Multisignal Biosensors Based on Mn Paramagnetic Relaxation and Nanocatalysis for Norovirus Detection.

A multisignal method for the sensitive detection of norovirus based on Mn paramagnetic relaxation and nanocatalysis was developed. This dual-modality sensing platform was based on the strong relaxation generated by cracked Au@MnO2 nanoparticles (NPs) and their intrinsic enzyme-like activity. Ascorbic acid rapidly cracked the MnO2 layer of Au@MnO2 NPs to release Mn(II), resulting in the relaxation modality being in a "switch-on" state. Under the optimal conditions, the relaxation modality exhibited a wide working range (6.02 × 103-3.01 × 107 copies/µL) and a limit of detection (LOD) of 2.29 × 103 copies/µL. Using 4,4',4″,4″'-(porphine-5,10,15,20-tetrayl) tetrakis (benzenesulfonic acid) (tpps)-ß-cyclodextrin (tpps-ß-CD) as a T1 relaxation signal amplification reagent, a lower LOD was obtained. The colorimetric modality exploited the "peroxidase/oxidase-like" activity of Au@MnO2 NPs, which catalyzed the oxidation of colorless 3,3',5,5'-tetramethylbenzidine (TMB) to blue oxidized TMB, which exhibited a working range (6.02 × 104-6.02 × 106 copies/µL) and an LOD of 2.6 × 104 copies/µL. In addition, the rapid amplification reaction of recombinase polymerase enabled the detection of low norovirus levels in food samples and obtained a working range of 101-106 copies/mL and LOD of 101 copies/mL (relaxation modality). The accuracy of the sensor in the analysis of spiked samples was consistent with that of the real-time quantitative reverse transcription polymerase chain reaction, demonstrating the high accuracy and practical utility of the sensor.

CRISPR/Cas12a-based technology: A powerful tool for biosensing in food safety.

BACKGROUND: In the context of the current pandemic caused by the novel coronavirus, molecular detection is not limited to the clinical laboratory, but also faces the challenge of the complex and variable real-time detection fields. A series of novel coronavirus events were detected in the process of food cold chain packaging and transportation, making the application of molecular diagnosis in food processing, packaging, transportation, and other links urgent. There is an urgent need for a rapid detection technology that can adapt to the diversity and complexity of food safety. SCOPE AND APPROACH: This review introduces a new molecular diagnostic technology-biosensor analysis technology based on CRISPR-Cas12a. Systematic clarification of its development process and detection principles. It summarizes and systematically organizes its applications in viruses, food-borne pathogenic bacteria, small molecule detection, etc. In the past four years, which provides a brand-new and comprehensive solution for food detection. Finally, this article puts forward the challenges and the prospects for food safety. KEY FINDINGS AND CONCLUSIONS: The novel coronavirus hazards infiltrated every step of the food industry, from processing to packaging to transportation. The biosensor analytical technology based on CRISPR-Cas12a has great potential in the qualitative and quantitative analysis of infectious pathogens. CRISPR-Cas12a can effectively identify the presence of the specific nucleic acid targets and the small changes in sequences, which is particularly important for nucleic acid identification and pathogen detection. In addition, the CRISPR-Cas12a method can be adjusted and reconfigured within days to detect other viruses, providing equipment for nucleic acid diagnostics in the field of food safety. The future work will focus on the development of portable microfluidic devices for multiple detection. Shao et al. employed physical separation methods to separate Cas proteins in different microfluidic channels to achieve multiple detection, and each channel simultaneously detected different targets by adding crRNA with different spacer sequences. Although CRISPR-Cas12a technology has outstanding advantages in detection, there are several technical barriers in the transformation from emerging technologies to practical applications. The newly developed CRISPR-Cas12a-based applications and methods promote the development of numerous diagnostic and detection solutions, and have great potential in medical diagnosis, environmental monitoring, and especially food detection.

CRISPR/Cas13a analysis based on NASBA amplification for norovirus detection.

