key: cord-0717931-hzznnhe7 authors: Jang, Ah Seong; Praveen Kumar, Panangattukara Prabhakaran; Lim, Dong-Kwon title: Attomolar Sensitive Magnetic Microparticles and a Surface-Enhanced Raman Scattering-Based Assay for Detecting SARS-CoV-2 Nucleic Acid Targets date: 2021-12-16 journal: ACS Appl Mater Interfaces DOI: 10.1021/acsami.1c17028 sha: bd206ffb8f67b6be5e15c6068493576d161986c0 doc_id: 717931 cord_uid: hzznnhe7 [Image: see text] Highly sensitive, reliable assays with strong multiplexing capability for detecting nucleic acid targets are significantly important for diagnosing various diseases, particularly severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The nanomaterial-based assay platforms suffer from several critical issues such as non-specific binding and highly false-positive results. In this paper, to overcome such limitations, we reported sensitive and remarkably reproducible magnetic microparticles (MMPs) and a surface-enhanced Raman scattering (SERS)-based assay using stable silver nanoparticle clusters for detecting viral nucleic acids. The MMP–SERS-based assay exhibited a sensitivity of 1.0 fM, which is superior to the MMP-fluorescence-based assay. In addition, in the presence of anisotropic Ag nanostructures (nanostars and triangular nanoplates), the assay exhibited greatly enhanced sensitivity (10 aM) and excellent signal reproducibility. This assay platform intrinsically eliminated the non-specific binding that occurs in the target detection step, and the controlled formation of stable silver nanoparticle clusters in solution enabled the remarkable reproducibility of the results. These findings indicate that this assay can be employed for future practical bioanalytical applications. The outbreak of coronavirus disease 2019 (COVID- 19) , which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused an insidious global pandemic. 1−3 Early diagnosis is essential to control the COVID-19 outbreak as it helps in reducing the potential spread of the virus. 4 Various assays, such as reverse transcription-polymerase chain reaction (RT-PCR), reverse transcription loop-mediated isothermal amplification, 5 and clustered regularly interspaced short palindromic repeats, 6 have been utilized for the detection of SARS-CoV-2 viral nucleic acids. Among them, the RT-PCRbased assay is the most commonly used method. RT-PCR involves the conversion of viral RNA into complementary DNA using RNA-dependent DNA polymerase, which is amplified through thermal cycling. The RT-PCR-based assay relies on enzymatic amplification to attain attomolar sensitivity; however, this amplification leads to high falsepositive results owing to the presence of artificial signals from sequence interference by oligonucleotides and cross-reactivity. 7 Therefore, it is essential to develop a new robust assay platform that does not rely on enzymatic amplification for the diagnosis of COVID-19. There are three different nucleic acid targets required to verify the infection of SARS-CoV-2 for the diagnosis of COVID- 19, 4, 8 which are the RNA-dependent RNA polymerase (RdRp), the envelope protein (E), and the nucleocapsid protein (N) genes of SARS-CoV-2. However, there are many variants of these nucleic acid targets, indicating the importance of accurate and high throughput assay platforms. 9 Several assay platforms, which are based on unique signal amplification strategies using nanomaterials, have been developed to achieve highly sensitive and reproducible results. Electrical, 10,11 electrochemical, 12−14 magnetic, 15 and optical 16, 17 signals have been widely investigated as signal transduction modes for the diagnosis of SARS-CoV-2 infection. Among them, assays based on optical signals, such as fluorescence and Raman scattering, have been extensively utilized. Although fluorescence signals are well-established optical signals in various assay platforms, the significant photobleaching, broad emission spectrum, and lack of molecular information of this signal have limited their further application. 18 In contrast, Raman signals have emerged as a promising alternative because they are not susceptible to photobleaching. In addition, owing to their molecular information and signal amplification via plasmonic nanomaterials, the surface-enhanced Raman scattering (SERS) phenomenon and the bioanalytical applications of Raman signals have attracted significant research attention. 19−23 To obtain an enhanced Raman signal intensity, the formation of nanogaps (ideally less than 1.0 nm), also known as a "hot spot", using plasmonic nanoparticles (NPs) is required. 