Dynamic Aqueous Multiphase Reaction System for One-Pot CRISPR-Cas12a-Based Ultrasensitive and Quantitative Molecular Diagnosis

Abstract

Recently, CRISPR-Cas technology has opened a new era of nucleic acid-based molecular diagnostics. However, current CRISPR-Cas-based nucleic acid biosensing has a lack of the quantitative detection ability and typically requires separate manual operations. Herein, we reported a dynamic aqueous multiphase reaction (DAMR) system for simple, sensitive and quantitative one-pot CRISPR-Cas12a based molecular diagnosis by taking advantage of density difference of sucrose concentration. In the DAMR system, recombinase polymerase amplification (RPA) and CRISPR-Cas12a derived fluorescent detection occurred in spatially separated but connected aqueous phases. Our DAMR system was utilized to quantitatively detect human papillomavirus (HPV) 16 and 18 DNAs with sensitivities of 10 and 100 copies within less than 1 h. Multiplex detection of HPV16/18 in clinical human swab samples were successfully achieved in the DAMR system using 3D-printed microfluidic device. Furthermore, we demonstrated that target DNA in real human plasma samples can be directly amplified and detected in the DAMR system without complicated sample pretreatment. As demonstrated, the DAMR system has shown great potential for development of next-generation point-of-care molecular diagnostics.

Figure 1.

Schematic illustration of dynamic aqueous multiphase reaction (DAMR) system for simple, sensitive and quantitative DNA detection by one-pot RPA/CRISPR-Cas12a assay. Inset is an image of aqueous multiphase system with different sucrose concentrations.

Figure 2.. Optimization of one-pot RPA/CRISPR-Cas12a detection in the DAMR system.

A: Fluorescent monitoring of the one-pot RPA/CRISPR-Cas12a reactions at different incubation times in the DAMR and one-phase system, respectively. B: Series sucrose concentration (0%, 5%, 10%, 20%, 30%, 40%, and 50%) were added to 20 μL RPA bottom phase and tested in the DAMR system. C: Effect of the volume ratios (1: 1:5, 1:2, 1:1, 2:1, 5:1) of the RPA bottom phase and CRISPR-Cas12a top phase on DNA detection in the DAMR system. Error bars denote s.d. (n=3).

Figure 3.. Quantitative detection of HPV DNA using one-pot RPA/CRISPR-Cas12a in the DAMR system.

A-D. Ten-fold serial dilution of HPV 16 DNA (0, 1, 10, 10², 10³, 10⁴, 10⁵ copies/reaction) was added into 20 μL RPA reaction bottom phase (with 10% sucrose) in the DAMR system and incubated at 37 °C for 1h. A. Endpoint fluorescent image taken by ChemiDocTM MP Imaging System. B. Endpoint fluorescent image taken by smartphone camera under blue light. C. Real-time fluorescent signal monitored by PCR machine. D. Linear relationship between the threshold time and HPV16 concentration (copies/reaction). (n=3) E. The selective detection of 10⁴ copies HPV 16 DNA over HPV18 and HPV31 using RPA/CRISPR-Cas12a detection in the DAMR system.

Figure 4.. Multiple HPV DNA detection in clinical human swab sample.

A. Top: Schematic outlining of the detection of HPV DNA from human swab samples by RPA/CRISPR-Cas12a assay in the DAMR system. Bottom: HPV 16/18 detection results of 15 clinical samples using qPCR (left) and RPA/CRISPR-Cas12a assay in the DAMR system (right), respectively. B. HPV16/18 detection in separate PCR tubes by RPA/CRISPR-Cas12a in the DAMR system. C. 3D-printed microfluidic device in 96-well microplate for multiplexed detection of HPV DNA in the DAMR system. D. Three chambers of 3D-printed device with or without specific crRNA (1: without crRNA, 2: with HPV16 crRNA and 3: with HPV18 crRNA). E. Endpoint fluorescent image of multiplex detection of HPV DNA with 3D printed device in the DAMR system.

Figure 5.. Direct DNA detection in untreated plasma sample in the DAMR system.

A. Schematic illustration of three-phase system (plasma/RPA/CRISPR-Cas12a) for direct DNA detection using untreated plasma sample. B. Comparison of three-phase system and two-phase system for detection of HPV16 DNA in the untreated plasma. C. Optimization of sucrose concentration in the plasma bottom phase. D. Schematic illustration of two-phase system for DNA detection using untreated plasma samples by LAMP or qPCR method. E. Comparison of LAMP-based two-phase system and one-phase system for DNA detection in plasma samples. F. Comparison of PCR-based two-phase system and one-phase system for detection of DNA in plasma samples.