Dual-target nucleic acid sequences responsive electrochemiluminescence biosensor using single type carbon dots as probe for SARS-CoV-2 detection based on series catalytic hairpin assembly amplification.

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) is rampant all over the world, and rapid and effective virus detection is the best auxiliary to curb the spread of the epidemic. A diagnosis can only be made if two or more different nucleic acid sequences are confirmed at the same time, and in most of traditional detection technologies, these target sequences have been detected separately. In this work, an electrochemiluminescent (ECL) biosensor employing a single ECL probe as signal output and responding to dual-target simultaneously is proposed for the first time. Taking the two sequences located in ORF 1ab region and N region of SARS-CoV-2 gene sequence as the model target and nitrogen doped carbon quantum dots (CDs) as ECL beacon, supplemented with catalytic hairpin assembly (CHA) reaction for signal amplification, the presented strategy has been successfully applied to the rapid detection of SARS-CoV-2. The developed SARS-CoV-2 biosensor based on the series CHA systems can realize the quantitative determination of SARS-CoV-2 in the range of 50 fM to 200 pM within 40 min. Moreover, the clinical validity of this method has been verified by the high consistency between the detection results of using this method and those using RT-qPCR for seven clinical pharyngeal swab samples.

Target DNA- and pH-responsive DNA hydrogel-based capillary assay for the optical detection of short SARS-CoV-2 cDNA.

DNA is recognized as a powerful biomarker for clinical diagnostics because its specific sequences are closely related to the cause and development of diseases. However, achieving rapid, low-cost, and sensitive detection of short-length target DNA still remains a considerable challenge. Herein, we successfully combine the catalytic hairpin assembly (CHA) technique with capillary action to develop a new and cost-effective method, a target DNA- and pH-responsive DNA hydrogel-based capillary assay, for the naked eye detection of 24 nt short single-stranded target DNA. Upon contact of target DNA, three individual hairpin DNAs hybridize with each other to sufficiently amplify Y-shaped DNA nanostructures (Y-DNA) until they are completely consumed via CHA cycling reactions. Each arm of the resultant Y-DNA contains sticky ends with i-motif DNA structure-forming sequences that can be self-assembled in an acidic environment (pH 5.0) to form target DNA- and pH-responsive DNA hydrogels by means of i-motif DNA-driven crosslinking. When inserting a capillary tube in the resultant solution, the liquid level inside clearly reduces due to the decrease in capillary force induced by the gels. In this way, the developed assay demonstrates sensitive and quantitative detection, with a detection limit of approximately 10 pM of 24 nt short complementary DNA (cDNA) targeting SARS-CoV-2 RNA genes at room temperature within 1 h. The assay is further shown to successfully detect target cDNA in serum, and it is also applied to detect several types of target sequences. Requiring no analytic equipment, precise temperature control, or enzymatic reactions, the developed DNA hydrogel-based capillary assay has potential as a promising naked eye detection platform for target DNA in resource-limited clinical settings.

Catalytic hairpin DNA assembly-based chemiluminescent assay for the detection of short SARS-CoV-2 target cDNA.

Colorimetric sensors are recognized as a promising means for target molecule detection as they provide rapid, cost-effective, and facile sensing visible to the naked eye. Challenges remain though in terms of their detection sensitivity and specificity for short-length target genes. Herein, we demonstrate the successful combination of the catalytic hairpin DNA assembly (CHA) approach with enzyme-linked immunosorbent assay (ELISA)-mimicking techniques for a simple, sensitive, and sequence-specific colorimetric assay to detect short SARS-CoV-2 target cDNA. In the developed CHA-based chemiluminescent assay, a low concentration of target cDNA is continuously recycled to amplify dimeric DNA probes from two biotinylated hairpin DNA until the hairpin DNA is completely consumed. The dimeric DNA probes are effectively immobilized in a neutravidin-coated microplate well and then capture neutravidin-conjugated horseradish peroxidase via biotin-neutravidin interactions, resulting in a sensitive and selective colorless-to-blue color change. The developed sensing system exhibits a high sensitivity with a detection limit of ~1 nM for target cDNA as well as the ability to precisely distinguish a single-base mismatched mutant gene within 2 h. As the proposed system does not require complex protocols or expensive equipment to amplify target cDNA, it has the potential to be utilized as a powerful tool to improve the detection sensitivity of target genes for clinical diagnostics with colorimetric detection.

An electrochemical biosensor for sensitive analysis of the SARS-CoV-2 RNA.

The pandemic of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) is continuously worsening globally, herein we have proposed an electrochemical biosensor for the sensitive monitoring of SARS-CoV-2 RNA. The presence of target RNA firstly triggers the catalytic hairpin assembly circuit and then initiates terminal deoxynucleotidyl transferase-mediated DNA polymerization. Consequently, a large number of long single-stranded DNA products can be produced, and these negatively charged DNA products will bind a massive of positively charged electroactive molecular of Ru(NH3)63+ due to the electrostatic adsorption. Therefore, significantly amplified electrochemical signals can be generated for sensitive analysis of SARS-CoV-2 RNA in the range of 0.1-1000 pM with the detection limit as low as 26 fM. Besides the excellent distinguishing ability for SARS-CoV-2 RNA against single-base mismatched RNA, the proposed biosensor can also be successfully applied to complex matrices, as well as clinical patient samples with high stability, which shows great prospects of clinical application.

Rapid point-of-care testing for SARS-CoV-2 virus nucleic acid detection by an isothermal and nonenzymatic Signal amplification system coupled with a lateral flow immunoassay strip.

An outbreak of a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), began in December 2019. Accurate, rapid, convenient, and relatively inexpensive diagnostic methods for SARS-CoV-2 infection are important for public health and optimal clinical care. The current gold standard for diagnosing SARS-CoV-2 infection is reverse transcription-polymerase chain reaction (RT-PCR). However, RTPCR assays are designed for use in well-equipped laboratories with sophisticated laboratory infrastructure and highly trained technicians, and are unsuitable for use in under-equipped laboratories and in the field. In this study, we report the development of an accurate, rapid, and easy-to-implement isothermal and nonenzymatic signal amplification system (a catalytic hairpin assembly (CHA) reaction) coupled with a lateral flow immunoassay (LFIA) strip-based detection method that can detect SARSCoV-2 in oropharyngeal swab samples. Our method avoids RNA isolation, PCR amplification, and elaborate result analysis, which typically takes 6-8 h. The entire CHA-LFIA detection method, from nasopharyngeal sampling to obtaining test results, takes less than 90 min. Such methods are simple and require no expensive equipment, only a simple thermostatically controlled water bath and a fluorescence reader device. We validated our method using synthetic oligonucleotides and clinical samples from 15 patients with SARS-CoV-2 infection and 15 healthy individuals. Our detection method provides a fast, simple, and sensitive (with a limit of detection (LoD) of 2000 copies/mL) alternative to the SARS-CoV-2 RT-PCR assay, with 100 % positive and negative predictive agreements.