Human norovirus (HuNoV) is a leading cause of foodborne diseases worldwide, making rapid and accurate detection crucial for prevention and control. In recent years, the CRISPR/Cas13a system, known for its single-base resolution in RNA recognition and unique collateral cleavage activity, is particularly suitable for sensitive and rapid RNA detection. However, isothermal amplification-based CRISPR/Cas13 assays often require an external transcription step, complicating the detection process. In our study, an efficient diagnostic technique based on the NASBA/Cas13a system was established to identify conserved regions at the ORF1-ORF2 junction of norovirus. The RNA amplification techniques [Nucleic Acid Sequence-Based Amplification (NASBA)] integrates reverse transcription and transcription steps, enabling sensitive, accurate, and rapid enrichment of low-abundance RNA. Furthermore, the CRISPR/Cas13a system provides secondary precise recognition of the amplified products, generating a fluorescence signal through its activated accessory collateral cleavage activity. We optimized the reaction kinetics parameters of Cas13a and achieved a detection limit as low as 51pM. The conditions for the cascade reaction involving CRISPR analysis and RNA amplification were optimized. Finally, we validated the reliability and accuracy of the NASBA/Cas13a method by detecting norovirus in shellfish, achieving results comparable to qRT-PCR in a shorter time and detecting viral loads as low as 10 copies/µL.

Ni-Pt nanozyme-mediated relaxation and colorimetric sensor for dual-modality detection of norovirus.

An NiPt nanozyme-mediated relaxation and colorimetric sensor is developed for dual-modality detection of norovirus (NoV). The relaxation modality is based on the "catalase-like" activity of the NiPt nanozyme, which adjusts the hydrogen peroxide (H2O2) mediated Fe (II)/Fe(III) conversion, thereby changing the relaxation signal. Poly-γ-glutamic acid (MW ≈ 1w) can enhance the relaxivity of Fe(III) (r1 = 7.11 mM-1 s-1; r2 = 8.94 mM-1 s-1). The colorimetric modality exploits the "peroxidase-like" activity of the NiPt nanozyme, which can catalyze the oxidation of colorless 3, 3', 5, 5'-tetramethylbenzidine (TMB) to blue oxTMB in H2O2. Under optimal conditions, the relaxation modality exhibits a wide working range (1.0 × 101-1.0 × 104 fM) and a limit of detection (LOD) of 4.7 fM (equivalent to 2820 copies/µL). The spiked recoveries range from 99.593 to 106.442 %, and the relative standard deviation (RSD) is less than 5.124 %. The colorimetric modality exhibited the same working range with a lower LOD of 2.9 fM (equivalent to 1740 copies/µL) and an RSD of less than 2.611 %. Additionally, the recombinase polymerase amplification reaction enabled the detection of low NoV levels in food samples with a working range of 102-106 copies/mL and LOD of 102 copies/mL. The accuracy of the sensor in the analysis of spiked samples is consistent with the gold standard method (real-time quantitative reverse transcription-polymerase chain reaction), demonstrating the high accuracy and practical utility of the sensor.

Self-assembled DNA origami-based duplexed aptasensors combined with centrifugal filters for efficient and rechargeable ATP detection.

DNA origami technology has great potential for biosensor applications. Here, we described the construction of a self-assembled DNA origami biosensor for the precise localization of fluorescent aptamers. Due to the molecular weight difference between DNA origami and aptamer, centrifugal filters were used to quantitatively detect adenosine triphosphate (ATP). The ATP-specific aptamer labeled with fluorescence reporter 6-carboxyfluorescein FAM (FAM-aptamer) was selected as the recognition element and signal probe. ATP duplexed aptamers bound to triangular DNA origami by base-complementary pairing, resulting in high fluorescence signals on the origami arrays. The competitive binding of ATP toward the FAM-aptamer triggered the release of FAM-aptamer-ATP complexes from the surface of the origami array, resulting in weakened fluorescence signals. For ATP quantification, 100 kD centrifugal filters were employed, followed by measurement of the fluorescence signal trapped on the origami arrays of the filter device. The successful synthesis of origami-aptamer arrays was characterized by atomic force microscopy, laser confocal microscopy, and electrophoresis. Fluorescence measurements exhibited an excellent linear relationship with logarithms of ATP concentrations within 0.1-100 ng mL-1, with a detection limit of 0.29 ng mL-1. By replacing aptamers and complementary strands, we demonstrated the potential of this method for 17ß-estradiol detection. Considering that the detection mechanism is based on the hybridization and displacement of DNA strands, the detection system had the potential for recharging. Our study provides new insights into applying DNA origami technology in small molecule detection.

