Bioinspired CRISPR-Mediated Cascade Reaction Biosensor for Molecular Detection of HIV Using a Glucose Meter

Abstract

HIV molecular detection plays a significant role in early diagnosis and antiretroviral therapy for HIV patients. CRISPR technology has recently emerged as a powerful tool for highly sensitive and specific nucleic acid based molecular detection when used in combination with isothermal amplification. However, it remains a challenge to improve the compatibility of such a multienzyme reaction system for simple and sensitive molecular detection. Inspired by the multicompartment structures in a living cell, we present a nanoporous membrane-separated (compartmentalized), artificial, cascade reaction system to improve the compatibility of a CRISPR-mediated multienzyme reaction. We further integrated the multienzyme cascade reaction system with a microfluidic platform and glucose biosensing technology to develop a bioinspired, CRISPR-mediated cascade reaction (CRISPR-MCR) biosensor, enabling HIV molecular detection by a simple glucose meter, analogous to diabetes home testing. We applied the bioinspired CRISPR-MCR biosensor to detect HIV DNA and HIV RNA, achieving a detection sensitivity of 43 copies and 200 copies per test, respectively. Further, we successfully validated the bioinspired biosensor by testing clinical plasma samples of HIV, demonstrating its great application potential for point-of-care testing of HIV virus and other pathogens at home or in resource-limited settings.

Keywords: CRISPR-based nucleic acid detection; HIV molecular detection; electrochemical biosensor; microfluidic technology; nanoporous membrane.

Figure 1.. Bioinspired CRISPR-MCR biosensor overview.

A) Structure of a eukaryotic cell containing multi-compartment structures (e.g., subcellular organelles) for a multi-enzyme cascade reaction. B) Working principle of the nanoporous membrane-separated, CRISPR-mediated cascade reaction system for HIV nucleic acid detection inspired by the eukaryotic cell. The cascade reaction system is separated by a hydrophilic nanoporous membrane (right bottom inset) into two compartments: i) an RPA or RT-RPA reaction chamber, and ii) a CRISPR reaction chamber. Due to the concentration gradient (difference), the short DNA amplicons generated by RPA exponential amplification can diffuse into the CRISPR reaction chamber through the nanoporous membrane to specifically initiate the downstream CRISPR cleavage reaction for HIV nucleic acid biosensing. C) Schematic illustration of the bioinspired CRISPR-MCR biosensor integrating the CRISPR-mediated cascade reaction system, catalytic reaction chamber, and glucose biosensing strip. Left bottom inset: catalytic chamber where pre-stored sucrose is hydrolyzed into fructose and glucose in the presence of the released invertase enzyme from the MB-ssDNA-invertase in the CRISPR reaction chamber. D) Detection of HIV nucleic acid using a personal glucose meter (PGM).

Figure 2.

Nanoporous membrane-separated, CRISPR-mediated cascade reaction system for HIV nucleic acid detection. A) Effect of the RPA reaction solution volume on the electrochemical detection of HIV nucleic acid by the glucose meter. * = 0.001 < P ≤ 0.05, ** = 0.0001 < P ≤ 0.001, *** = 0.00001 < P ≤ 0.0001. B) Comparison of real-time fluorescent detection of the RPA/CRISPR reaction with and without membrane separation. Both PES membrane and PVDF membrane were tested for comparison. C) Real-time fluorescent signal in the CRISPR-mediated cascade reaction system embedded with PES membranes of different pore sizes (0.03, 0.2, 1.2, and 8.0 μm). D) Scanning electron microscope (SEM) image of cross-sections of the PES membranes with different pore sizes (0.03, 0.2, 1.2, and 8.0 μm). E) Relative diffusion rate of different components in the CRISPR-MCR assay through the PES membranes with various pore sizes (0.03, 0.2, 1.2, and 8.0 μm). n.s., not significant with p > 0.05. The asterisks (*, **, ***, ****) denote significant differences with p values (* = 0.001 < P ≤ 0.05, ** = 0.0001 < P ≤ 0.001, *** = 0.00001 < P ≤ 0.0001, **** = P ≤ 0.00001).

Figure 3.

Bioinspired CRISPR-MCR biosensor and its optimization. A) Photograph of the bioinspired CRISPR-MCR biosensor fabricated by 3D-printing technology. The glucose biosensing strip was incorporated into the biosensor for electrochemical detection of HIV nucleic acids by a glucose meter. B) Optimization of the usage of magnetic beads to synthesize the MB-ssDNA-invertase reporter. C) Effect of the amount of MB-ssDNA-invertase reporter on CRISPR-mediated electrochemical detection. n.s., not significant with p > 0.05. The asterisks (*, **, ***, ****) denote significant differences with p values (* = 0.001 < P ≤ 0.05, ** = 0.0001 < P ≤ 0.001, *** = 0.00001 < P ≤ 0.0001, **** = P ≤ 0.00001). D) Representative SEM image of the MB-ssDNA-invertase reporter for glucose biosensing detection in the bioinspired CRISPR-MCR biosensor. E) TEM image of the MB-ssDNA-Invertase reporter.

Figure 4.

Detection of HIV nucleic acids in the bioinspired CRISPR-MCR biosensor by using a glucose meter. A) HIV DNA detection in the biosensor by using a glucose meter after various catalytic reaction times of 5, 15, and 30 min. B) HIV RNA detection in the biosensor by using a glucose meter after a catalytic time of 10 and 20 min. NC, negative control. n.s., not significant with p > 0.05. The asterisks (*, **, ***, ****) denote significant differences with p values (* = 0.001 < P ≤ 0.05, ** = 0.0001 < P ≤ 0.001, *** = 0.00001 < P ≤ 0.0001, **** = P ≤ 0.00001).

Figure 5.

Clinical validation of the bioinspired CRISPR-MCR biosensor to detect HIV virus in clinical plasma samples. A) Procedures of the CRISPR-MCR biosensor and the RT-qPCR assay for HIV detection in clinical samples. B) Representative HIV test results of 15 clinical samples and positive/negative controls by the bioinspired CRISPR-MCR biosensor. C) Cq values of the clinical plasma samples and positive/negative controls by RT-PCR. D) Detection results of HIV in clinical samples by using the CRISPR-MCR biosensor and glucose meter. PC and NC are, respectively, positive control and negative control. The cut-off threshold value was set as 14 mg/dL according to the 3σ criterion.