Skip to main content
NCBI home page
ACS AuthorChoice logoLink to ACS AuthorChoice
. 2024 Jul 13;96(29):12022–12029. doi: 10.1021/acs.analchem.4c02087

CRISPR/Cas13a-Responsive and RNA-Bridged DNA Hydrogel Capillary Sensor for Point-of-Care Detection of RNA

Hui Wang †,‡, Honghong Wang †,‡, Hongru Pian †, Fengxia Su †, Fu Tang §, Desheng Chen †, Junjie Chen †, Yongqiang Wen †, X Chris Le ‡,*, Zhengping Li †,*
PMCID: PMC11270516  PMID: 39001804

Abstract

graphic file with name ac4c02087_0005.jpg

Disease diagnostics and surveillance increasingly highlight the importance of portable, cost-effective, and sensitive point-of-care (POC) detection of nucleic acids. Here, we report a CRISPR/Cas13a-responsive and RNA-bridged DNA hydrogel capillary sensor for the direct and visual detection of specific RNA with high sensitivity. The capillary sensor was simply prepared by loading RNA-cross-linking DNA hydrogel film (∼0.2 mm ± 0.02 mm) at the end of a capillary. When CRISPR/Cas13a specifically recognizes the target RNA, the RNA bridge in the hydrogel film is cleaved by the trans-cleavage activity of CRISPR/Cas13a, increasing the permeability of the hydrogel film. Different concentrations of target RNA activate different amounts of Cas13a, cleaving different amounts of the RNA bridge in the hydrogel and causing corresponding changes in the permeability of the hydrogel. Therefore, samples containing different amounts of the target RNA travel to different distances in the capillary. Visual reading of the distance provides quantitative detection of the RNA target without the need for any nucleic acid amplification or auxiliary equipment. The technique was successfully used for the determination of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in clinical nasopharyngeal (NP) swab and saliva samples. Easily quantifiable distance using a ruler eliminates the need for any optical or electrochemical detection equipment, making this assay potentially useful for POC and on-site applications.

Rapid and sensitive detection of specific RNA sequences is important in many areas, ranging from clinical diagnostics and monitoring to biotechnological applications.1−3 For instance, RNA-based nucleic acid testing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is considered the gold standard for the diagnosis of coronavirus disease (COVID-19).4 The widely used RNA detection techniques rely mainly on reverse transcription-polymerase chain reaction (RT-PCR) due to its high sensitivity and specificity.2,5−7 RT-PCR requires equipment for thermal cycling and usually fluorescence detection, which is not readily available in resource-limited settings.

As attractive alternatives to the RT-PCR method, isothermal amplification strategies, such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP), have been developed and applied to the quantitative detection of RNA. These isothermal amplification techniques do not require a thermal cycling instrument, and they can be easily integrated with clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas) systems.1,8−17 The main benefit of this integration is high sensitivity due to the exponential amplification of nucleic acids and high specificity offered by CRISPR/Cas systems. These integrated methods usually require multiple enzymes, primers, and probes, which increase the cost of assays. The discovery of the trans-cleavage activity of the CRISPR/Cas13 system has stimulated much interest in the development of techniques for direct RNA assay without reverse transcription or nucleic acid amplification.18−25 However, the detection limit of these amplification-free methods is usually at the pM level, which cannot meet the testing requirements of most clinical samples. The cascade CRISPR/Cas (casCRISP) system26 and multiple crRNAs-CRISPR/Cas13 assay24 improved the detection limit of direct RNA analysis to the fM level. However, the additional introduction of another CRISPR/Cas system or multiple crRNAs increases the analysis costs and design complexity. In addition, most of these assays require optical (e.g., fluorescence) or electrochemical detection equipment.

The objective of our research is to develop a sensitive RNA assay that does not require nucleic acid amplification and does not rely on optical or electrochemical detection equipment. Our strategy takes advantage of the trans-cleavage activity of CRISPR/Cas13a, the permeability of RNA-bridged DNA hydrogel in response to RNA cleavage, and the easily measurable distance traveled by the sample solution in the permeable hydrogel. We report here a CRISPR/Cas13a-responsive and RNA-bridged DNA hydrogel capillary sensor for potential POC detection of RNA. The CRISPR/Cas13a-responsive RNA-bridged DNA hydrogel is introduced into a capillary to form a thin film inside it. When the crRNA-guided CRISPR/Cas13a recognizes and binds with the target RNA, the trans-cleavage activity of CRISPR/Cas13a is activated. Subsequently, the RNA bridge in the DNA hydrogel film is cleaved by the activated CRISPR/Cas13a, leading to an increase in the permeability of the hydrogel. The changes in the hydrogel permeability result in corresponding differences in the distance traveled by the sample solution inside the capillary.27,28 Quantitative readout is through simple measurement of the distance traveled by the solution inside the capillary. Thus, quantitative analysis of RNA is achieved without the need for any optical or electrical equipment.

Experimental Section

Materials and Reagents

Recombinant CRISPR-Cas13a protein (LwaCas13a) was supplied by Shanghai HuicH Biotech Co. Ltd. (Shanghai, China). 10× NEBuffer r2.1 (100 mM Tris-HCl, 500 mM NaCl, 100 mM MgCl2, 1 μg/μL Recombinant Albumin, pH 7.9 @25 °C), T7 RNA polymerase, 10× T7 RNAPol reaction buffer (400 mM Tris-HCl, 60 mM MgCl2, 20 mM spermidine, 1 mM DTT, pH 7.9), DNase I, and NTP mixture (10 mM) were obtained from New England Biolabs (Beijing, China). RNase inhibitor (RRI) (40 U/μL), 6× loading buffer, 20 bp DNA Marker, DL 5,000 DNA Marker, and SMART MMLV Reverse Transcriptase were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China). JumpStart Taq DNA polymerase and 10× PCR buffer (100 mM Tris-HCl, 500 mM KCl, 15 mM MgCl2) were purchased from Sigma-Aldrich (Shanghai, China). RNA Viral Sample Collection Tubes (Inactivated), EZ-10 Spin Column Viral Total RNA Extraction Kit, Acrylamide, N,N,N′,N′-tetramethylethylenediamine (TEMED), ammonium persulfate (APS), SanPrep Column PCR Product Purification Kit, and 4S Red Plus Nucleic Acid Stain (10,000×, aqueous solution) were purchased from Shanghai Sangon Biotech Co., Ltd. (Shanghai, China). Pseudovirus of SARS-CoV-2-abMEN, HPLC-purified acrydite-modified DNAs (A-DNA-X and A-DNA-Y), DRCL-ssRNA, FQ-ssRNA reporter, DNA primers, and crRNA1 were synthesized and supplied by Shanghai Sangon Biotech Co., Ltd. DNA plasmids (including pUC57-2019-nCov-N, pUC57-bat-SL-CoVZC45-N, pUC57-SARS-N, and pUC57-HCoV-HKU1-N) and crRNA2 were supplied by Genscript Biotech Corporation (Nanjing, China). The sequences of the oligonucleotides used in this work are listed in Table S1. All reagents were analytically pure and could be used without further purification. All of the solutions used in this work were prepared in RNase-free water (Thermo Fisher Scientific).