23 Accordingly, two major strategies have been utilized for the formation of hot spots in most SERS-based assays. One of these strategies involves the formation of random NP aggregates via salt-induced or specific target binding events. 24−26 Although this strategy can be used to detect a single molecule in dry-state analysis, single-molecule detection is not a realistic practical assay platform for detecting biomolecules because of the unavoidable non-specific binding between NPs or NPs and biomolecules. 27, 28 The other strategy involves the use of NPs as a label, known as "SERS-tag", which is composed of specific nanostructures with nanogaps and Raman reporters. 29−31 Highly bright SERS tags with Raman reporters can detect multiple nucleic acids or protein targets at aM sensitivity. 32−34 However, the poor reproducibility of most SERS-based assay platforms that rely on the use of NPs has limited the further application of these platforms. 20, 22, 28, 35 This is because of the non-specific binding of NPs that occurs in the target detection step. 28 Moreover, the required conditions for measuring SERS signals in the dry state or the formation of NP aggregates in the solution state significantly enhance the Raman signals and background signals. These factors significantly affect the efficiency of SERS-based assay platforms, particularly at a low target concentration (T m ) ( Figure S2 ). 37 The SERS-based detection was enabled by the fluorescence molecules in the released signal probes and the PBS-induced stable AgNP clusters in the solution ( Figure S3) . Consequently, the performances of the assay based on two different optical signals (i.e., fluorescence and Raman scattering) could be accurately compared. As the NPs were not involved in the target detection step (first step), the possibility of non-specific binding between the NPs and the target was eliminated. In the second step, the detection of optical signals (i.e., fluorescence or Raman responses) from the MMPs or AgNP clusters in solution was performed to compare the assay performances of both platforms (Scheme 1). For Raman analysis, two different experimental setups such as solution-state analysis or dry-state analysis (Raman mapping) were performed ( Figure S3 ). Formation of Stable Silver Nanoparticle Clusters in Solution. The formation of hot spots using plasmonic NPs is essential to obtain a significantly enhanced Raman scattering signal intensity. 22, 23 However, the SERS measurement in the dry state showed a high background signal and poor reproducibility in the assay results. The solution-state analysis also showed a low signal reproducibility because of the random aggregation of NPs and rapidly changing aggregated states in the solution. 22, 23 Therefore, in this study, we focused on solution-state Raman analysis and investigated the electrolyte conditions that enable the production of uniform and stable AgNP clusters in solution for more than 30 min at room temperature ( Figure 1 ). The four representative electrolyte compositions such as phosphate buffer (PB), NaCl, 0.3 M PBS, and 0.15 M PBS were investigated. As shown in Figure 1a , the addition of PB alone (10, 1.0, and 0.1 mM) did not induce the formation of AgNP clusters in the AgNP mixture and signal probe sequence, which could be attributed to the weak adsorption affinity of PB on the AgNPs. 41 (Figure 1c, (3, 4) ). The results indicate that the instant formation of aggregates and low colloidal stability is not a desirable condition to obtain a reproducible Raman (Figure 2a) . The color of the AgNP solution (yellowish green) readily changed to gray, indicating the slight aggregation of AgNPs. In addition, a strong fluorescence intensity could only be obtained from the MMP solutions in the presence of a specific target gene sequence, such as RdRp, E, and N, whereas a low fluorescence intensity was observed in the absence of a target (Figure 2b) . Moreover, similar results were observed in the fluorescence images of the MMPs. In addition, the released signal probe solution exhibited a strong fluorescence intensity in the presence of targets (Figure 2c) . Furthermore, the SERS spectra of the solution containing the signal probes and AgNPs were consistent with the reference Raman spectra (dotted line) of ATTO 488, ATTO 565, and ATTO 647N dyes obtained at a laser excitation of 532 nm (Figure 2d ). The two characteristic peaks at 1348, 1643 cm −1 for ATTO 488 (RdRp gene), 1502, 1651 cm −1 for ATTO 565 (E gene), and 1426, 1631 cm −1 for ATTO 647N (E gene) were clearly identified. As a higher local electromagnetic field was obtained with the excitation of AgNPs at a shorter wavelength (532 vs 633 nm), a wavelength of 532 nm was considered as a more preferable laser source for the Raman analysis ( Figure S3 ). 