key: cord-0691353-5yldxc6x
authors: Chen, Hao; Park, Sung-Kyu; Joung, Younju; Kang, Taejoon; Lee, Mi-Kyung; Choo, Jaebum
title: SERS-based dual-mode DNA aptasensors for rapid classification of SARS-CoV-2 and influenza A/H1N1 infection
date: 2021-12-30
journal: Sens Actuators B Chem
DOI: 10.1016/j.snb.2021.131324
sha: fe6fc84e95b9e4d453c3c8284f13a32372017df5
doc_id: 691353
cord_uid: 5yldxc6x

We developed a dual-mode surface-enhanced Raman scattering (SERS)-based aptasensor that can accurately diagnose and distinguish severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A/H1N1 at the same time. Herein, DNA aptamers that selectively bind to SARS-CoV-2 and influenza A/H1N1 were immobilized together on Au nanopopcorn substrate. Raman reporters (Cy3 and RRX), attached to the terminal of DNA aptamers, could generate strong SERS signals in the nanogap of the Au nanopopcorn substrate. Additionally, the internal standard Raman reporter (4-MBA) was immobilized on the Au nanopopcorn substrate along with aptamer DNAs to reduce errors caused by changes in the measurement environment. When SARS-CoV-2 or influenza A virus approaches the Au nanopopcorn substrate, the corresponding DNA aptamer selectively detaches from the substrate due to the significant binding affinity between the corresponding DNA aptamer and the virus. As a result, the related SERS intensity decreases with increasing target virus concentration. Thus, it is possible to determine whether a suspected patient is infected with SARS-CoV-2 or influenza A using this SERS-Based DNA aptasensor. Furthermore, this sensor enables a quantitative evaluation of the target virus concentration with high sensitivity without being affected by cross-reactivity. Therefore, this SERS-based diagnostic platform is considered a conceptually new diagnostic tool that rapidly discriminates against these two respiratory diseases to prevent their spread.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus have many clinical similarities [1] [2] [3] . Both viruses are transmitted through the respiratory tract and show mild symptoms in the initial infection state but lead to death when the virus reaches a severe condition. In addition, they are primarily airborne and have similar symptoms such as fever, cough, fatigue, and muscle pain [4] [5] [6] . Therefore, if the SARS-CoV-2 pandemic situation is prolonged and favorable conditions for influenza virus are created due to the seasonal characteristics, we cannot exclude the possibility for the simultaneous outbreaks of SARS-CoV-2 and influenza virus. Pharmaceutical companies such as Pfizer and Moderna, which have J o u r n a l P r e -p r o o f successfully developed an RNA vaccine against SARS-CoV-2 [7] [8] [9] , have also started developing a vaccine that can act simultaneously against these two respiratory infectious diseases.

However, it is urgent to develop a diagnostic technology rapidly discriminating these two respiratory diseases to prevent their spread. In preparation for such a situation, dual reversetranscription polymerase chain reaction (RT-PCR) reagents to simultaneously diagnose SARS-CoV-2 and influenza virus from clinical nasopharyngeal samples were already commercialized [10, 11] . Furthermore, dual-mode rapid immunodiagnostic kits for SARS-CoV-2 and influenza virus using antibody-antigen interactions were also developed [12, 13] . Scheme 1a shows the symptoms that appear when infected with SARS-CoV-2 or influenza A and the RT-PCR and Rapid antigen kit that can diagnose them.

As reported so far, RT-PCR shows high sensitivity and specificity, but it takes a long time for diagnosis since it needs a viral RNA extraction and 20-40 PCR amplification steps. Therefore, the research into shortening the diagnosis time using isothermal PCR or gene scissors is actively underway in RT-PCR diagnosis [14] [15] [16] . In the case of an immunoassay-based rapid kit, the diagnostic accuracy of the initial or asymptomatic infection patient is very low due to the limit of its detection sensitivity. As a result, the false-negative diagnosis of infected patients is recognized as the most severe problem [17, 18] . To resolve this problem, many researchers have made efforts to improve the detection sensitivity of a rapid kit through optical measurements such as fluorescence and chemiluminescence [19] [20] [21] [22] [23] . Our research group has been trying to improve its diagnostic sensitivity and accuracy by using a surface-enhanced Raman scattering (SERS)-based detection different from the fluorescence or chemiluminescence detections used in conventional molecular diagnostics or immunoassays [24] [25] [26] [27] [28] . The SERS detection is a method J o u r n a l P r e -p r o o f of measuring the high-sensitivity Raman scattering signals of incident light amplified by the localized surface plasmon effects of molecules present in the nanogaps of the Au nanocomposite [29] [30] [31] [32] . We recently reported a novel SERS-based DNA aptasensor platform that can detect SARS-CoV-2 [33] or influenza virus [34] with high sensitivity. Herein, Au nanopopcorn substrates were used as a susceptible and reproducible SERS platform for the virus assays.

