Imunocapture Magnetic Beads Enhanced and Ultrasensitive CRISPR-Cas13a-Assisted Electrochemical Biosensor for Rapid Detection of SARS-CoV-2

The rapid and ongoing spread of the coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emphasizes the urgent need for an easy and sensitive virus detection method. Here, we describe an immunocapture magnetic bead-enhanced electrochemical biosensor for ultrasensitive SARS-CoV-2 detection based on clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, collectively known as CRISPR-Cas13a technology. At the core of the detection process, low-cast and immobilization-free commercial screen-printed carbon electrodes are used to measure the electrochemical signal, while streptavidin-coated immunocapture magnetic beads are used to reduce the background noise signal and enhance detection ability by separating the excessive report RNA, and a combination of isothermal amplification methods in the CRISPR-Cas13a system is used for nucleic acid detection. The results showed that the sensitivity of the biosensor increased by two orders of magnitude when the magnetic beads were used. The proposed biosensor required approximately 1 h of overall processing time and demonstrated an ultrasensitive ability to detect SARS-CoV-2, which could be as low as 1.66 aM. Furthermore, owing to the programmability of the CRISPR-Cas13a system, the biosensor can be flexibly applied to other viruses, providing a new approach for powerful clinical diagnostics.


Introduction
Rapid and accurate point-of-care testing (POCT) for nucleic acid detection is crucial for emergency disease confirmation and treatment and epidemic prevention and control [1]. Currently, the most widely used method for nucleic acid detection is polymerase chain reaction (PCR), owing to its high sensitivity and specificity. However, PCR analysis requires well-equipped laboratories, large and expensive equipment, a professional tester, and long turnover times, which limits its ability to be used in POCT [2,3]. Finding a simple, rapid, and accurate detection system, which can satisfy the needs of a variety of application scenarios, has therefore become a popular trend in the fields of clinical diagnosis and pathogen detection.
Recently, there have been significant developments in using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins (e.g., Cas9, Cas12a, Cas13a) in the detection of nucleic acids by exploiting the trans-cleavage activity of the CRISPR-Cas system, which can indiscriminately cleave surrounding reporter RNA/DNA (near and around the target RNA/DNA). Together with isothermal amplification technology (e.g., rolling circle amplification; loop-mediated isothermal amplification; Leptotrichia wadei) was purchased from GenScript (Nanjing, China). Magnesium chloride (MgCl 2 ), sodium chloride (NaCl), Tris-hydrochloride (Tris-HCl), chloroform (CHCl 3 ), and Ethylene Diamine Tetraacetic Acid (EDTA) were purchased from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China, and used without further processing. MB was obtained from Sigma-Aldrich. HEPES buffer solution was purchased from GIBCO (Invitrogen). A COVID-19 Nucleic Acid Detection Kit (CRISPR flow immunochromatographic method) was purchased from Zhongtest Biotechnology Co., Ltd. (Hangzhou, China). A COVID-19 Nucleic Acid Detection Kit (fluorescence PCR method) was purchased from Da and Gene Co., Ltd. (Guangzhou, China). The ribonucleotide solution mix and T7 RNA polymerase were purchased from New England Biolabs LTD (Beijing). Novel Coronavirus Nucleic Acid Standard (High Concentration) was purchased from the Research and Management Center of Standard Substances, Chinese Academy of Metrology.

RT-RAA Amplification
For target amplification, the RAA reaction was performed using a Novel Coronavirus 2019-nCoV Nucleic Acid Test Kit (CRISPR Immunoassay) following the manufacturer's instructions (product no. 211104006), which included freeze-drying the primer sets and other necessary substances in a reaction tube. Briefly, lyophilized reagent pellets containing 0.4 µM of forward and reverse primers in the kit were suspended in 17.5 µL of the supplied rehydration buffer, 30 µL of target RNA template, and 2.5 µL of magnesium acetate. The reaction mixture was then subjected to amplification at 42 • C for 30 min.
For real-time fluorescence monitoring, the reactions were incubated for 60 min at 37 • C in a LightCycler ® 96 System (Roche, Switzerland). The fluorescence of the trans-cleavage reaction was measured every 2 min with excitation at 495 nm and emission at 520 nm.