Preparation of Linear-Chain Polyacrylamide-DNA

First, 100 μL of 500 μM Acrydite-DNA X (A-DNA-X) and 500 μM Acrydite-DNA Y (A-DNA-Y) were separately mixed with 20 μL of 25% (wt/vol) acrylamide and 78 μL of RNase-free water in two 1.5 mL DNA LoBind Tubes (Eppendorf). After brief vortexing and centrifugation, the two tubes (open lid) were degassed in a vacuum desiccator at room temperature for 10 min. Then, 1.0 μL of freshly prepared 20% (wt/vol) APS and 1.0 μL of 20% (v/v) TEMED were added separately to the two tubes. After brief mixing and centrifugation, the tubes were immediately placed in a vacuum desiccator again at room temperature for 15 min to polymerize linear-chain polyacrylamide-DNA (PA-X and PA-Y). Unpolymerized DNA-X, DNA-Y, and acrylamides were eliminated by using a 100 K NMWL Amicon Ultra-0.5 Centrifugal Filter Device (Millipore) according to the User Guide. Eventually, the PA-X and PA-Y were cycled again separately in 100 μL RNAase-free water in which the concentration of DNA X and DNA Y was about 500 μM.

Preparation of CRISPR/Cas13a-Responsive and RNA-Bridged DNA Hydrogel

Ten microliters of 500 μM PA-X, 10 μL of 500 μM PA-Y, 4 μL of 10× NEBbuffer r2.1, 2−10 μL of 500 μM cross-linking RNA, 0.8 μL of RRI, and appropriate RNase-free water were mixed for a final volume of 40 μL. The mixture was gently mixed with the tip of a pipet at 65 °C to guarantee the homogeneity of the hydrogel, then allowed to cool naturally to room temperature, and kept for 5 min to form the CRISPR/Cas13a-responsive RNA-bridged DNA hydrogel.

Treatment of the Capillary Tube

The glass capillary tubes (length of ∼10 cm and inner diameter of ∼0.3 mm) were treated with piranha solution (a mixture of 7:3 (v/v) 98% H2SO4 and 30% H2O2) at 90 °C for 2 h and followed by ultrasonication for 10 min in ethanol. Then, the glass capillary tubes were rinsed three times with Milli-Q water and blown dry with nitrogen gas.

Preparation of DNA Hydrogel Capillary Sensor

The CRISPR/Cas13a-responsive and RNA-bridged DNA hydrogel capillary sensor was prepared by filling capillary tubes with plugs of RNA-bridged DNA hydrogel.28,29 Briefly, centrifuge tubes containing CRISPR/Cas13a-responsive RNA-bridged DNA hydrogel were heated to 58 °C in a water bath. The DNA hydrogel turned into a liquid of high viscosity and little fluidity. Capillary tubes were inserted vertically into the heated gel solution within the centrifuge tubes for 3 s and then removed. The gel solution was loaded into the capillary tubes by capillary action, forming a DNA hydrogel film inside each capillary tube at room temperature. The capillary tubes were inspected under a microscope: the hydrogel film thickness was 0.2 mm ± 0.02 mm. The RNA-bridged DNA hydrogel capillary was fixed on a ruler for subsequent use (Figure 1).

Figure 1.

Figure 1

Open in a new tab

(A) Illustration of the design and working principle of the RNA-bridged DNA hydrogel capillary sensor for the detection of RNA. A target RNA sequence, e.g., the N gene of SARS-CoV-2, activates the CRISPR/Cas13a system through specific interaction with the crRNA. The activated CRISPR/Cas13a cleaves the RNA bridge in the DNA hydrogel, resulting in increases in permeability. (B) Preparation and use of the CRISPR/Cas13a-responsive DNA hydrogel capillary sensor. The distance traveled by the reaction solution in the capillary depends on the permeability changes of the hydrogel, which are determined by the amount of target that activates the CRISPR/Cas13a system.

Collection and Preparation of the Clinical Samples

Collection and analysis of nasopharyngeal (NP) swab and saliva samples were approved by the Research Ethics Board of the University of Science and Technology Beijing (2023-1-107). Samples were collected from adult volunteers in Beijing during June and July 2023, and no personal information was recorded. All swabs were prepared in RNA Viral Sample Collection Tubes (Inactivated). The viral RNA was extracted from the NP swab samples using an EZ-10 Spin Column Viral Total RNA Extraction Kit according to the manufacturer’s instructions. The input sample solution of the NP swab was 200 μL, and the viral RNA was extracted and collected in 50 μL of RNAase-free water. Five microliters of the RNA extract was used for SARS-CoV-2 detection with the CRISPR/Cas13a-responsive and RNA-bridged DNA hydrogel capillary sensor. One microliter of the RNA extract was used for SARS-CoV-2 detection with RT-PCR.

Saliva samples were self-collected in 1.5 mL centrifuge tubes. Subsequently, 2 μL of saliva samples were transferred into 8 μL of phosphate-buffered saline (PBS) solution containing 8 U RRI and 2.5% Trion-X 100. Then, the saliva solution sample was heated to 95 °C for 5 min, and 2 μL of heat-treated saliva sample was immediately detected by the capillary sensor-based assay.