Upconversion-mediated CRISPR-Cas12a biosensing for sensitive detection of ochratoxin A.

The CRISPR-Cas system has achieved breakthrough applications in the field of molecular diagnostics. CRISPR/Cas12a can accurately identify subtle changes in a target nucleic acid sequence and has a wide range of applications such as in highly sensitive detection methods. In this study, an upconversion-magnetic probe-DNA-Fe3O4 probe was designed to replace traditional fluorescent probes using nucleic acid aptamers to design a biosensing method powered by CRISPR/Cas12a. The CRISPR/Cas12a technology can be widely used for sensitive, rapid, and stable detection of ochratoxin A. In our sensing strategy, the ochratoxin A capture probe was used to capture ochratoxin A and release the Cas protein activation chain, further triggering the single-stranded DNA degradation activity of CRISPR/Cas12a and removal of the fluorescent probe to generate a fluorescent signal. Compared with traditional commercial kits, our method was more rapid and exhibited comparable detection capability.

Simple and programmed three-dimensional DNA tweezer for simultaneous one-step detection of ochratoxin A and zearalenone.

Three-dimensional (TD) deoxyribonucleic acid (DNA) tweezers were programmed for one-step identification and detection of ochratoxin A (OTA) and zearalenone (ZEN). The unfolding of the TD-DNA tweezers by aptamers specific to these two mycotoxins "turned" the fluorescent signals "on." The bonding of the aptamers to their corresponding targets in OTA and ZEN "turned" the fluorescent signals and the DNA tweezers "off." The detection limit of the TD-DNA tweezers for OTA and ZEN was 0.032 and 0.037 ng mL-1, respectively. The feasibility of this method was tested using two samples. Detection via this method increased the recovery of OTA and ZEN from 95.8% to 110.2%. Spike recovery and certified food products were used to detect applicability in actual situations. Analyte detection in complex samples using TD-DNA tweezers is rapid, as the process involves a single operational step. This proposed design has considerable potential for application in mycotoxin detection.

Development of Fe3O4@Au nanoparticles coupled to Au@Ag core-shell nanoparticles for the sensitive detection of zearalenone.

Agricultural products are frequently contaminated by mycotoxins; thus, the accurate detection of mycotoxins is important to food safety. Zearalenone (ZEN), a mycotoxin produced by certain Fusarium and Gibberella species, is a group III carcinogen. We developed a universal surface-enhanced Raman scattering (SERS) aptasensor for the detection of ZEN. The SERS biosensor consists of two functional nanomaterials: sulfhydryl (SH)-ZEN aptamer complementary DNA-modified Fe3O4@Au was used as a capture probe and SH-ZEN aptamer-modified Au@Ag core-shell nanoparticles served as reporter probes. In the absence of ZEN, the highest Raman signal was obtained owing to the SERS effects of Fe3O4@Au and Au@Ag core-shell nanoparticles. Conversely, the addition of ZEN triggered the release of Au@Ag core-shell nanoparticles from Fe3O4@Au, leading to a decrease in SERS intensity after magnetic separation. Hybridization of the ZEN aptamer and its complementary strand generated a strong SERS signal from the reporter probe. Moreover, preferential binding of the ZEN aptamer to ZEN was observed. The signal intensity in SERS decreased linearly when the capture probes released the reporter. For ZEN detection, a linear range from 0.005 to 500 ng mL-1, with an R2 of 0.9981, was obtained. The detection limit was 0.001 ng mL-1. The SERS aptasensor showed excellent performance for analytical applications with real-world samples (beer and wine). This study presents a new model for the detection of mycotoxins based on simple changes in aptamers.