2†).
This study provides direct and quantitative virus detection by a simple process without any pretreatment for the target virus and would be helpful in developing a direct and easy-to-use virus detection kit in the form of POCT in the near future.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra21665f |
This journal is © The Royal Society of Chemistry 2016 |