Viral RNA Detection

The determination procedure comprised the Cas13a/crRNA-RNA reaction and the DNA hydrogel capillary sensor-based detection. First, 10 μL of Cas13a/crRNA-RNA reaction mixture, composed of 500 nM Cas13a protein, 250 nM crRNA1, 250 nM crRNA2, 8 U RRI (RNase inhibitor), 1× NEBbuffer r2.1, and the RNA sample, was prepared in 0.2 mL centrifuge tubes. After incubation at 37 °C for 5 min, the centrifuge tubes were placed horizontally and the capillary tubes were inserted into the sample solution within the centrifuge tubes. Then, the distance traveled by the sample solution inside the capillary was measured using a ruler. The results were viewed by the naked eye or photographed using a smartphone camera.

Results and Discussion

Principle and Design of the CRISPR/Cas13a-Responsive and RNA-Bridged DNA Hydrogel Capillary Sensor

The CRISPR/Cas13a-responsive and RNA-bridged DNA hydrogel was prepared using the oligonucleotide cross-linking strategy. As shown in Figure 1A, two acrydite-modified DNA strands (A-DNA-X and A-DNA-Y) were each copolymerized with acrylamide monomers to form linear-chain polyacrylamide-DNA, PA-X and PA-Y, respectively. A dual-role cross-linking single-stranded RNA (DRCL-ssRNA) was designed to contain three sequence regions: two sequences at the 3′- and 5′-terminals were, respectively, complementary to DNA-X and DNA-Y, and a U-rich motif in the middle served as the substrate of Cas13a trans-cleavage. Hybridization of DRCL-ssRNA at its two ends with the DNA-X and DNA-Y strands formed a DNA hydrogel that had porous structures at room temperature (Figure S2A). The U-rich motif in the middle of the DRCL-ssRNA was cleavable by the active CRISPR/Cas13a. Cleavage of the DRCL-ssRNA increased the permeability of the hydrogel.

Activation of the CRISPR/Cas13a system is achieved by the specific interaction of a target RNA with the crRNA-Cas13a ribonucleoprotein (RNP). Once activated by the RNA target, the CRISPR/Cas13a system has both cis-cleavage (target-specific) and trans-cleavage (collateral cleavage) functions. Our technique utilizes the trans-cleavage activity of the target-activated CRISPR/Cas13a system because the trans-cleavage is multiple turnover, with a kcat of about 2 s−1.30,31 Each target-activated CRISPR/Cas13a cleaves multiple RNA bridge sequences in the hydrogel, which breaks the cross-link between DNA-X and DNA-Y and increases the permeability of the DNA hydrogel (Figure 1A). Different concentrations of the target RNA will activate different amounts of the CRISPR/Cas13a enzyme to cleave the RNA bridge, resulting in differences in the permeability of the hydrogel. Because the distance that the reaction solution travels in the capillary depends on the permeability of the hydrogel, the amounts of the target in the reaction solution can be estimated from this distance.

Feasibility of the CRISPR/Cas13a-Responsive and RNA-Bridged DNA Hydrogel Capillary Sensor

As a proof of principle, we tested two segments of the nucleocapsid (N) gene of SARS-CoV-2, corresponding to those selected for the RT-PCR assays by the United States Centers for Disease Control and Prevention (U.S. CDC) and the China CDC.35 We designed two N gene-specific crRNAs, crRNA1 and crRNA2, to recognize the corresponding target sequences (Figure S4). We used crRNA1 and crRNA2 separately to form crRNA-Cas13a ribonucleoproteins and monitored the trans-cleavage of the fluorophore-quencher-labeled reporter (FAM-UUUUUC-BHQ1) after the activation of the crRNA-Cas13a ribonucleoprotein by the target N gene. Figure 2A,B shows that both crRNA1 and crRNA2 performed similarly and that both segments of the N gene can be detected using the CRISPR/Cas13a system. Additional results from the nondenaturing polyacrylamide gel electrophoresis (PAGE) analysis confirmed that the target N gene of SARS-CoV-2 activated the CRISPR/Cas13a system to trans-cleave the DRCL-ssRNA sequence (lanes 2 and 4 in Figure 2C). Because the DRCL-ssRNA sequence serves as the bridge (cross-linking) in the DNA hydrogel, the target-activated cleavage of this bridge sequence increases the permeability of the hydrogel. Thus, in the presence of the target N gene of SARS-CoV-2, the resulting reaction solution migrated in the CRISPR/Cas13a-responsive hydrogel capillary to 40 mm (Figure 2D), whereas the migration was minimal (<3 mm) when the reaction solution did not contain the target N gene. The migration distance in the CRISPR/Cas13a-responsive hydrogel capillary was readily measured for visual detection. These experiments did not require any nucleic acid amplification. These results demonstrate the feasibility of the CRISPR/Cas13a-responsive hydrogel capillary sensor for the amplification-free and visual detection of specific nucleic acid sequences.

Figure 2.

Figure 2

Open in a new tab

Feasibility evaluation of the CRISPR/Cas13a-responsive and RNA-bridged DNA hydrogel capillary sensor. (A) and (B) Real-time measurement of fluorescence generated by the trans-cleavage of an RNA reporter (FAM-UUUUUC-BHQ1). The CRISPR/Cas13a system was activated by the N gene of SARS-CoV-2 (1, 10, and 100 pM). The concentrations of Cas13a, crRNA, and the RNA reporter were 500 nM, 250 nM, and 500 nM, respectively: (A) using crRNA1 to recognize the N2 region of the U.S. CDC RT-PCR target and (B) using crRNA2 to recognize the N gene segment of the China CDC assay target. (C) Nondenaturing PAGE analysis of the CRISPR/Cas13a trans-cleavage reaction. All samples were prepared in 1× NEBbuffer r2.1. A-DNA-X: 500 nM; A-DNA-Y: 500 nM; DRCL-ssRNA: 500 nM; crRNA1: 250 nM; crRNA2: 250 nM; Cas13a: 500 nM; the N gene sequence of SARS-CoV-2 RNA: 100 pM; reaction time: 30 min. (D) Amplification-free and visual detection of the N gene of the SARS-CoV-2 RNA using the DNA hydrogel capillary sensor. The concentration of the RNA was 100 fM and the reaction time was 20 min.