42 These results indicate that the MMPs exhibited a sequence-specific binding capability toward their single DNA target. Comparison of the Sensitivity of the Fluorescence and SERS Signals. (Figure 3a ). This could be attributed to the fact that the signal-to-noise ratio was not sufficiently large at ranges between 10 −10 and 0 M. Moreover, the MMPs were not visible in the fluorescence images at target concentrations below 10 −10 M (Figure 3b ). However, distinguishable Raman spectra were observed in the SERS spectra from the mixture of released solution at a target concentration of 10 −15 M ( Figure 3c ) and AgNPs. The SERS mapping images for the dried spot of the mixture, which was based on the Raman shift at 1348 cm −1 , could be observed at a target concentration of 10 −15 M (Figure 3d) . For the E gene target, the limit of detection (LOD) of the fluorescence intensity of the MMPs and the released signal probes was observed at 10 −10 M (Figure 3e,f) , whereas a signal intensity was observed in the SERS spectrum and SERS mapping image (based on the Raman shift at 1502 cm −1 ) at a target concentration of 10 −15 M (Figure 3g,h) . For the N gene target, the LOD values of the fluorescence intensity of the MMPs and released signal probes were observed at 10 −10 M (Figure 3i,j) , whereas a signal intensity was observed in the SERS spectra and SERS mapping images (based on 1426 cm −1 ) at a target concentration of 10 −12 M (Figure 3k,l) , which was lower than those of ATTO 488 and ATTO 565. This could be attributed to the non-resonant effect of ATTO 647N dyes at an excitation wavelength of 532 nm. In summary, the LOD of the MMP−SERS-based system for the detection of RdRp and E gene targets was 1.0 fM and that of the N gene target was 1.0 pM with detection ranges from 10 −9 to 10 −15 M and 10 −9 to 10 −12 M, respectively, in the solution and dry-state analysis, thereby demonstrating its excellent performance as a highly sensitive assay platform. 43 In addition to the high sensitivity, the signal reproducibility of the SERS-based assay is also a critical parameter for practical application. As shown in the Raman spectrum (Figure 3c,g,k) , the Raman spectral patterns were consistent with a decreasing target concentration from 10 −9 to 10 −12 M. However, small new peaks could be observed in the case of the 10 −15 M target concentration and no target. Since the Raman signal is obtained from the solution state, these fluctuating signals are expected to be the background noise signal from the random orientation of Raman reporters in the silver nanoparticle clusters. This kind of transiently fluctuating signal becomes more clearly visible in the case of the low target concentration because of a decreased intensity of characteristic Raman peaks. This is the reason why the small unusual peak could be seen in the 10 −15 M target concentration. However, the small transient peak is not greatly problematic in determining the assay results because of the observed two characteristic peaks of Raman reporters (Figure 3c,g,k) . To further examine the signal reproducibility of the SERS-based assay platform, three independent assays were performed for the targets at varying concentrations (10 −9 , 10 −10 , 10 −12 , and 10 −15 M and no target) using MMP, and the Raman responses were measured. No significant difference in the SERS spectral patterns and intensities in the three independently performed assays demonstrates the excellent signal reproducibility of the MMP−SERS assay platform ( Figure S4) . Selectivity of the MMP-Based Assay. The capability of an assay to selectively detect a specific target sequence in the presence of multiple sequences in an analyte is an essential parameter for the practical application of an assay. 