Nucleic Acid Biosensor Synthesis of an All-in-One Universal Blocking Linker Recombinase Polymerase Amplification with a Peptide Nucleic Acid-Based Lateral Flow Device for Ultrasensitive Detection of Food Pathogens

Abstract

In this study, a whole-course nucleic-acid-constructed biosensor that combines the all-in-one concepts of the universal blocking linker recombinase polymerase amplification (UBLRPA) and a peptide nucleic acid (PNA)-based lateral flow device (PLFD) has been developed for the ultrasensitive detection of food pathogens. Using the preamplification UBLRPA principle, a universal linker and C3 space blocker were utilized to produce the universal linker single-duplex DNA products. The developed amplification system was employed to convert duplex products to a single strand. In the signal recognition strategy, a special PNA probe was successfully employed in the portable PLFD. The UBLRPA products were identified visually using the PLFD through dual hybridization (a PNA probe on the gold nanoparticle (Au-NP) was combined with a universal linker on the end of the products; a PNA capture probe was used on the test line and a universal linker on the other end of the products). The accumulation of Au-NPs produced a characteristic red band, enabling the visual detection of a food pathogen without further testing. To demonstrate the value of the all-in-one biosensor, Salmonella enterica subsp. enterica serovar typhimurium was used as a model organism. The biosensor showed high selectivity and extraordinary repeatability using S. typhimurium, and the limit of detection was 4 CFU mL. Furthermore, when milk samples artificially contaminated with S. typhimurium were analyzed, the analysis was completed within 30 min without complicated instrumentation. The results exhibited good precision and recovery. This portable all-in-one biosensor demonstrates great promise for the screening of pathogens in food and environmental samples.

Multiplexed Instrument-Free Bar-Chart SpinChip Integrated with Nanoparticle-Mediated Magnetic Aptasensors for Visual Quantitative Detection of Multiple Pathogens

“…The sensitivity of our method is even comparable to that of DNA amplification-based lateral flow devices with the LOD of 4 CFU/mL. 68…”

MXenes-Based Bioanalytical Sensors: Design, Characterization, and Applications

“…Immunosensors combining immunochemical reactions based on the antibody (Ab)-antigen (Ag) recognition with an appropriate electrochemical transducer provide significant advantages for bioanalysis, including cost-effectiveness, low reagent and sample volume, portability, high-specificity and sensitivity, and high-throughput analysis [ 76 ]. Immunosensors have applications in various fields of analysis, such as clinical medicine [ 77 , 78 ], environmental pollutant evaluation [ 79 , 80 , 81 ], food inspection [ 82 , 83 ], and pathogenic microorganism detection [ 84 , 85 ]. The incorporation of MXenes in the fabrication of immunosensors provide opportunities for increasing bioactivity of immobilized Abs on electrode surfaces, increasing surface area, and improving detection sensitivity.…”

Rapid point‐of‐care testing methods/devices for meat species identification: A review

“…The enhancement of signals at T‐ or C‐line also improves the detection sensitivity (Ding et al., 2018; Jauset‐Rubio et al., 2016; Xu et al., 2018). The sensitivity of LFA can be enhanced using multilayer surface functionalized nanospheres (Lou, Fan, Ji, Gu, & Zhang, 2019).…”