Analytical Performance of the CRISPR/Cas13a-Responsive Hydrogel Capillary Sensor

We evaluated the CRISPR/Cas13a-responsive RNA-bridged hydrogel capillary sensor, under optimal experimental conditions (Figures S5−S7), for visual detection of the specific N gene sequence of SARS-CoV-2 RNA. The naked-eye observation results (Figure 3A) show that the distance traveled by the solution in the capillary increased proportionally with the increase of the target RNA concentration from 100 aM (∼60 copies/μL) to 100 fM (6 × 104 copies/μL). The difference in the travel distance between the 100 aM target (1.2 cm) and the negative control (0.5 cm) was easily distinguishable. The distances (D) traveled by the sample solution in the capillary are linearly correlated with the logarithm of the target RNA concentration in the range of 100 aM to 100 fM (Figure 3B). The correlation equation was D = 38.75 + 2.35 log CRNA and the correlation coefficient was 0.9960, indicating that the sensor can provide quantitative results. Furthermore, the sensor can specifically detect the N gene of SARS-CoV-2 with no cross-reaction against the related N gene from other SARS-like coronaviruses, including bat-SARS-like coronavirus ZC45 (bat-SL-COVZC45), SARS coronavirus (SARS-CoV), and human coronavirus HKU1 (Human-COV-HKU1) (Figures 3C and S8).

Figure 3.

Figure 3

Open in a new tab

Analytical performance of the CRISPR/Cas13a-responsive hydrogel capillary sensor. (A) The naked-eye observation results were produced from the analysis of the N gene of SARS-CoV-2 RNA (negative control, 100 aM, 1 fM, 5 fM, 20 fM, and 100 fM). The capillaries for the blank and 100 aM sample were removed from the centrifuge tubes and placed on a ruler, with the ends of the capillaries at the 2 cm mark. For the other samples, the ends of the capillaries were at the bottom of the centrifuge tubes, at the 0 cm mark on the ruler. (B) The linear relationship between the flow distance (D) of sample solutions and the logarithm of RNA concentrations (log(CRNA)). (C) Specificity assessment of the assay. crRNA1 and crRNA2 were designed to specifically target the N gene of SARS-CoV-2. Results represent the average of three replicate analyses, and the error bars indicate one standard deviation. All samples were prepared in 1× NEBbuffer r2.1, and the concentrations of crRNA1 and crRNA2 were 250 nM each, and Cas13a was 500 nM. The incubation time was 45 min.

Two unique features are important to the enhanced sensitivity of this CRISPR/Cas13a-responsive RNA-bridged hydrogel capillary sensor, compared to the previously reported DNA hydrogel sensors that were based on macroscopic shape changes.32−34 First, the CRISPR/Cas13a-responsive RNA-bridged DNA hydrogel is confined to an ultrasmall volume (∼0.01 μL) in the capillary. Once the CRISPR/Cas13a is activated by a single target molecule, the trans-cleavage activity by the active CRISPR/Cas13a is multiple turnover (kcat ∼ 2 s−1),30,31 resulting in the cleavage of multiple RNA bridges within the hydrogel. Breaking multiple RNA bridges within the small volume of the hydrogel significantly increases its permeability. Therefore, only a small amount of the target is needed to activate the trans-cleavage activity and to cause significant changes in the permeability of the hydrogel. Second, the high local concentration of the substrate (RNA bridge) in relation to the Cas13a enzyme improves the enzymatic reaction with the substrate. Both of these features significantly enhance the analytical sensitivity and shorten the detection time compared to a typical fluorescence assay using CRISPR/Cas13a (Figure S9 and Table S2).

As a consequence of the significant enhancement in the sensitivity, the CRISPR/Cas13a-responsive RNA-bridged hydrogel capillary sensor can detect levels as low as 100 aM (∼60 copies/μL) of the target N gene of SARS-CoV-2. Without the need for nucleic acid amplification, this assay has sufficient sensitivity for direct determination of SARS-CoV-2 RNA in clinical specimens, in which the concentrations of SARS-CoV-2 RNA are typically ∼103−106 copies/μL.36 Compared to the previously reported amplification-free CRISPR/Cas13 platforms,3,20−26,37−40 which are either not quantitative or require detection instruments (Table S2), our capillary sensor can provide quantitative measurements using the naked eye without relying on any optical or electrical equipment. These features suggest promising potential for point-of-care and resource-limited settings.

Analysis of Nasopharyngeal Swab and Saliva Samples Using the CRISPR/Cas13a-Responsive Hydrogel Capillary Sensor

To evaluate practical applications of the CRISPR/Cas13a-responsive hydrogel capillary sensor, we analyzed clinical nasopharyngeal (NP) swab and saliva samples (Figure 4A). Viral RNA was extracted from ten confirmed SARS-CoV-2 positive and six negative NP swab samples. As shown in Figures 4B and S10, all positive samples from SARS-CoV-2-infected patients generated measurable travel distances of the sample solutions in the capillaries farther than those of the negative samples and the blank control. These results indicate that the developed CRISPR/Cas13a-responsive hydrogel capillary sensor is able to provide sensitive and specific detection of the specific viral RNA. Importantly, quantification results can be achieved without the need for any optical or electrochemical measurement equipment. According to the calibration and correlation (Figure 3B) and the dilution factors in the sample preparation steps, we estimated the copies of viral RNA (the N gene) in our tested NP samples, which ranged from 40 copies/μL to 16,720 copies/μL. The copies of the SARS-CoV-2 RNA in these positive NP samples were also measured using RT-PCR. As shown in Figure 4C, the quantification results obtained using the standard RT-PCR and our CRISPR/Cas13a-responsive hydrogel capillary sensor method are in good agreement.

Figure 4.