Additionally, specific DNA aptamers [34] [35] [36] [37] [38] [39] that selectively bind to the virus target protein biomarkers were used as the receptors (Scheme 1b).

To quickly distinguish whether a suspected patient with similar symptoms is infected with SARS-CoV-2 or influenza virus, we developed a dual-mode virus assay platform that rapidly and accurately differentiates the virus type in this study. Herein, SARS-CoV-2 and influenza A aptamers were simultaneously immobilized on the same Au nanopopcorn substrate and then selectively reacted according to the kind of virus approaching the substrate (Scheme 1c). This simultaneous dual-mode DNA aptasensor provides an efficient diagnostic tool that can accurately determine which virus is infected and respond quickly when a patient has cold symptoms during the changing seasons. Working principle of dual aptamer-immobilized Au nanopopcorn substrate for virus assays.

Ethanol (99.5%), tris(2-carboxyethyl) phosphine hydrochloride (TCEP), 6-mercapto-1- were of analytical reagent grade and were used without further purification.

The Raman images and SERS spectra were acquired using an in Via Renishaw Raman microscope system (Renishaw, New Mills, UK). A He-Ne laser operated at 632.8 nm was used as the excitation source. The Raman spectral data were collected using a charge-coupled device (CCD) camera. The Raman mapping images were obtained by using a 20 × (NA 0.4) objective lens with a diffraction limit of around 0.9 μm. The baseline correction of Raman spectra was performed using the WiRE V 4.0 software (Renishaw, New Mills, UK). The spectral analysis was performed using the Spectragryph software and Origin Pro V8 software (OriginLab Corporation, Northants, USA). J o u r n a l P r e -p r o o f

The fabrication procedure of the Au nanopopcorn surface has been previously reported [33, 34] . Before the functionalization, this plasmonic substrate was cleaned with ethanol and deionized water. Before the hybridization, equal molar concentrations of the capture DNA 1 and the aptamer probe 1 were mixed and heated to 90 °C for 10 min to unfold their strain and then cooled to room temperature. The identical process was performed for the capture DNA 2 and the aptamer probe 2. Then the DNAs were treated with TCEP to activate the thiol groups at the end of capture DNA sequences at pH 4 for 1 h. PBS buffer solution was added to adjust the concentration of the mixing two aptamer probes to be 2 μM. Subsequently, the plasmonic substrate was incubated for 2 h in the mixing aptamers DNA solution and then immersed in 2 mM MCH containing a 0.1 mM 4-MBA solution at room temperature for 2 h. After the functionalization on the surface of the Au nanopopcorn, the aptasensors were dried with nitrogen gas. The Raman image of the fabricated SERS-based aptasensor (5 mm×5 mm size) was measured using a mapping tool with a 20× objective lens. A computer-controlled microscope stage was used to acquire 36 Raman spectra with a step size of 20 μm (total size 120 μm ×120 μm). All spectra were measured with an exposure time of 5 s. Raman mapping images for five random areas on the same Au substrate were measured to investigate spot-to-spot fluctuations of Raman signal intensity. In addition, Raman mapping images for 6 different substrates were also measured to investigate the substrate-to-substrate fluctuations due to the change of the substrate.

J o u r n a l P r e -p r o o f

For the single viral assays, 5 μL of sample solution (SARS-CoV-2, 0 to 1000 PFU/mL) or (H1N1, 0 to 403 HAU/mL) was dropped onto the single aptamer-immobilized Au nanopopcorn substrate, followed by incubation for 15 min in a humid chamber at room temperature. Then the aptasensors were instantly rinsed with a washing buffer to remove non-specific binding species between the specific aptamers and target proteins. After being dried with nitrogen gas, the aptame sensors were measured using the Raman mapping technique. For the cross-reactivity tests, 5 μL of sample solution containing (SARS-CoV-2, 0.32 to 200 PFU/mL) or (H1N1, 0.13 to 80.6 HAU/mL) was dropped onto the duplex SERS-based aptasensor, followed by incubation for 15 min in a humid chamber at room temperature. After the aptasensors were instantly rinsed with a washing buffer, they were dried with nitrogen gas. Then corresponding Raman mapping images were measured and analyzed.

