An integrated biosensor platform for extraction and detection of nucleic acids

Abstract

The development of portable systems for analysis of nucleic acids (NAs) is crucial for the evolution of biosensing in the context of future healthcare technologies. The integration of NA extraction, purification, and detection modules, properly actuated by microfluidics technologies, is a key point for the development of portable diagnostic systems. In this paper, we describe an integrated biosensor platform based on a silicon–plastic hybrid lab‐on‐disk technology capable of managing NA extraction, purification, and detection processes in an integrated format. The sample preparation process is performed by solid‐phase extraction technology using magnetic beads on a plastic disk, while detection is done through quantitative real‐time polymerase chain reaction (qRT‐PCR) on a miniaturized silicon device. The movement of sample and reagents is actuated by a centrifugal force induced by a disk actuator instrument. The assessment of the NA extraction and detection performance has been carried out by using hepatitis B virus (HBV) DNA genome as a biological target. The quantification of the qRT‐PCR chip in the hybrid disk showed an improvement in sensitivity with respect to the qRT‐PCR commercial platforms, which means an optimization of time and cost. Limit of detection and limit of quantification values of about 8 cps/reaction and 26 cps/reaction, respectively, were found by using analytical samples (synthetic clone), while the results with real samples (serum with spiked HBV genome) indicate that the system performs as well as the standard methods.

CRISPR/Cas12-Based Ultra-Sensitive and Specific Point-of-Care Detection of HBV

“…In the prevention and control of hepatitis B transmission, the most suitable detection technology is still being studied [ 21 ]. Importantly, qPCR and ELISA assays may not be excellent choices for HBV testing in developing countries in the western Pacific and African regions, where the prevalence of hepatitis B is high ( Table 2 ) [ 22 , 23 ]. Therefore, rapid, accurate, and economical diagnostic tests that can be deployed at point-of-care are needed to prevent and control hepatitis B transmission.…”

A fully automated microfluidic PCR-array system for rapid detection of multiple respiratory tract infection pathogens

“…The Onestart system is compact and easy to operate and thus can be performed by non-skilled personnel in field conditions. Compared with POC devices with a limited number of targets [38,39], the Onestart respiratory panel can detect and distinguish up to 21 pathogens in a single run. Because the entire analytical process is performed in an enclosed microchip, there is a low risk of laboratory contamination.…”

An Overview on Microfluidic Systems for Nucleic Acids Extraction from Human Raw Samples

“…(1) mechanical-by grinding, freeze-thawing, potterization, centrifugation, sonication or bead-beating; (2) chemical-by detergents, solubilizing membrane lipids, or by chaotropic agents, perforating cell membranes with the denaturation of trans-membrane proteins, by Both types, mostly based on a magnetic beads extraction or, less commonly, on silica filtration, are developed onto a rotating platform where sample and reagents motion is enabled by centrifugal and related forces (Coriolis and Euler) that, usually in combination with pneumatic forces, handle the fluidic system and enable sample processing. This principle is applied in traditional rectangular LOCs [36,71,86] and in LOD systems [18,41,51,53,54,60]. These devices generally manage the sample by changing speed and sense of rotation: a particular example of a LOD is represented by LabDisk analyzer [28,64] which implements an automated lyse-bind-wash-elute protocol by means of a gas-phase transition magnetophoresis principle, which combines magnetic and centrifugal forces to carry NA-complexed magnetic beads through the separation liquid/gas interfaces between each reaction chamber.…”

“…It can be utilized alone, but it is often coupled with harder compounds, like glass or thermoplastic polymers, in layering the main structure of the chip. Because thermoplastic polymers are easily workable and less expensive than glass, several of them are very recurrent: PMMA (polymethylmethacrylate) is the most used alone [ 26 , 54 , 61 ], but more often it is coupled with PDMS [ 29 , 42 , 45 , 46 , 51 , 60 , 73 ]; PC (polycarbonate) was utilized, for example, by Ritzi-Lehnert et al [ 71 ], who developed a PC cassette to be inserted into chip analyzers, by Hwang et al [ 68 ], who developed a microfluidic device composed of four layers of PC, PDMS and glass, and by other authors [ 52 , 66 ]. Although less common, also PET (polyethylene terephthalate) and PS (polystyrene) have been used [ 50 , 62 ].…”

“…After binding, beads are separated from contaminating cellular components, washed, and exposed to ethanol to obtain a purified DNA elution [ 101 ]. The majority of LOCs based on a magnetic beads NA extraction utilize beads with silica coatings ( Table 1 ); they are added and mixed to sample and lysis buffers [ 28 , 49 , 50 , 51 , 54 , 60 , 61 , 63 , 64 , 67 , 70 , 71 , 73 ]. After cell lysis, DNA-bound beads are driven and collected by a magnetic coil, while the unbound debris is removed by a stream of washing buffers.…”