Figure 4

Open in a new tab

Amplification-free and visual detection of SARS-CoV-2 in clinical samples using the CRISPR/Cas13a-responsive RNA-bridged DNA hydrogel capillary sensor. (A) Schematic showing the assay procedures. (B) Results from the analysis of 16 nasopharyngeal (NP) swab samples and a negative control showing distances traveled by the sample solution in capillaries. Error bars represent one standard deviation from triplicate analyses. (C) Concentrations of SARS-CoV-2 N gene RNA copies in 10 positive NP samples, measured using both the capillary sensor and the standard RT-PCR assay. (D) Detection of SARS-CoV-2 in ten heat-treated saliva samples. After the saliva samples were added to the respective tubes containing CRISPR/Cas13a reagents and the reactions took place, the distances traveled by the solutions in the capillaries were visualized and measured. For clear visualization, the capillaries in saliva samples 4 and 6−10 were removed from the reaction tubes and placed against a ruler at the 2 cm mark. The capillaries in samples 1−3 and 5 were kept in the reaction tubes.

We also applied the CRISPR/Cas13a-responsive hydrogel capillary sensor to the detection of SARS-CoV-2 RNA in saliva samples because previous work has demonstrated the promising potential of saliva for POC diagnostic applications.41−43 We analyzed 10 heat-treated saliva samples, comprising five samples (saliva 1−5) from confirmed SARS-CoV-2-infected patients and five specimens (saliva 6−10) from healthy individuals. As displayed in Figure 4D, all the SARS-CoV-2 positive saliva sample solutions generated significant travel distances in the capillaries compared to the negative saliva samples and the blank control. These results suggest that the developed CRISPR/Cas13a-responsive hydrogel capillary sensor method has great potential for POC, on-site, and at-home testing applications.

Conclusions

We have developed a novel CRISPR/Cas13a-responsive and RNA-bridged DNA hydrogel capillary sensor for amplification-free and visual detection of target RNA. Two main reasons contribute to the high sensitivity without the need for any nucleic acid amplification. First, the technique takes advantage of the multiple turnover trans-cleavage activity of the CRISPR/Cas13a system. When activated by a single RNA target molecule, the CRISPR/Cas13a system cleaves multiple RNA bridges within the hydrogel, resulting in significant increases in the permeability of the hydrogel and thus the diffusion of solutions through the hydrogel. Second, the hydrogel is confined to a very small volume (∼10 nL) in the capillary as a thin film. The high local concentration of the RNA bridge (the substrate) relative to the CRISPR/Cas13a enzyme favors the enzymatic reaction kinetics. Therefore, the fast kinetics and repeated cleavage of the RNA bridge in the hydrogel produce signal amplification (permeability and diffusion distance) without the need for nucleic acid amplification.

The sequence-specific recognition by the crRNA ensures the high specificity of the assay. We have demonstrated a successful application of our technique to the sensitive and selective detection of SARS-CoV-2 RNA in nasopharyngeal swab and saliva samples. This technique can be applied to the detection of diverse nucleic acid targets. For the detection of new target sequences, a minor modification of the technique involves the design and use of the corresponding crRNA that recognizes the specific target sequences. The programmability of the crRNA makes our technique versatile for various RNA assays and point-of-care molecular diagnosis.

The CRISPR/Cas13a-responsive RNA-bridged DNA hydrogel capillary sensor provides visual detection with the naked eye and quantifiable distances measured using a ruler. This technique does not require any photoelectric equipment for detection. This technique has great potential for point-of-care, onsite, and at-home applications.

Acknowledgments

We gratefully acknowledge the financial support from the National Natural Science Foundation of China (22234001), the Central Guidance on Local Science and Technology Development Fund of Hebei Province (236Z2402G), and Fundamental Research Funds for the Central Universities (FRF-TP-20-043A2, QNXM20230030, and QNXM20220051). Hui Wang and Honghong Wang were supported by China Scholarship Council (CSC) scholarships to visit the University of Alberta.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.4c02087.

  • Sequences of primers, probes, and crRNAs in relation to the N gene of SARS-CoV-2; in vitro transcription (IVT) of SARS-CoV-2 N gene RNA (Figure S1); characterization of DNA hydrogel film (Figure S2); melting temperature of DNA-X/DRCL-ssRNA and DNA-Y/DRCL-ssRNA (Figure S3); schematic of the crRNAs targeting the N gene regions of the SARS-CoV-2 genome (Figure S4); investigation of experimental conditions (Figures S5−S7); specificity assessment of the CRISPR/Cas13a-responsive hydrogel capillary sensor (Figure S8); typical results of a CRISPR/Cas13a assay using fluorescence detection of the N gene of SARS-CoV-2 RNA (Figure S9); amplification-free and visual detection of SARS-CoV-2 in nasopharyngeal swab samples using the CRISPR/Cas13a-responsive hydrogel capillary sensor (Figure S10); the sequences of oligonucleotides used in this work (Table S1); comparison between the performance of the developed hydrogel capillary sensor and other amplification-free CRISPR/Cas13a methods for RNA detection (Table S2) (PDF)

Author Contributions

# Hui Wang and Honghong Wang have contributed equally to this work.

The authors declare no competing financial interest.

Supplementary Material

ac4c02087_si_001.pdf (2.2MB, pdf)