CRISPR/Cas13a Electrochemical Assay
For the electrochemical analysis of trans-cleaved reporters, the cleavage assay was performed using previously described fluorescence CRISPR methods [27], with slight modifications. The working concentrations of the components used in the electrochemical CRISPR activation assay were as follows: 25 mM HEPES, 2.5 mM rNTP, 75 nM Cas13a, 50 nM crRNA, 1 µM biotin-RNA-MB reporter, 25 mM MgCl 2 , 1 IU/µL T7 RNA polymerase, 2 µL nucleic acid amplification products, and RNase-free water to a final volume of 40 µL. The reactions were incubated for 30 min at 37 • C.
Electrochemical measurements were performed using a CHI 660E electrochemical workstation (CH Instruments Co., Shanghai, China). For SWV tests, 40 µL of the supernatant was added dropwise to the screen-printed electrode surface and scanned by electrochemical methods. SWV was performed at 50 Hz with an amplitude of 50 mV and a potential increment of 4 mV in the range of −0.5 to 0 V. The supernatant was tested using the electrochemical method after being dropped onto the screen-printed electrode for about 1 min. Finally, we observed the current value (peak current value after subtracting the background) from SWV and analyzed the data.
A cutoff method was used to judge the detection result. The cutoff current value was defined by the average current value plus three times the standard deviation (SD) of the negative control (NC) group for determining positivity and negativity [28].

Immunocapture Separation
First, after mixing thoroughly, 30 µL of commercial SA-coated magnetic bead turbidity in a tube was taken and placed on a magnetic stand for 1 min; the supernatant was discarded. Resuspended SA-coated magnetic beads were then added in 40 µL of ddH 2 O, the tube was placed on a magnetic stand for 1 min again, and then the supernatant was discarded. The electrochemical CRISPR reaction mixture was added to the SA-coated magnetic beads and mixed. Finally, the tube was placed on a magnetic stand for 1 min before the supernatant was collected for square wave voltammetry (SWV).

RT-qPCR Assay
For comparison of the detection limit and clinical sample testing of the different methods, Reverse transcription quantitative real-time PCR (RT-qPCR) was performed using a commercial RT-qPCR kit (Da An Gene Co., Ltd., Sun Yat-sen University, Guangzhou, China). Although this kit can detect both SARS-CoV-2 ORF1ab and N genes, the sensitivity analysis focused on the N gene. The RT-qPCR reaction system and PCR conditions were as described in the kit instructions. A cycle threshold (Ct) value ≤40 was defined as a positive result, whereas a Ct value >40 was defined as negative.

Statistical Analysis
A Student's t-test or Welch's t-test was performed to compare different groups, and all data were presented as the mean ± SD of at least three independent experiments.

Design of the Electrochemical Biosensor
The principle of the developed electrochemical biosensor is shown in Figure 1. The strategy involves using guide CRISPR RNA (crRNA) designed to specifically recognize a target RNA, for example, SARS-CoV-2 RNA. After the Cas protein recognizes the target RNA, trans-cleavage activity is activated, resulting in the cleavage of reporter RNA (termed reRNA), which is a 20 nucleotide (nt) ssRNA (poly U) that modified with electrochemically activated biotin on the 5 end and MB on the 3 end (termed biotin-ssRNA-MB). After trans-cleavage activity, SA-coated magnetic beads are used to bind the reRNA through the biotin moiety and remove any uncleaved reRNA and biotin-oligonucleotide in the reaction solution by magnetic separation, leaving the MB-oligonucleotide in the solution. Subsequently, an electrochemical workstation and a three-electrode commercial screenprinted carbon electrode were applied to detect the MB electrochemical signal in the mixture, leading to the ultrasensitive and specific detection of the target RNA.
Using SARS-CoV-2 RNA as the target, the trans-cleavage activity of Cas13a was activated by RT-RAA-amplified and T7 transcribed target nucleic acid, resulting in the cleavage of the reRNA. The reRNA was cleaved into MB-oligonucleotide and biotin-oligonucleotide. The remaining uncleaved reRNA and biotin-oligonucleotide were removed by the SAcoated magnetic beads, leaving the MB-oligonucleotide in solution. At this stage, an electrochemical signal is acquired by SWV, and the redox peak signal associated with the MB concentration is recorded. In turn, in the absence of target SARS-CoV-2 RNA, an MB redox peak signal is not observed. In combination with RT-RAA, this novel electrochemical CRISPR biosensor could be used as a low-cost, easy-to-fabricate, ultrasensitive, and highly specific biosensor for the detection of SARS-CoV-2 as well as other pathogens.
MB redox peak signal is not observed. In combination with RT-RAA, this novel electrochemical CRISPR biosensor could be used as a low-cost, easy-to-fabricate, ultrasensitive, and highly specific biosensor for the detection of SARS-CoV-2 as well as other pathogens. The main process of the electrochemical CRISPR biosensor. When the target RNA is present, the function of Cas13a-crRNA is activated, and the reRNA is cleaved into MB-oligonucleotide and biotin-oligonucleotide. The extra uncleaved reRNA and biotin-oligonucleotide are removed by SA-coated magnetic beads, leaving the MB-oligonucleotide in the solution, which could be detected by electrochemical measurement. When the target RNA is absent, the function of Cas13a-crRNA is not activated. No cleavage of reRNA occurs in the solution, and the reRNA is removed by streptavidin (SA)-coated magnetic beads. Therefore, almost no electrochemical signal can be observed.

