key: cord-0746601-zb96ke3r
authors: López-Valls, María; Escalona-Noguero, Carmen; Rodríguez-Díaz, Ciro; Pardo, Demian; Castellanos, Milagros; Milán-Rois, Paula; Martínez-Garay, Carlos; Coloma, Rocío; Abreu, Melanie; Cantón, Rafael; Galán, Juan Carlos; Miranda, Rodolfo; Somoza, Álvaro; Sot, Begoña
title: CASCADE: Naked eye-detection of SARS-CoV-2 using Cas13a and gold nanoparticles
date: 2022-03-22
journal: Anal Chim Acta
DOI: 10.1016/j.aca.2022.339749
sha: 16bc8e95e5753c5870cfc457abf6b3d59012a93c
doc_id: 746601
cord_uid: zb96ke3r

The COVID-19 pandemic has brought to light the need for fast and sensitive detection methods to prevent the spread of pathogens. The scientific community is making a great effort to design new molecular detection methods suitable for fast point-of-care applications. In this regard, a variety of approaches have been developed or optimized, including isothermal amplification of viral nucleic acids, CRISPR-mediated target recognition, and read-out systems based on nanomaterials. Herein, we present CASCADE (CRISPR/CAS-based Colorimetric nucleic Acid DEtection), a sensing system for fast and specific naked-eye detection of SARS-CoV-2 RNA. In this approach, viral RNA is recognized by the LwaCas13a CRISPR protein, which activates its collateral RNase activity. Upon target recognition, Cas13a cleaves ssRNA oligonucleotides conjugated to gold nanoparticles (AuNPs), thus inducing their colloidal aggregation, which can be easily visualized. After an exhaustive optimization of functionalized AuNPs, CASCADE can detect picomolar concentrations of SARS-CoV-2 RNA. This sensitivity is further increased to low femtomolar (3 fM) and even attomolar (40 aM) ranges when CASCADE is coupled to RPA or NASBA isothermal nucleic acid amplification, respectively. We finally demonstrate that CASCADE succeeds in detecting SARS-CoV-2 in clinical samples from nasopharyngeal swabs. In conclusion, CASCADE is a fast and versatile RNA biosensor that can be coupled to different isothermal nucleic acid amplification methods for naked-eye diagnosis of infectious diseases.

) were prepared using a H6/H8 DNA/RNA Synthesizer 154 (K&A). After solid-phase synthesis, the solid support was transferred to a screw-cap glass 155 vial and incubated at room temperature for 16 hours with 2 mL of ammonia / ethanol 156 solution (3:1). Then, the samples were dried and purified using standard DNA/RNA min. Then, 12 µL of a 5 M NaCl solution were added every 20 min (5 times) until 0.3 M 164 salt concentration was reached in the solution. The mixture was incubated for 16h in the 165 dark at room temperature. The gold nanoparticles were then centrifuged at 13200 rpm at 166 4 °C for 35 min, the supernatant was removed, and the pellet was resuspended in water. 167 This process was repeated twice to remove the unbound oligonucleotides. To obtain the 168 0.7 and 2.3 pmol µL -1 AuNP loadings we employed 1.1 pmol µL -1 and 4.5 pmol µL -1 169 oligonucleotide initial concentration, respectively. 170 To quantify the attached oligonucleotide, the supernatants were collected, concentrated, 171 and quantified by spectroscopy at 260 nm. published works based on Cas12a and ssDNA-AuNPs, which constitutes a major 283 advantage. 284 We performed several control experiments to further confirm that the mechanism 311 ssRNA-functionalized AuNPs play an essential role in CASCADE, as they act as a 312 substrate for Cas13a's trans-cleavage activity and provide its colorimetric read-out. Thus, 313 we decided to assess the main critical parameters that might control this process. We three loadings tested (0.7, 1.5 and 2.3 pmol µL -1 ) were suitable for detecting the target 340 sequence (Fig. 3C, Fig. S5 ). However, AuNPs loaded with 0.7 pmol µL -1 ssRNA did not contain enough oligonucleotide to stabilize the AuNPs (Fig. S5B) . Even in the absence 342 of target, they aggregated and showed a color shift, making them unsuitable for reliable 343 detection. On the other hand, the 2.3 pmol µL -1 loading yielded the most stable 344 nanoparticles, which required longer destabilization times. Therefore, the 1.5 pmol µL -1 345 loading was selected since it provided stable nanostructures and the fastest read-out. 346 Finally, we evaluated the effect of AuNP concentration on detection. Three different 347 AuNP concentrations were assessed: 11.7, 23.4 and 46.8 nM. At 11.7 nM the AuNP 348 suspension was so diluted that changes in absorbance were hardly noticeable. At 46.8 349 nM, no significant differences could be observed between the samples with and without 350 target. However, at 23.4 nM the decrease in absorbance was readily apparent at the three 351 time points evaluated (Fig. 3D, Fig. S6) . Furthermore, at this concentration, color changes 352 were easily detectable by the naked eye (Fig. S6B) . 353 After the optimization of the system, we evaluated the temporal response of our sensor to Specificity is an essential feature for diagnostic tools to be reliable. To determine the 364 specificity of CASCADE we confronted RNP R1 with the S target and RNP S1 with the 365 Orf1ab target (Fig. S7) . In the presence of the unspecific target, there was no detection, demonstrating that the AuNP colloidal destabilization is only produced by specific target 367 recognition. Furthermore, there was specific detection even when both target sequences 368 were mixed, meaning that the presence of unspecific sequences did not hinder target 369 recognition. This is a necessary feature for sensors, since they must recognize specific 370 sequences in complex samples.

