Electric field-enhanced electrochemical CRISPR biosensor for DNA detection

Abstract

DNA detection plays an important role in the rapid screening of cancers and early diagnosis of infectious diseases. Here, we developed a simple, versatile, electric field-enhanced (EFE), electrochemical CRISPR biosensor to detect DNA targets in a homogeneous solution phase. To improve the detection sensitivity, we applied a pulsed electric field to enrich nucleic acids on the electrode surface. The EFE electrochemical CRISPR biosensor takes advantage of the diffusivity difference between electrochemical oligonucleotide probes and CRISPR-cleaved probes toward a negatively charged working electrode, enabling simple and sensitive electrochemical detection of DNA without the need for complicated immobilization processing of electrochemical probes. Our developed CRISPR biosensor directly detects unamplified human papillomavirus-16 (HPV-16) DNA with a sensitivity of 1 pM. Further, the EFE electrochemical CRISPR biosensor coupled with recombinase polymerase amplification (RPA) successfully detects HPV-16 DNA in clinical samples. Thus, the EFE electrochemical CRISPR biosensor provides a simple, robust, and sensitive detection method for nucleic acid-based molecular diagnostics.

Keywords: CRISPR-Cas12a; Electrochemical DNA biosensor; Human papillomavirus (HPV)-associated cancer screening; Nucleic acid enrichment; Pulsed electric field.

Figure 1.

Working principle of the EFE, immobilization-free electrochemical CRISPR biosensor for DNA detection. A) Trans-cleavage and cis-cleavage activities of CRISPR-Cas12a protein in the presence of crRNA, DNA target, and ssDNA (non-target). B) Operation procedures of the EFE, electrochemical CRISPR biosensor. (i) A pulsed electric field is applied to attract nucleic acids (e.g., electroactive ssDNA probe, dsDNA target) on the positively charged working electrode surface due to the static electric force. (ii) Electrochemical detection in the absence and presence of the target DNA. The trans-cleavage activity of activated Cas12a releases less negative MB-labeled probe, resulting in an increased electrochemical current during DPV detection.

Figure 2.

Optimization of the ssDNA-MB probe concentration for the EFE electrochemical CRISPR biosensor. A) Electrochemical current of the positive and negative samples at different ssDNA-MB probe concentrations (1, 2, 5, and 10 μM) (i-iv). PC is the positive control with 500 pM HPV-16 DNA target. NC is the negative control without HPV-16 DNA target. B) Comparison of the electrochemical current difference at ssDNA-MB concentrations ranging from 1 μM to 10 μM. Error bars represent the means ± s.d. from three replicates (n = 3).

Figure 3.

Optimization of the pulsed electric field for CRISPR-based DNA detection. A) Fluorescence images of the reaction chambers of the electrochemical CRISPR biosensor at different incubation times (0, 20, 40, and 60 min). B) Effect of the peak-to-peak amplitude (0, 1, 10, and 100 mV) on the CRISPR-based DNA detection. C) Effect of the frequency (1, 10, and 100 Hz) on the CRISPR-based DNA detection. The relative fluorescence intensities were recorded at the endpoint of the CRISPR reaction. PC is the positive control with 500 pM HPV-16 DNA target. NC is the negative control without HPV-16 DNA target. Error bars represent the means ± s.d. from three replicates (n = 3).

Figure 4.

Comparison of HPV-16 DNA detection by the electrochemical CRISPR biosensor with and without applying a pulsed electric field. Statistical analysis was performed using a one-way ANOVA test with Tukey’s comparison test, where n.s. = not significant with p > 0.05 and the asterisks (*, **, ***, ****) denote significant differences with p values (* = 0.001

Figure 5.

HPV-16 DNA detection in clinical samples by using the EFE electrochemical CRISPR biosensor after RPA pre-amplification. A) Electrochemical response curves of the EFE electrochemical CRISPR biosensor for HPV-16 DNA detection in clinical samples. B) Normalized electrochemical current of the EFE electrochemical CRISPR biosensor for HPV-16 DNA detection in clinical samples. S1, S2, S4, and S5 are positive clinical samples. S3 and S6 are negative samples. PC and NC are, respectively, the positive and negative controls. Error bars represent the means ± s.d. from three replicates (n = 3).