Verification of the Electrochemical CRISPR Biosensor
To validate the feasibility of this electrochemical CRISPR biosensor, the SA-coated magnetic beads were used to enrich the amount of trans-cleavage-associated electrochemically active molecule (MB in this study). This was achieved through the addition of abundant reRNA, followed by the separation of uncleaved reRNA. The reRNA separation ability of the SA-coated magnetic beads was evaluated by measuring the SWV electrochemical signals of MB and reRNA in solution and the reRNA solution after treatment with SAcoated magnetic beads (all in a NaCl solution) and compared with the signals obtained using an NC sample (NaCl solution only) (Figure 2a). A significant difference in electrochemical signal was observed between the MB and NC samples (p < 0.001), indicating the participation of MB in the redox reaction on the surface of the screen-printed carbon electrode. The reRNA sample also exhibited a strong electrochemical signal response, but slightly less than the pure MB sample. This was mainly due to a reduction in diffusion velocity with increasing molecule size because MB is much smaller than MB-ssRNA-biotin and therefore diffuses faster [29]. Almost no electrochemical signal response was observed for the reRNA sample after separation with the magnetic beads, demonstrating The main process of the electrochemical CRISPR biosensor. When the target RNA is present, the function of Cas13a-crRNA is activated, and the reRNA is cleaved into MB-oligonucleotide and biotin-oligonucleotide. The extra uncleaved reRNA and biotin-oligonucleotide are removed by SAcoated magnetic beads, leaving the MB-oligonucleotide in the solution, which could be detected by electrochemical measurement. When the target RNA is absent, the function of Cas13a-crRNA is not activated. No cleavage of reRNA occurs in the solution, and the reRNA is removed by streptavidin (SA)-coated magnetic beads. Therefore, almost no electrochemical signal can be observed.

Verification of the Electrochemical CRISPR Biosensor
To validate the feasibility of this electrochemical CRISPR biosensor, the SA-coated magnetic beads were used to enrich the amount of trans-cleavage-associated electrochemically active molecule (MB in this study). This was achieved through the addition of abundant reRNA, followed by the separation of uncleaved reRNA. The reRNA separation ability of the SA-coated magnetic beads was evaluated by measuring the SWV electrochemical signals of MB and reRNA in solution and the reRNA solution after treatment with SA-coated magnetic beads (all in a NaCl solution) and compared with the signals obtained using an NC sample (NaCl solution only) (Figure 2a). A significant difference in electrochemical signal was observed between the MB and NC samples (p < 0.001), indicating the participation of MB in the redox reaction on the surface of the screen-printed carbon electrode. The reRNA sample also exhibited a strong electrochemical signal response, but slightly less than the pure MB sample. This was mainly due to a reduction in diffusion velocity with increasing molecule size because MB is much smaller than MB-ssRNA-biotin and therefore diffuses faster [29]. Almost no electrochemical signal response was observed for the reRNA sample after separation with the magnetic beads, demonstrating that intact reRNA could be efficiently removed from the solution by the SA-coated magnetic beads. that intact reRNA could be efficiently removed from the solution by the SA-coated magnetic beads. replicates. An unpaired two-tailed Welch's t-test was used to analyze statistical significance (*** p < 0.001, **** p < 0.0001).
To further examine the feasibility of the biosensor for nucleic acid detection, we selected the N gene of SARS-CoV-2 as the target RNA. Cas13a trans-cleavage activity was activated in the presence of target RNA, and the cleavage of reRNA could be monitored by real-time fluorescence monitoring ( Figure 2b) and electrochemical measurements (Figure 2c,d) by reading a significant fluorescence signal or electrochemical peak. The fluorescence and electrochemical signals barely changed in the absence of Cas13a, crRNA, or 10,000 pM target RNA (Figure 2b-d). The amplitude of the electrochemical peak reflected the degree of reRNA cleavage. The average electrochemical peak current of the Cas13a + crRNA + target RNA group reached 1398.0 nA after CRISPR/Cas13a trans-cleavage activity, which is 30-fold higher than the average peak current of the Cas13a + crRNA group (46.1 nA), indicating excellent feasibility for this detection platform (Figure 2d).