Finally, we tested the long-term colloidal stability of CASCADE´s main components. 372 We stored ssRNA3-AuNPs for 5 months at 4ºC and used them for CASCADE detection.

They showed an adequate stability when the target was not present, and a detection 374 performance comparable to that of freshly prepared AuNPs (Fig S8A,B, C) . Their 375 colloidal stability was further tested by comparing the plasmon spectra of stored and 376 freshly made ssRNA3-AuNPs ( Figure S8D ). Their plasmon spectra were similar, 377 meaning that they were the same size. In conclusion, our RNA-coated AuNPs can be 378 safely kept at 4ºC for months. In addition, it has been previously observed that Cas13a- 397 Next, we studied CASCADE's sensitivity, another essential feature for PoC diagnosis.

Guide RNA design crucially influences Cas13a's activity, and therefore its overall 399 sensitivity. To assess the role of the crRNA sequences, two guides were used to recognize 400 a specific region of the Orf1ab target RNA (R1 and R2) and another two to recognize the 401 S target RNA (S1 and S2) (Table S1 ). R1 and S1 were described by Zhang et al.[19] , 402 while R2 and S2 were carefully designed using RNA secondary structure prediction 403 software. R1, R2 and S1 yielded comparable results, enabling the detection of 0.5 nM of 404 their respective target RNAs at 15-30 minutes (Fig. 4, Fig. S9 -S11), determined by the 405 statistical significance of the absorbance measurements in comparison with the negative 406 control. Moreover, R1 and S1 allowed the detection of 0.1 nM of their target after a one-407 hour incubation (Fig. S9, Fig. S11 ). The detection with S2, however, was notably less 408 sensitive (Fig. S12) . Although S1 and S2 target sites are not very distant, their behavior is markedly different. This highlights the importance of crRNA design. crRNAs should 410 be carefully selected so that they target an accessible RNA region with minimal secondary 411 structure. Furthermore, several guides should be tested to choose the one with optimal 412 performance.

The combination of two spectral changes, the blue-shift and the decrease in intensity due (Table S2) . [27] . We then evaluated whether using this strategy for CASCADE would 437 improve the sensitivity of the sensor in comparison with single-guide targeting. We 438 combined RNP R1 with RNP R2 and RNP S1 with RNP S2 to detect Orf1ab or S RNA 439 targets, respectively. These targets have a size similar to that of amplicons resulting from 440 isothermal amplification. However, the RNP combinations did not result in improved 441 sensitivity (Fig. S13 ) as compared to the use of single RNPs (Fig. 4) . It is likely that the 442 binding sites in the target for the two guides tested were too close (Fig. S13A ). This might both the retro-and T7 transcriptions. Strikingly, it has barely been applied before to 487 CRISPR-based detection. To assess whether this amplification method could be coupled 488 to CASCADE-based detection, an RNA template (RNA template NASBA, Table S1 ) was 489 generated via RT-PCR and T7 transcription using T7 S_200 5' PCR and S_200 3' PCR 490 primers (Table S1 ). Different concentrations of this RNA were subjected to NASBA 491 amplification using NASBA FW and RV primers (Table S1) described by Wu et al.[20] .

After a 90-minute incubation at 41 °C, samples were added to CASCADE. 15 minutes 493 later, we observed a significant decrease in absorbance for all the template concentrations 494 tested (Fig. S15) . After 30 minutes of incubation, absorbance continued to decrease, and 495 samples also displayed a color change visible to the naked eye ( Fig. 6 and Fig. S15 ).

NASBA-CASCADE was able to detect RNA sequences from SARS-CoV-2 with 497 attomolar sensitivity (40 aM) in two hours.

Taken together, these results suggest that our AuNP-Cas13a sensor is a versatile detection 499 system that can be coupled to different isothermal amplification methods, which further 500 highlights its potential for PoC applications. Remarkably, the sensitivity measured by the 501 absorbance decrease correlates with naked-eye detection ( Figure S14 and S15), 502 supporting the use of CASCADE as a reliable naked-eye sensor. for S1) ± SD. *P <0.05; **P < 0.01; ***P < 0.001; ns: non-significant. (n=3) ± SD. *P <0.05; **P < 0.01; ***P < 0.001; ns: non-significant. Figure S18 shows that the presence of RNA from other coronaviruses did 569 not impair SARS-CoV-2 detection by CASCADE. In conclusion, CASCADE is a highly 570 specific detection method able to detect all positive samples tested (Cts 16-28), while 571 showing no changes for negative (Ct>35) ( Table 1) CASCADE is characterized by its inexpensive straightforward read-out, whether by using 597 a simple spectrophotometer or even the naked eye. In contrast to other similar methods 598 that require thermocyclers, centrifuges or fluorimeters, CASCADE only needs a 599 heatblock (Table S2 ). CASCADE's sensing, which involves Cas13a target recognition 600 and subsequent AuNP aggregation, requires very short times (15-30 min). Additionally, 601 all its components are commercially available and it can be carried out by non-specialized 602 staff. These features reinforce its feasibility and future practical application. 

PRE/IND-4438)

R thanks the Ministry of Economy, Industry and competitiveness of Spain for the 635 FPI fellowship

IMDEA Nanociencia acknowledges support from the 'Severo Ochoa' Programme for 637 Centres of Excellence in R&D (MINECO, Grant SEV-2016-0686

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