References

  1. Gootenberg J. S.; Abudayyeh O. O.; Lee J. W.; Essletzbichler P.; Dy A. J.; Joung J.; Verdine V.; Donghia N.; Daringer N. M.; Freije C. A.; Myhrvold C.; Bhattacharyya R. P.; Livny J.; Regev A.; Koonin E. V.; Hung D. T.; Sabeti P. C.; Collins J. J.; Zhang F. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science 2017, 356 (6336), 438–442. 10.1126/science.aam9321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Zhao Y.; Zuo X.; Li Q.; Chen F.; Chen Y.-R.; Deng J.; Han D.; Hao C.; Huang F.; Huang Y.; Ke G.; Kuang H.; Li F.; Li J.; Li M.; Li N.; Lin Z.; Liu D.; Liu J.; Liu L.; Liu X.; Lu C.; Luo F.; Mao X.; Sun J.; Tang B.; Wang F.; Wang J.; Wang L.; Wang S.; Wu L.; Wu Z.-S.; Xia F.; Xu C.; Yang Y.; Yuan B.-F.; Yuan Q.; Zhang C.; Zhu Z.; Yang C.; Zhang X.-B.; Yang H.; Tan W.; Fan C. Nucleic Acids Analysis. Sci. China: Chem. 2021, 64 (2), 171–203. 10.1007/s11426-020-9864-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Tian T.; Shu B.; Jiang Y.; Ye M.; Liu L.; Guo Z.; Han Z.; Wang Z.; Zhou X. An Ultralocalized Cas13a Assay Enables Universal and Nucleic Acid Amplification-Free Single-Molecule RNA Diagnostics. ACS Nano 2021, 15 (1), 1167–1178. 10.1021/acsnano.0c08165. [DOI] [PubMed] [Google Scholar]
  4. Centers for Disease Control and Prevention, COVID-19 Testing. https://www.cdc.gov/coronavirus/2019-ncov/symptoms-testing/testing.html#types-of-tests, 2023.
  5. Chen C.; Ridzon D. A.; Broomer A. J.; Zhou Z.; Lee D. H.; Nguyen J. T.; Barbisin M.; Xu N. L.; Mahuvakar V. R.; Andersen M. R.; Lao K. Q.; Livak K. J.; Guegler K. J. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res. 2005, 33 (20), e179 10.1093/nar/gni178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Nolan T.; Hands R. E.; Bustin S. A. Quantification of mRNA using real-time RT-PCR. Nat. Protoc. 2006, 1 (3), 1559–1582. 10.1038/nprot.2006.236. [DOI] [PubMed] [Google Scholar]
  7. Vogels C. B. F.; Brito A. F.; Wyllie A. L.; Fauver J. R.; Ott I. M.; Kalinich C. C.; Petrone M. E.; Casanovas-Massana A.; Catherine Muenker M.; Moore A. J.; Klein J.; Lu P.; Lu-Culligan A.; Jiang X.; Kim D. J.; Kudo E.; Mao T.; Moriyama M.; Oh J. E.; Park A.; Silva J.; Song E.; Takahashi T.; Taura M.; Tokuyama M.; Venkataraman A.; Weizman O.-E.; Wong P.; Yang Y.; Cheemarla N. R.; White E. B.; Lapidus S.; Earnest R.; Geng B.; Vijayakumar P.; Odio C.; Fournier J.; Bermejo S.; Farhadian S.; Dela Cruz C. S.; Iwasaki A.; Ko A. I.; Landry M. L.; Foxman E. F.; Grubaugh N. D. Analytical sensitivity and efficiency comparisons of SARS-CoV-2 RT--qPCR primer-probe sets. Nat. Microbiol. 2020, 5 (10), 1299–1305. 10.1038/s41564-020-0761-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Zhang M.; Wang H.; Wang H.; Wang F.; Li Z. CRISPR/Cas12a-Assisted Ligation-Initiated Loop-Mediated Isothermal Amplification (CAL-LAMP) for Highly Specific Detection of microRNAs. Anal. Chem. 2021, 93 (22), 7942–7948. 10.1021/acs.analchem.1c00686. [DOI] [PubMed] [Google Scholar]
  9. Pang B.; Xu J.; Liu Y.; Peng H.; Feng W.; Cao Y.; Wu J.; Xiao H.; Pabbaraju K.; Tipples G.; Joyce M. A.; Saffran H. A.; Tyrrell D. L.; Zhang H.; Le X. C. Isothermal Amplification and Ambient Visualization in a Single Tube for the Detection of SARS-CoV-2 Using Loop-Mediated Amplification and CRISPR Technology. Anal. Chem. 2020, 92 (24), 16204–16212. 10.1021/acs.analchem.0c04047. [DOI] [PubMed] [Google Scholar]
  10. Xiong E.; Jiang L.; Tian T.; Hu M.; Yue H.; Huang M.; Lin W.; Jiang Y.; Zhu D.; Zhou X. Simultaneous Dual-Gene Diagnosis of SARS-CoV-2 Based on CRISPR/Cas9-Mediated Lateral Flow Assay. Angew. Chem., Int. Ed. 2021, 60 (10), 5307–5315. 10.1002/anie.202014506. [DOI] [PubMed] [Google Scholar]
  11. Broughton J. P.; Deng X.; Yu G.; Fasching C. L.; Servellita V.; Singh J.; Miao X.; Streithorst J. A.; Granados A.; Sotomayor-Gonzalez A.; Zorn K.; Gopez A.; Hsu E.; Gu W.; Miller S.; Pan C.-Y.; Guevara H.; Wadford D. A.; Chen J. S.; Chiu C. Y. CRISPR−Cas12-based detection of SARS-CoV-2. Nat. Biotechnol. 2020, 38 (7), 870–874. 10.1038/s41587-020-0513-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ding X.; Yin K.; Li Z.; Lalla R. V.; Ballesteros E.; Sfeir M. M.; Liu C. Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay. Nat. Commun. 2020, 11 (1), 4711. 10.1038/s41467-020-18575-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jiang Y.; Hu M.; Liu A. A.; Lin Y.; Liu L.; Yu B.; Zhou X.; Pang D. W. Detection of SARS-CoV-2 by CRISPR/Cas12a-Enhanced Colorimetry. ACS Sens. 2021, 6 (3), 1086–1093. 10.1021/acssensors.0c02365. [DOI] [PubMed] [Google Scholar]