44 To this end, the selectivity of the MMP−SERS-based assay was evaluated in the presence of dual-target sequences ( Figure S5 ) and triple-target sequences ( Figure 4) . First, in the presence of dual-target sequences, the assay was performed by adding the MMPs to binary mixtures of RdRp and E gene target sequences (concentration: 10 −9 M), where the ATTO 488modified sequence was used for the RdRp gene, the ATTO 565-modified sequence was used for the E gene, and the ATTO 647N-modified sequence was used for the N gene. The binding of the ATTO 488 and ATTO 565 signal probe sequences to their target sequences was observed; however, the binding of ATTO 647N was not observed owing to the absence of its target sequence in the mixture ( Figure S5 ). Similar results were observed in the fluorescence images of the MMPs, released signal probe solution, and SERS spectra, indicating the excellent selectivity of the signal probe sequences with no interference in the presence of dual-target sequences ( Figure S5 ). The selectivity of the MMP-based assay was further investigated in the presence of triple-target sequences ( Figure 4a ). For this, MMPs were added to tertiary mixtures of the RdRp, E, and N gene target sequences (concentration: 10 −9 M) containing the same three signal probe sequences. Strong and discriminable fluorescence intensity was observed for the (Figure 4b,c) . In addition, a high background fluorescence intensity was observed for the RdRp gene target, which could be attributed to the relatively high fluorescence signals of the MMPs. 45 The results in Figure 4b −d indicate the absence of mutual sequence interference and the excellent selectivity of the designed assay. Improved Sensitivity Using Anisotropic Ag Nanostructures. In this study, the SERS-based assay using AgNPs achieved a sensitivity of 1.0 fM for RdRp and E gene targets and a sensitivity of 1.0 pM for the N gene target (Figure 3) . However, to enhance the possibility of replacing the PCRbased assay with the SERS-based assay, it is essential to further improve the sensitivity of the SERS-based assay to the attomolar range. In our recent study, 37 we reported an MMP−SERS assay, which exhibited a sensitivity of 10 fM for the detection of an Enterococcus faecalis target using AuNPs (Cys-AuNPs, 30 nm). In this study, the sensitivity of SERSbased detection was only slightly improved using AgNPs instead of AuNPs. Accordingly, we anticipated that the sensitivity of SERS-based detection can be further improved by utilizing anisotropic Ag nanostructures because of the enhanced localized electromagnetic field in their tips and edges. 46, 33, 47 Therefore, AgNSs, AgTPs, and AgNCs were synthesized to compare the effects of various anisotropic Ag nanostructures on the sensitivity of the SERS-based assay ( Figure S6 ). The sizes of the AgNSs, AgTPs, and AgNCs were 45 ± 5 nm with 5−6 nm tips, 65 ± 5 nm, and 55 ± 5 nm, respectively, with peak extinctions at 390, 477, and 432 nm, respectively ( Figure S6a−d) . These sizes were selected to accurately compare the effect of the shape of the nanostructures on the signal enhancement compared to that of spherical AgNPs (40−45 nm) . Since all silver nanostructures in this study are negatively charged, the salt conditions to produce Ag nanoclusters in solution could be applied for all structures. The changes of AgNPs as a representative were investigated in detail for the behavior in PB, NaCl, and PBS ( Figure 1 ). AgTPs also showed the same trends of cluster formation with that of AgNPs as observed in solution color changes, TEM, and D/F images as well as SERS responses ( Figure S7 ). To compare the effects of anisotropic Ag nanostructures on the sensitivity of the SERS-based assay, the SERS intensity performances of the three signal probe sequences (i.e., ATTO 488, ATTO 565, and ATTO 647N) in the presence of the various Ag nanostructures were compared by obtaining their SERS mapping images. Spots of the mixtures on glass were imaged using SERS mapping at 1348 cm −1 for ATTO 488, 1502 cm −1 for ATTO 565, and 1426 cm −1 for ATTO 647N with 532 nm excitation. When AgNPs were used, signals were observed in the SERS mapping images of the ATTO 488 and ATTO 565 signal probe sequences at a concentration of 10 −15 M ( Figure S6e−g) , whereas signals were observed in the SERS mapping image of the ATTO 647N probe sequence at 10 −12 M ( Figure S6g ). In addition, the SERS mapping results revealed that, when AgNSs were used, the mapping images of ATTO 488, ATTO 565, and ATTO 647N signal probe sequences were observed at 10 −18 ( Figure S6h Based on these results, we next performed the MMP−SERSbased assay for the RdRp gene target at varying target concentrations (10 −12 , 10 −15 , 10 −16 , 10 −17 , 10 −18 , and 0 M) using AgNPs, AgNSs, AgTPs, and AgNCs ( Figure 5a) . The difference in the Ag nanostructures did not affect the Raman spectra of the ATTO 488 dye (Figure 5b ). The LOD was measured using SERS mapping at a Raman shift of 1348 cm −1 . When AgNPs, AgNSs, AgTPs, and AgNCs were used, the target was detected at concentrations of 10 −15 , 10 −17 , 10 −17 , and 10 −12 M, respectively (Figure 5c−f) . This indicates that AgNSs or AgTPs were significantly more sensitive than the other structures. The significantly enhanced SERS intensity when AgNSs or AgTPs were used could be attributed to the high degree of anisotropy and a large number of edges in the AgNS and AgTP structures. 