Recent advancements in nucleic acid detection with microfluidic chip for molecular diagnostics

Abstract

The coronavirus disease 2019 (COVID-19) has extensively promoted the application of nucleic acid testing technology in the field of clinical testing. The most widely used polymerase chain reaction (PCR)-based nucleic acid testing technology has problems such as complex operation, high requirements of personnel and laboratories, and contamination. The highly miniaturized microfluidic chip provides an essential tool for integrating the complex nucleic acid detection process. Various microfluidic chips have been developed for the rapid detection of nucleic acid, such as amplification-free microfluidics in combination with clustered regularly interspaced short palindromic repeats (CRISPR). In this review, we first summarized the routine process of nucleic acid testing, including sample processing and nucleic acid detection. Then the typical microfluidic chip technologies and new research advances are summarized. We also discuss the main problems of nucleic acid detection and the future developing trend of the microfluidic chip.

Keywords: Amplification; CRISPR; Digital; Microfluidic chip; Nucleic acid.

Fig. 1

Outline of microfluidics technology for the nucleic acid test.

Fig. 2

Diagram of nucleic acid extraction method. (A) Chitosan-modified magnetic microspheres for pH-induced nucleic acid extraction and integrated into a centrifugal microfluidic chip. Reproduced with permission from Ref. [44]. (B) Rapid plant DNA extraction method using disposable polymeric microneedle patches. Reproduced with permission from Ref. [46]. (C) Using ME chip with thermal gel electrophoresis to directly separate and quantify multiple miRNAs. Reproduced with permission from Ref. [51]. (D) A micro-pipette tip-based nucleic acid test (MTNT) for high-throughput sample-to-answer detection of both DNA and RNA from crude samples including cells, bacteria, and solid plants. Reproduced with permission from Ref. [53].

Fig. 3

Diagram of integrated microfluidic chips with nucleic acid amplification. (A) dPCR chip for high-throughput, high-sensitivity quantitative measurements of SARS-CoV-2 viral genes. Reproduced with permission from Ref. [66]. (B) Microcapillary LAMP for the detection of nucleic acids. Reproduced with permission from Ref. [73]. (C) Dual-mode LAMP incorporating magnetic bead separation to determine the methylated Septin9 gene in colorectal cancer. Reproduced with permission from Ref. [75]. (D) A CRISPR/Cas12a-based SNP detection genotyping method based on the centrifugal microfluidic device. Reproduced with permission from Ref. [16].

Fig. 4

Diagram of signal read-out method. (A) An integrated device for in situ fluorescence detection for SARS-CoV-2 RNA detecting. Reproduced with permission from Ref. [26].(B) Fluorescence-based digital warm-start CRISPR assay for sensitive, quantitative detection of SARS-CoV-2 Reproduced with permission from Ref. [88]. (C) A 3D-printed lab-on-a-chip that simultaneously detects SARS-CoV-2 RNA in saliva and anti-SARS-CoV-2 immunoglobulin within 2 h via multiplexed electrochemical output. Reproduced with permission from Ref. [22]. (D) A face mask with a lyophilized CRISPR sensor and a colorimetric sensing platform for wearable, noninvasive detection of SARS-CoV-2. Reproduced with permission from Ref. [98].

Fig. 5

Diagram of centrifugal chip and valves chip. (A) An automated, integrated centrifugal chip that can complete the process from sample pre-processing to detection. Reproduced with permission from Ref. [17]. (B) A centrifugal chip used for RT-LAMP amplification detection has 20 independent reaction chambers, which can identify 6 influenza virus subtypes at the same time. Reproduced with permission from Ref. [102]. (C) A multifunctional microfluidic device based on a monolayer membrane valve consisting of three parts, including a CTCs capture region, a monolayer membrane flap region, and a microchamber nucleic acid-based dPCR detection and analysis region. Reproduced with permission from Ref. [109]. (D) A rotary valve-assisted fluidic chip coupled with CRISPR/Cas12a for fully integrated nucleic acid detection. Reproduced with permission from Ref. [110].

Fig. 6

Diagram of digital droplet chip. (A) A ddPCR chip can achieve multiplex screening of genes in transgenic maize lines. Reproduced with permission from Ref. [111]. (B) A droplet chip with a water phase entrance and two oil phase entrances, the negative pressure formed by the piston syringe at the end exit to promote liquid flowing to form droplets. Reproduced with permission from Ref. [116]. (C) A droplet array chip platform CARMEN can detect 4000–5000 targets simultaneously. Reproduced with permission from Ref. [23]. (D) A "cross" junction droplet chip with hybrid detection technology to realize attomolar sensitivity of HPV virus without amplification. Reproduced with permission from Ref. [19].

Fig. 7

Diagram of paper-based chip and ME chip. (A) The paper-based barcode assay system. Reproduced with permission from Ref. [118]. (B) Multiplexed barcode paper-based inspection that is compatible with mobile devices. Reproduced with permission from Ref. [125]. (C) CRISPR-Cas12a-mediated SERS to improve the sensitivity and specificity of LFA-based nucleic acid detection. Reproduced with permission from Ref. [127]. (D) A portable all-in-one microfluidic device for rapid diagnosis of pathogens based on an integrated CF-PCR and electrophoresis biochip. Reproduced with permission from Ref. [135]. (E) A ME device combining microfluidics, on-chip electric field control, and CRISPR to rapidly detect SARS-CoV-2 RNA in clinical samples. Reproduced with permission from Ref. [136].