  14. Ganguli A.; Mostafa A.; Berger J.; Aydin M. Y.; Sun F.; Ramirez S. A. S. D.; Valera E.; Cunningham B. T.; King W. P.; Bashir R. Rapid isothermal amplification and portable detection system for SARS-CoV-2. Proc. Natl. Acad. Sci. U. S. A. 2020, 117 (37), 22727-22735. 10.1073/pnas.2014739117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Arizti-Sanz J.; Freije C. A.; Stanton A. C.; Petros B. A.; Boehm C. K.; Siddiqui S.; Shaw B. M.; Adams G.; Kosoko-Thoroddsen T. F.; Kemball M. E.; Uwanibe J. N.; Ajogbasile F. V.; Eromon P. E.; Gross R.; Wronka L.; Caviness K.; Hensley L. E.; Bergman N. H.; MacInnis B. L.; Happi C. T.; Lemieux J. E.; Sabeti P. C.; Myhrvold C. Streamlined inactivation, amplification, and Cas13-based detection of SARS-CoV-2. Nat. Commun. 2020, 11 (1), 5921. 10.1038/s41467-020-19097-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Zhou M.; Fan C.; Wang L.; Xu T.; Zhang X. Enhanced Isothermal Amplification for Ultrafast Sensing of SARS-CoV-2 in Microdroplets. Anal. Chem. 2022, 94 (10), 4135–4140. 10.1021/acs.analchem.2c00008. [DOI] [PubMed] [Google Scholar]
  17. Feng W.; Newbigging A. M.; Tao J.; Cao Y.; Peng H.; Le C.; Wu J.; Pang B.; Li J.; Tyrrell D. L.; Zhang H.; Le X. C. CRISPR technology incorporating amplification strategies: Molecular assays for nucleic acids, proteins, and small molecules. Chem. Sci. 2021, 12 (13), 4683–4698. 10.1039/D0SC06973F. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. East-Seletsky A.; O’Connell M. R.; Knight S. C.; Burstein D.; Cate J. H. D.; Tjian R.; Doudna J. A. Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection. Nature 2016, 538 (7624), 270–273. 10.1038/nature19802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Shan Y.; Zhou X.; Huang R.; Xing D. High-Fidelity and Rapid Quantification of miRNA Combining crRNA Programmability and CRISPR/Cas13a trans-Cleavage Activity. Anal. Chem. 2019, 91 (8), 5278–5285. 10.1021/acs.analchem.9b00073. [DOI] [PubMed] [Google Scholar]
  20. Bruch R.; Baaske J.; Chatelle C.; Meirich M.; Madlener S.; Weber W.; Dincer C.; Urban G. A. CRISPR/Cas13a-Powered Electrochemical Microfluidic Biosensor for Nucleic Acid Amplification-Free miRNA Diagnostics. Adv. Mater. 2019, 31 (51), 1905311 10.1002/adma.201905311. [DOI] [PubMed] [Google Scholar]
  21. Qin P.; Park M.; Alfson K. J.; Tamhankar M.; Carrion R.; Patterson J. L.; Griffiths A.; He Q.; Yildiz A.; Mathies R.; Du K. Rapid and Fully Microfluidic Ebola Virus Detection with CRISPR-Cas13a. ACS Sens. 2019, 4 (4), 1048–1054. 10.1021/acssensors.9b00239. [DOI] [PubMed] [Google Scholar]
  22. Chen D.; Liang Y.; Wang H.; Wang H.; Su F.; Zhang P.; Wang S.; Liu W.; Li Z. CRISPR-Cas-Driven Single Micromotor (Cas-DSM) Enables Direct Detection of Nucleic Acid Biomarkers at the Single-Molecule Level. Anal. Chem. 2023, 95 (13), 5729–5737. 10.1021/acs.analchem.2c05767. [DOI] [PubMed] [Google Scholar]
  23. Li H.; Yang J.; Wu G.; Weng Z.; Song Y.; Zhang Y.; Vanegas J. A.; Avery L.; Gao Z.; Sun H.; Chen Y.; Dieckhaus K. D.; Gao X.; Zhang Y. Amplification-Free Detection of SARS-CoV-2 and Respiratory Syncytial Virus Using CRISPR Cas13a and Graphene Field-Effect Transistors. Angew. Chem., Int. Ed. 2022, 61 (32), e202203826 10.1002/anie.202203826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Fozouni P.; Son S. D.; de Leon Derby M.; Knott G. J.; Gray C. N.; D’Ambrosio M. V.; Zhao C.; Switz N. A.; Kumar G. R.; Stephens S. I.; Boehm D.; Tsou C. L.; Shu J.; Bhuiya A.; Armstrong M.; Harris A. R.; Chen P. Y.; Osterloh J. M.; Meyer-Franke A.; Joehnk B.; Walcott K.; Sil A.; Langelier C.; Pollard K. S.; Crawford E. D.; Puschnik A. S.; Phelps M.; Kistler A.; DeRisi J. L.; Doudna J. A.; Fletcher D. A.; Ott M. Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy. Cell 2021, 184 (2), 323–333 e9. 10.1016/j.cell.2020.12.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Li H.; Xie Y.; Chen F.; Bai H.; Xiu L.; Zhou X.; Guo X.; Hu Q.; Yin K. Amplification-free CRISPR/Cas detection technology: challenges, strategies, and perspectives. Chem. Soc. Rev. 2023, 52 (1), 361–382. 10.1039/D2CS00594H. [DOI] [PubMed] [Google Scholar]
  26. Sha Y.; Huang R.; Huang M.; Yue H.; Shan Y.; Hu J.; Xing D. Cascade CRISPR/cas enables amplification-free microRNA sensing with fM-sensitivity and single-base-specificity. Chem. Commun. 2021, 57 (2), 247–250. 10.1039/D0CC06412B. [DOI] [PubMed] [Google Scholar]
  27. Li Y.; Ma Y.; Jiao X.; Li T.; Lv Z.; Yang C. J.; Zhang X.; Wen Y. Control of capillary behavior through target-responsive hydrogel permeability alteration for sensitive visual quantitative detection. Nat. Commun. 2019, 10 (1), 1036. 10.1038/s41467-019-08952-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wang H.; Wang H.; Li Y.; Jiang C.; Chen D.; Wen Y.; Li Z. Capillarity self-driven DNA hydrogel sensor for visual quantification of microRNA. Sens. Actuat. B: Chem. 2020, 313, 128036. 10.1016/j.snb.2020.128036. [DOI] [Google Scholar]
  29. Jiang C.; Li Y.; Wang H.; Chen D.; Wen Y. A portable visual capillary sensor based on functional DNA crosslinked hydrogel for point-of-care detection of lead ion. Sens. Actuat. B: Chem. 2020, 307, 127625. 10.1016/j.snb.2019.127625. [DOI] [Google Scholar]