For the dual assays, 5 sample solutions containing (SARS-CoV-2, 0 to 200 PFU/mL) and (H1N1, 0 to 80.6 HAU/mL) were mixed with different molar ratios, and then 5 μL of sample solution was dropped onto the duplex SERS-based aptasensor, followed by incubation for 15 min in a humid chamber at room temperature. The Raman mapping images were measured after the aptasensors were instantly rinsed with a washing buffer and dried with nitrogen gas. Meanwhile, Fig. 1b and c, the characteristic Raman peak intensity of MBA at 1075 cm -1 has a constant peak intensity regardless of the presence of viruses. As the concentration of J o u r n a l P r e -p r o o f SARS-CoV-2 increases, the number of DNA aptamers that escape from the Au nanopopcorn substrate increases, so the characteristic Raman peak intensity of Cy3 at 1470 cm -1 decreases. As the concentration of influenza A/H1N1 increases, the peak intensity of RRX at 1650 cm -1 decreases by the same principle. Therefore, it is possible to quantify SARS-CoV-2 and influenza A/H1N1 concentrations by monitoring changes in Raman peak intensity of Cy3 and RRX. Cy3 (SARS-CoV-2), and RRX (influenza A/H1N1), used in this work. Among the Raman reporter molecules used here, 4-MBA has a much smaller Raman cross-section than Cy3 or RRX, so the enhancement factor is small when the same concentration of molecules is used for measurement. Therefore, we used a much higher molar concentration of 4-MBA than that of Cy3 or RRX for SERS measurements. shows the photograph of an Au nanopopcorn substrate and Raman mapping method used in this study. A 5×5 mm 2 Au nanopopcorn substrate was used as the substrate for a dualmode SERS-based aptasensor. A 120×120 μm 2 area was randomly selected on the substrate, and its Raman mapping image was measured at 20 μm intervals (Fig. 3a) . The upper figure of Raman reporter molecule is improved when each Raman intensity is corrected using the peak of the internal standard 4-MBA. When we used the internal standard, the relative standard deviations (RSDs) were improved from 8.0% to 5.2% for SARS-CoV-2 and 9.1% to 5.0% for Influenza A, respectively.

Herein, it needs to evaluate the substrate-to-substrate reproducibility since many pieces of Au nanopopcorn substrates are used in actual virus assays. Fig. S2a and b show photographs of six different substrate pieces and the average Raman spectra for 36 point pixels of each substrate, respectively. Fig. S2c shows histograms of the RSDs for their normalized Raman peak intensity ratios. This figure shows that the substrate-to-substrate reproducibility is excellent since RSDs for Cy and RRX are 1.7% and 2.1%, respectively. In addition, we also evaluated spot-to-spot reproducibility for the same substrate. As shown in Fig. S3a , five areas were selected on the same substrate, and their corresponding Raman spectra were measured for six random points J o u r n a l P r e -p r o o f chosen for each location (Fig. S3b) . According to our experimental data, the spot-to-spot reproducibility is also nice because the RSDs for the five testing areas were estimated to be 9.7% (I 1470 ) and 11.9% (I 1650 ), respectively. decreases concomitantly as the concentration increases. This is because the spike protein of SARS-CoV-2 lysate induces the DNA aptamer to detach from the substrate and reduces its Raman intensity. Fig. 4c shows the calibration curve for the I 1470 /I 1075 ratio for the increase of SARS-CoV-2 concentration. The Raman peak intensity ratio for each concentration was determined from the average value of Raman peak intensities for 36 mapping area pixels, which showed a good correlation of R 2 =0.9938 and LOD=0.78 PFU/mL for SARS-CoV-2 in Fig. 3.   Fig. 4d , e and f also show the process of assaying influenza A/H1N1 after immobilizing DNA aptamers for hemagglutinin, the change in Raman spectra in the 0-2046 HAU/mL concentration range, and corresponding calibration curve, respectively. At this time, the LOD and R 2 were estimated to be 0.62 HAU/mL and 0.9939, respectively. J o u r n a l P r e -p r o o f protein DNA aptamer selectively binds to SARS-CoV-2, and the characteristic Raman peak intensity of Cy3 at 1470 cm -1 decreases (Fig. 5b) . On the other hand, the characteristic Raman peak intensity at 1650 cm -1 of RRX attached to the hemagglutinin DNA aptamer is maintained at a constant intensity regardless of the concentration of SARS-CoV-2 (Fig. 5c) . Conversely, as shown in Fig. 5d , when influenza A/H1N1 lysate was added to Au nanopopcorn substrate, only hemagglutinin DNA aptamers selectively combined with influenza A/H1N1 and the characteristic Raman peak intensity of RRX at 1650 cm -1 decreases (Fig. 5e) . In this case, the Raman peak intensity at 1470 cm -1 is kept constant (Fig. 5f ). From these experimental results. we can conclude that the spike protein-and hemagglutinin-compatible DNA aptamers, fixed together on the Au nanopopcorn substrate, have good cross-reactivity against SARS-CoV-2 and influenza A/H1N1 without any interference. Therefore, it is possible to quickly and accurately distinguish SARS-CoV-2 and influenza A/H1N1 using this dual-mode DNA aptasensor platform.