48, 47 As reproducibility in a low target concentration is crucial for SERS-based assays, three independent assays were performed for the detection of RdRp gene targets using AgNSs and AgTPs to examine their signal reproducibility in both the dry and solution states ( Figure S8 ). The three independent assays performed using AgNSs ( Figure S8a −c) or AgTPs ( Figure S8d −f) showed a detectable Raman response at a concentration of 10 aM in both analyses. However, the SERS mapping images performed in the dry-state analysis were not consistent in the result (10 aM in Figure S8c ,e) because of the random distribution of hot spots in the dry-state analysis. In contrast, the results obtained from the solution state clearly showed the difference Raman spectrum between 10 −17 and 10 −18 M. These repeated assay results indicate that the use of anisotropic structures, such as AgNSs and AgTPs, greatly amplified the SERS signal and excellent signal reproducibility from the stable NP clusters in solution. In this study, we demonstrated a highly sensitive and reproducible MMP−SERS-based assay platform for nucleic acid target detection. The DNA sequences designed to detect the RdRp, E, and N genes of SARS-CoV-2 exhibited reliable sensitivity and reproducible assay results. The sensitivity of the assay platform using two different optical signals (i.e., fluorescence and SERS) was rigorously compared. The SERS-based detection using AgNPs exhibited significantly higher sensitivity (1.0 fM for the RdRp and E genes and 1.0 pM for the N gene) than the fluorescence-based detection for all the three target genes. In addition, the use of anisotropic Ag nanostructures (AgNSs and AgTPs) for the MMP−SERSbased assay significantly improved the sensitivity of the assay for the detection of the RdRp gene target (10 aM). The number of target sequences in 10 aM LOD is calculated to be 114, which corresponds to ca. 376 copies/mL. Therefore, the sensitivity of the MMP−SERS-based assay is comparable with that of current commercial kits (200−500 copies/mL). 53 It is important to note that the possibility of false-positive results from the use of NPs, particularly at low target concentrations, was significantly minimized in this platform owing to exclusion of the NPs in the target detection step. In addition, the ensemble-averaged measurement of the SERS response from the highly stable Ag clusters in solution significantly improved the signal reproducibility of this platform. These results indicate that the method demonstrated in this study can effectively address the two key issues of NPs and the SERSbased assay. Therefore, we expect that the MMP−SERS-based method will be a promising high-throughput platform technology with attomolar sensitivity and robust assay results. The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsami.1c17028. Measurement of the quantity of the capture probes on the magnetic microparticle (MMP); experimental setup of the filter set; surface-enhanced Raman scattering (SERS) spectra of the three different signal probes with ATTO 488, ATTO 565, and ATTO 647N dyes and experimental setups for solution-state or dry-state Raman analysis; Raman spectra of the three independent assays for the detection of RNA-dependent RNA polymerase (RdRp) gene target DNA; performance of the magnetic microparticle (MMP)-based assay with fluorescenceand surface-enhanced Raman scattering (SERS)-based detection of dual DNA targets; characterization of the various Ag nanostructures; results of the salt-induced formation of AgTPs clusters in solutions; and reproducibility of the three independent SERSbased assays for the detection of RdRp gene target DNA using AgNSs and AgTPs (PDF) A New Coronavirus Associated with Human Respiratory Disease in China A Novel Coronavirus from Patients with Pneumonia in China COVID-19 Diagnosis and Management: a Comprehensive Review SARS-CoV-2 Detection with CRISPR Diagnostics Detection of a Novel Human Coronavirus by Real-Time Reverse-Transcription Polymerase Chain Reaction Drosten, C. 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