  30. Huyke D. A.; Ramachandran A.; Bashkirov V. I.; Kotseroglou E. K.; Kotseroglou T.; Santiago J. G. Enzyme Kinetics and Detector Sensitivity Determine Limits of Detection of Amplification-Free CRISPR-Cas12 and CRISPR-Cas13 Diagnostics. Anal. Chem. 2022, 94 (27), 9826–9834. 10.1021/acs.analchem.2c01670. [DOI] [PubMed] [Google Scholar]
  31. Feng W.; Zhang H.; Le X. C. Signal Amplification by the trans-Cleavage Activity of CRISPR-Cas Systems: Kinetics and Performance. Anal. Chem. 2023, 95 (1), 206–217. 10.1021/acs.analchem.2c04555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lu C. H.; Guo W.; Hu Y.; Qi X. J.; Willner I. Multitriggered Shape-Memory Acrylamide-DNA Hydrogels. J. Am. Chem. Soc. 2015, 137 (50), 15723–15731. 10.1021/jacs.5b06510. [DOI] [PubMed] [Google Scholar]
  33. Guo W.; Lu C. H.; Orbach R.; Wang F.; Qi X. J.; Cecconello A.; Seliktar D.; Willner I. pH-stimulated DNA hydrogels exhibiting shape-memory properties. Adv. Mater. 2015, 27 (1), 73–78. 10.1002/adma.201403702. [DOI] [PubMed] [Google Scholar]
  34. Zhu Z.; Wu C.; Liu H.; Zou Y.; Zhang X.; Kang H.; Yang C. J.; Tan W. An aptamer cross-linked hydrogel as a colorimetric platform for visual detection. Angew. Chem., Int. Ed. 2010, 49 (6), 1052–1056. 10.1002/anie.200905570. [DOI] [PubMed] [Google Scholar]
  35. Udugama B.; Kadhiresan P.; Kozlowski H. N.; Malekjahani A.; Osborne M.; Li V. Y. C.; Chen H.; Mubareka S.; Gubbay J. B.; Chan W. C. W. Diagnosing COVID-19: The Disease and Tools for Detection. ACS Nano 2020, 14 (4), 3822–3835. 10.1021/acsnano.0c02624. [DOI] [PubMed] [Google Scholar]
  36. Wolfel R.; Corman V. M.; Guggemos W.; Seilmaier M.; Zange S.; Muller M. A.; Niemeyer D.; Jones T. C.; Vollmar P.; Rothe C.; Hoelscher M.; Bleicker T.; Brunink S.; Schneider J.; Ehmann R.; Zwirglmaier K.; Drosten C.; Wendtner C. Virological assessment of hospitalized patients with COVID-2019. Nature 2020, 581 (7809), 465–469. 10.1038/s41586-020-2196-x. [DOI] [PubMed] [Google Scholar]
  37. Myhrvold C.; Freije Catherine A.; Gootenberg Jonathan S.; Abudayyeh Omar O.; Metsky Hayden C.; Durbin Ann F.; Kellner Max J.; Tan Amanda L.; Paul Lauren M.; Parham Leda A.; Garcia Kimberly F.; Barnes Kayla G.; Chak B.; Mondini A.; Nogueira Mauricio L.; Isern S.; Michael Scott F.; Lorenzana I.; Yozwiak Nathan L.; MacInnis Bronwyn L.; Bosch I.; Gehrke L.; Zhang F.; Sabeti Pardis C. Field-deployable viral diagnostics using CRISPR-Cas13. Science 2018, 360 (6387), 444–448. 10.1126/science.aas8836. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Shinoda H.; Taguchi Y.; Nakagawa R.; Makino A.; Okazaki S.; Nakano M.; Muramoto Y.; Takahashi C.; Takahashi I.; Ando J.; Noda T.; Nureki O.; Nishimasu H.; Watanabe R. Amplification-free RNA detection with CRISPR-Cas13. Commun. Biol. 2021, 4 (1), 476. 10.1038/s42003-021-02001-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sun Y.; Yang C.; Jiang X.; Zhang P.; Chen S.; Su F.; Wang H.; Liu W.; He X.; Chen L.; Man B.; Li Z. High-intensity vector signals for detecting SARS-CoV-2 RNA using CRISPR/Cas13a couple with stabilized graphene field-effect transistor. Biosens. Bioelectron. 2023, 222, 114979. 10.1016/j.bios.2022.114979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Li J.; Tang L.; Li T.; Li K.; Zhang Y.; Ni W.; Xiao M.-M.; Zhao Y.; Zhang Z.-Y.; Zhang G.-J. Tandem Cas13a/crRNA-Mediated CRISPR-FET Biosensor: A One-for-All Check Station for Virus without Amplification. ACS Sens. 2022, 7 (9), 2680–2690. 10.1021/acssensors.2c01200. [DOI] [PubMed] [Google Scholar]
  41. Liu Y.; Kumblathan T.; Tao J.; Xu J.; Feng W.; Xiao H.; Hu J.; Huang C. V.; Wu Y.; Zhang H.; Li X. F.; Le X. C. Recent advances in RNA sample preparation techniques for the detection of SARS-CoV-2 in saliva and gargle. Trend. Anal. Chem. 2023, 165, 117107. 10.1016/j.trac.2023.117107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Lalli M. A.; Langmade J. S.; Chen X.; Fronick C. C.; Sawyer C. S.; Burcea L. C.; Wilkinson M. N.; Fulton R. S.; Heinz M.; Buchser W. J.; Head R. D.; Mitra R. D.; Milbrandt J. Rapid and Extraction-Free Detection of SARS-CoV-2 from Saliva by Colorimetric Reverse-Transcription Loop-Mediated Isothermal Amplification. Clin. Chem. 2021, 67 (2), 415–424. 10.1093/clinchem/hvaa267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tang Z.; Cui J.; Kshirsagar A.; Liu T.; Yon M.; Kuchipudi S. V.; Guan W. SLIDE: Saliva-Based SARS-CoV-2 Self-Testing with RT-LAMP in a Mobile Device. ACS Sens. 2022, 7 (8), 2370–2378. 10.1021/acssensors.2c01023. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ac4c02087_si_001.pdf (2.2MB, pdf)

Articles from Analytical Chemistry are provided here courtesy of American Chemical Society

RESOURCES