Optimization of the Reaction Conditions
In this electrochemical CRISPR biosensor, the separation and removal of extra, uncleaved reRNA are key to distinguishing positive and negative results. Therefore, the ability of SA-coated magnetic beads to bind the reRNA in the solution and then remove it was evaluated (1.7 mol/L NaCl served as the electrolyte) ( Figure S1a). The SA-coated magnetic replicates. An unpaired two-tailed Welch's t-test was used to analyze statistical significance (*** p < 0.001, **** p < 0.0001).
To further examine the feasibility of the biosensor for nucleic acid detection, we selected the N gene of SARS-CoV-2 as the target RNA. Cas13a trans-cleavage activity was activated in the presence of target RNA, and the cleavage of reRNA could be monitored by real-time fluorescence monitoring ( Figure 2b) and electrochemical measurements (Figure 2c,d) by reading a significant fluorescence signal or electrochemical peak. The fluorescence and electrochemical signals barely changed in the absence of Cas13a, crRNA, or 10,000 pM target RNA (Figure 2b-d). The amplitude of the electrochemical peak reflected the degree of reRNA cleavage. The average electrochemical peak current of the Cas13a + crRNA + target RNA group reached 1398.0 nA after CRISPR/Cas13a trans-cleavage activity, which is 30-fold higher than the average peak current of the Cas13a + crRNA group (46.1 nA), indicating excellent feasibility for this detection platform (Figure 2d).

Optimization of the Reaction Conditions
In this electrochemical CRISPR biosensor, the separation and removal of extra, uncleaved reRNA are key to distinguishing positive and negative results. Therefore, the ability of SA-coated magnetic beads to bind the reRNA in the solution and then remove it was evaluated (1.7 mol/L NaCl served as the electrolyte) ( Figure S1a). The SA-coated magnetic beads efficiently removed the reRNA in solution as indicated by a decrease in the electrochemical signal; after 1 min of incubation, the electrochemical signal was barely detected. Therefore, an incubation time of 1 min with SA-coated magnetic beads was used in this study. Divalent Mg ions not only play an important role in DNA polymerase catalysis during RT-RAA isothermal amplification [30], but they also affect the cleavage activity of Cas13a [26]. We analyzed a 0-30 mM Mg 2+ concentration gradient for verification and found that 25 mM Mg 2+ yielded the best detection performance ( Figure S1b). This result is close to the 20 mM Mg 2+ suggested in one previous Cas12a study [12]. Therefore, we selected 25 mM Mg 2+ for further investigation in this study. Different concentrations of Cas13a protein were also evaluated, revealing that the electrochemical current increased with an increase in Cas13a protein concentration. For cost considerations, we selected the concentration of 75 mM Cas13a protein for use with the electrochemical CRISPR biosensor ( Figure S1c).