J o u r n a l P r e -p r o o f When the concentrations of SARS-CoV-2 and influenza A/H1N1 were relatively changed, corresponding variations in SERS signal intensity were also measured (Figs. 6a, b) . In Fig. 6c , the relative changes in SERS signal intensity were plotted when the concentrations of SARS-CoV-2 and influenza A/H1N1 lysates ranged in 0.32-200 PFU/mL and 80.6-0.13 HAU/mL, respectively. When the concentration ratio of H1N1 and SARS-CoV-2 was 0.13/200, the SERS peak intensity of H1N1 was relatively strong but when it was 3.2/8, they had almost the same intensity. However, when the ratio is 80.6/0.32, the SERS intensity of SARS-CoV-2 was relatively strong. SERS spectra were also measured with the increase of both concentrations of SARS-CoV-2 and influenza A/H1N1 equally in the ranges of 0.2-200 PFU/mL and 0-80.6 HAU/mL (Fig. 6d) . As the concentration of each virus increased, the corresponding SERS intensity for both viruses concomitantly decreased in a similar pattern (Fig. 6e) . Thus, the concentration of each virus can be accurately determined using those calibration curves. To test the specificity of the dual-mode SERS-based aptasensor, four different viruses, SARS-CoV-2 (200 PFU/mL), Influenza A/H3N2 (1000 HAU/mL), Influenza A/ H1N1 (80 HAU/mL), and Influenza B (500 HAU/mL), were added to the substrate and corresponding SERS spectra were measured. Fig. 7a shows the normalized Raman spectra showing only the characteristic Raman peaks of 4-MBA, Cy3, and RRX for each virus. The full Raman spectra for related viruses are shown in Fig. S4. Fig. 7b shows the changes in relative SERS peak intensities Therefore, we can conclude that the spike protein and hemagglutinin DNA aptamers have good selectivity only for SARS-CoV-2 and influenza A/H1N1. 

There is a possibility that SARS-CoV-2 and influenza A will coincide in autumn or winter.

Since both respiratory infectious diseases have similar symptoms, it is critical to differentiate them through a prompt and accurate diagnosis. This study developed a conceptually new SERSbased aptasensor that can accurately distinguish them using Au nanopopcorn substrates coimmobilized with the spike protein and hemagglutinin DNA aptamers. When SARS-CoV-2 approaches the substrate, only the corresponding spike protein DNA aptamer binds to the SARS-CoV-2 virus and moves away from the substrate, and the Raman peak intensity of Cy3 at 1470 cm -1 decreases. When the influenza A/H1N1 virus approaches the substrate, the hemagglutinin DNA aptamer that binds to the influenza/H1N1 virus moves away from the substrate. As a result, the characteristic Raman peak intensity of RRX at 1650 cm -1 decreases. The characteristic Raman peak intensity at 1075 cm -1 of 4-MBA was used as an internal standard to reduce errors caused by changes in the measurement environment. It was possible to measure very minute changes in the concentrations of SARS-CoV-2 or influenza A/H1N1 without the influence of cross-reactivity using the SERS-based aptasensor developed in this study. Therefore, this SERSbased aptasensor is expected to be used as a new diagnostic tool that can quickly and accurately determine which virus is infected when a patient shows a symptom of infection.

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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Received his B.S. degree in 2011 from the Department of Chemistry at Shandong University, China. He is currently studying for his Ph.D. degree in the Department of

Received his Ph.D. degree in 2010 from the Department of Chemical Engineering at KAIST, South Korea. He is currently a Senior Researcher at the Korea Institute of Materials Science (KIMS) in South Korea

She is currently studying for her Ph.D. degree in the Department of

Received his Ph.D. degree in 2010 from the Department of Chemistry at KAIST, South Korea

Received her Ph.D. degree in 1999 from Medical School at Chung-Ang University, South Korea. She is currently a Professor in the

Received his Ph.D. degree in 1994 from the Department of Chemistry at

Conceptualization, Validation, Formal analysis, Investigation, Sung-Kyu Park: Funding acquisition, Methodology, Investigation, Younju Joung: Validation, Formal analysis, Investigation, Taejoon Kang: Funding acquisition, Conceptualization, Methodology, Visualization, Mi-Kyung Lee: Clinical Validation, Conceptualization, Methodology, Jaebum Choo: Funding acquisition, Conceptualization, Methodology, Writing-review & editing. declare the following financial interests/personal relationships which may be considered as potential competing interests