Electrochemical CRISPR Biosensor without Target RNA Amplification
Under optimized experimental conditions, we investigated the performance of the electrochemical CRISPR biosensor without target RNA amplification. The electrochemical current cutoff value was calculated to be 44.4 nA (average background value plus three times the standard deviation). Samples containing 10,000, 1000, 100, and 10 pM RNA had a higher electrochemical current than the cutoff value (Figure 3a,b), indicating that the target RNA in these samples was successfully detected. The average electrochemical current values between 10,000 pM and 1000 pM groups were almost equal; this result may be because all of the reRNA was successfully cleaved, and the highest electrochemical current value had been reached in both groups. Compared with other electrochemical CRISPR biosensors in detecting unamplified target RNA, the biosensor developed here showed an improved detection limit (down to 10 pM target RNA) [4,20]. The developed biosensor benefits from both an efficient reaction in a homogeneous solution and a significant reduction in the background signal by the removal of extra reRNA. More specifically, in our system, compared to heterogeneous electrochemical methods, analyte recognition and signal amplification transduction are performed in a homogeneous solution, which avoids steric hindrance of Cas13a interfacial cleavage [31]. In addition, our experimental procedure is simplified, effectively improving measurement reliability and repeatability of the biosensors. When SA-coated magnetic beads were used to remove the remaining reRNA, the background signal was greatly reduced, and the signal-to-noise ratio was improved, which was beneficial to signal output. Furthermore, we verified the assay's specificity by designing and testing two different SARS-CoV-2 variant sites (HV69-70del and D614G). Positive results were obtained only when mutant or wild-type SARS-CoV-2 sequences matched their respective crRNAs, and no obvious cross-reactivity was observed. The HV69-70del SARS-CoV-2 mutant strain was missing six bases compared to the wild-type strain ( Figure S2a), and the D614G SARS-CoV-2 mutant strain had a single base exchange ( Figure S2b). As indicated in Figure 3c, mutant-type SARS-CoV-2 with the corresponding crRNA induced specific electrochemical responses. When the crRNA of the mutant-type SARS-CoV-2 strains bound to the wildtype strain sequence, the wild-type samples showed a lower electrochemical signal. These results showed that the NucID strain had a single base exchange or missing six bases that could be distinguished by this biosensor, which demonstrates that the biosensor has high specificity. Additionally, owing to the programmability of crRNA, the electrochemical CRISPR biosensor allows for the development of a universal biosensing platform for any target RNA by easily changing the sequence of the crRNA guide region [32].  replicates. An unpaired two-tailed Welch's t-test was used to analyze the statistical significance.

Electrochemical CRISPR Biosensor with RT-RAA Amplification
The RT-RAA is an isothermal nucleic acid detection method that is both fast and robust; it is based on an enzyme reaction that has been successfully applied in the highly sensitive detection of pathogens [3,33,34]. Together with the signal amplification of Cas13a trans-cleavage activity [35], the RT-RAA-based CRISPR/Cas13a electrochemical biosensor described here exhibited very good sensitivity. When the electrochemical CRISPR biosensor was combined with RT-RAA isothermal amplification, T7 transcription was required for trans-cleavage activity, a time-consuming process. However, for the purpose of POCT, the whole operating time should not be very long. Based on our previous study [27], 30 min was sufficient for T7 transcription and trans-cleavage. In practice, the entire process required no more than 63 min, broken down as follows: RT-RAA isothermal amplification to amplify the target nucleic acid (30 min), T7 transcription and Cas13a cleaving time (30 min), removal of excess reRNA with SA-coated magnetic beans (1 min), and measurement of the electrochemical signal (2 min).
Under optimized experimental conditions and successful nucleic acid amplification ( Figure S3), we investigated the performance of the electrochemical CRISPR biosensor both with and without SA-coated magnetic bead enhancement against a range of different concentrations of target RNA. Coronavirus disease 2019 (COVID-19) RNA reference material (high concentration) containing the SARS-CoV-2 N gene sequence was used to compare the ability of the biosensor to detect RNA using a fluorescent CRISPR assay and RT- The red-filled dots (b) means the current value was larger than the cutoff current value, while the black-filled dots (b) means the current value was smaller than the cutoff current. Values represent means ± SD, where n = 3 replicates. An unpaired two-tailed Welch's t-test was used to analyze the statistical significance.

Electrochemical CRISPR Biosensor with RT-RAA Amplification
The RT-RAA is an isothermal nucleic acid detection method that is both fast and robust; it is based on an enzyme reaction that has been successfully applied in the highly sensitive detection of pathogens [3,33,34]. Together with the signal amplification of Cas13a trans-cleavage activity [35], the RT-RAA-based CRISPR/Cas13a electrochemical biosensor described here exhibited very good sensitivity. When the electrochemical CRISPR biosensor was combined with RT-RAA isothermal amplification, T7 transcription was required for trans-cleavage activity, a time-consuming process. However, for the purpose of POCT, the whole operating time should not be very long. Based on our previous study [27], 30 min was sufficient for T7 transcription and trans-cleavage. In practice, the entire process required no more than 63 min, broken down as follows: RT-RAA isothermal amplification to amplify the target nucleic acid (30 min), T7 transcription and Cas13a cleaving time (30 min), removal of excess reRNA with SA-coated magnetic beans (1 min), and measurement of the electrochemical signal (2 min).
Under optimized experimental conditions and successful nucleic acid amplification ( Figure S3), we investigated the performance of the electrochemical CRISPR biosensor both with and without SA-coated magnetic bead enhancement against a range of different concentrations of target RNA. Coronavirus disease 2019 (COVID-19) RNA reference material (high concentration) containing the SARS-CoV-2 N gene sequence was used to compare the ability of the biosensor to detect RNA using a fluorescent CRISPR assay and RT-qPCR. Figure 4a, the SA-coated magnetic beads demonstrated a remarkable ability to reduce the background noise signal, while the NC group with SA-coated magnetic beads enhancement demonstrated a lower level of background noise signal than the NC group without SA-coated magnetic beads enhancement. Moreover, the electrochemical CRISPR biosensor with SA-coated magnetic bead enhancement could detect down to 1.66 × 10 0 aM of COVID-19 RNA reference material, while the biosensor without magnetic bead enhancement could only detect down to 1.66 × 10 2 aM of reference material.

As shown in
Biosensors 2023, 13, x FOR PEER REVIEW 9 of 13 qPCR. As shown in Figure 4a, the SA-coated magnetic beads demonstrated a remarkable ability to reduce the background noise signal, while the NC group with SA-coated magnetic beads enhancement demonstrated a lower level of background noise signal than the NC group without SA-coated magnetic beads enhancement. Moreover, the electrochemical CRISPR biosensor with SA-coated magnetic bead enhancement could detect down to 1.66 × 10 0 aM of COVID-19 RNA reference material, while the biosensor without magnetic bead enhancement could only detect down to 1.66 × 10 2 aM of reference material. Currently, RT-qPCR is considered the "gold standard" for COVID-19 diagnosis by the United States Center for Disease Control and Prevention (CDC) [36]; thus, the ability Currently, RT-qPCR is considered the "gold standard" for COVID-19 diagnosis by the United States Center for Disease Control and Prevention (CDC) [36]; thus, the ability of RT-qPCR to detect the same amount of COVID-19 RNA reference material as that detected using the electrochemical CRISPR biosensor was also evaluated. However, RT-qPCR could not detect 1.66 × 10 0 aM; the detection limit was 1.66 × 10 1 aM of COVID-19 RNA reference material (Figure 4b), which is consistent with the results released by the CDC [36]. Notably, the detection limit using intact RT-qPCR methods for diagnosis may even be less than 0.83 aM (i.e., 0.5 copies/µL), especially when using a nucleic acid extraction procedure that can concentrate the samples about 5-10 times. However, since the samples in this study were COVID-19 RNA reference material, we did not need to extract the nucleic acid; hence these samples were not concentrated. This explains that the RT-qPCR detection limit of 1.66 × 10 1 aM for COVID-19 RNA reference material is reasonable. This experimental result also shows that the sensitivity of the electrochemical CRISPR biosensor was better than that of RT-qPCR, and the results were more stable than those using the fluorescent CRISPR method.
As a classic nucleic acid test, the fluorescent CRISPR assay was also used to detect COVID-19 RNA reference material. The results showed that in the 1.66 × 10 0 aM group, all but one of the five samples were positive, which demonstrates the fluorescent CRISPR assay could only detect down to 1.66 × 10 1 aM reference material (Figure 4c).
In addition to the flexible programmability of crRNA and the effective signal amplification efficiency of Cas13a, the high selectivity of the Cas13a/crRNA system is another important advantage. It was previously confirmed that the Cas13a/crRNA-based direct nucleic acid detection systems have single-base resolution [37]. In this study, the specificity of the electrochemical CRISPR biosensor with RT-RAA amplification was investigated using six different pathogens: SARS-CoV-2, human immunodeficiency virus (HIV), hepatitis B virus (HBV), hemagglutinin 1 neuraminidase 1 (H1N1), dengue virus (DENV), and Coxiella burnetii (CB). The target RNA extracted from SARS-CoV-2 exhibited a significant positive signal, whereas negative signals were obtained for the other pathogens, comparable to the blank control. These results indicate the superior specificity of the electrochemical CRISPR biosensor with RT-RAA amplification (Figure 4d).

Analysis of Clinical Samples
To achieve a better clinical diagnosis, we introduced the concept of a cutoff value, which was set to the background (based on the average current value of seven health clinic samples) plus three times the SD. The experimental results showed that the cutoff value for positivity was 55 nA (Figure 5a). When the current value of the electrochemical CRISPR biosensor with RT-RAA amplification was >55 nA, it was considered a positive result, and a current value of ≤55 nA was considered a negative result. A total of 39 clinical samples from the Beijing Institute of Microbiology and Epidemiology were analyzed using the electrochemical CRISPR biosensor and RT-qPCR (Figure 5b). Samples 2, 3, 5-13, 15, 17, and 19-21 were detected both positive by electrochemical CRISPR biosensor with RT-RAA amplification and real-time fluorescence RT-qPCR. Samples 4, 22-28, and 30-39 were detected as negative using an electrochemical CRISPR biosensor with RT-RAA amplification and real-time fluorescence RT-qPCR. However, samples 1, 14, and 29 were detected positive only by electrochemical CRISPR biosensor with RT-RAA amplification. So we can get the detailed numbers in Table 1. The electrochemical CRISPR biosensor yielded 19 positive samples and 20 negative samples, whereas real-time fluorescent RT-qPCR yielded 16 positive samples and 23 negative samples. The coincidence rate was 92.3% (Table 1). A total of three samples tested negative using RT-qPCR but positive using the electrochemical CRISPR biosensor. Further, these same three samples were additionally tested using Droplet Digital PCR, and all three samples tested positive (Table 2), which was consistent with the electrochemical CRISPR biosensor results. The curve is the SWV signal of the electrochemical CRISPR biosensor test results. The red curve indicates that the maximum current value exceeds the threshold and is judged as positive. Conversely, the black curve indicates that its maximum current value is less than the threshold value, and the result is judged negative.

Conclusions
In this study, we developed an immunocapture magnetic bead-enhanced biosensor that uses both isothermal amplification and CRISPR-based detection to test for SARS-CoV-2. This biosensor enables rapid, ultrasensitive (down to ~1.66 × 10 0 aM), and highly specific nucleic acid detection. We attribute the rapid detection speed and ultrahigh sensitivity of the biosensor to the following: (i) the immunocapture magnetic beads reduce Sample numbers 1-39 successively represent 39 clinical sample numbers. The horizontal dashed line indicates the threshold (value is 55nA). The curve is the SWV signal of the electrochemical CRISPR biosensor test results. The red curve indicates that the maximum current value exceeds the threshold and is judged as positive. Conversely, the black curve indicates that its maximum current value is less than the threshold value, and the result is judged negative.

Conclusions
In this study, we developed an immunocapture magnetic bead-enhanced biosensor that uses both isothermal amplification and CRISPR-based detection to test for SARS-CoV-2. This biosensor enables rapid, ultrasensitive (down to~1.66 × 10 0 aM), and highly specific nucleic acid detection. We attribute the rapid detection speed and ultrahigh sensitivity of the biosensor to the following: (i) the immunocapture magnetic beads reduce the background noise signal while allowing an abundant concentration of cleaved reRNA in the positive group, (ii) the system enables CRISPR-Cas13a cleavage within the homogeneous solution rather than at the electrode/heterogeneous solution interface, and (iii) RAA amplification and CRISPR-Cas13a-based activity can be combined. Additionally, the biosensor uses a commercial screen-printed carbon electrode, which does not require pre-treatment or molecular immobilization, making the biosensor easy to fabricate and operate while saving time overall. Based on the results of this study, the immunocapture magnetic bead-enhanced electrochemical biosensor for ultrasensitive SARS-CoV-2 detection based on CRISPR-Cas13a technology represents a promising electrochemical nucleic acid detection strategy for clinical application at the point of care.