Rapid, sensitive detection of intact SARS-CoV-2 using DNA nets and a smartphone-linked fluorimeter

We report a single-step, room-temperature, 5-10 minute SARS-CoV-2 saliva self-monitoring method that overcomes the limitations of existing approaches through the use of fluorophore-releasing Designer DNA Nanostructures (DDNs) that bind with the multivalent pattern of spike proteins on the exterior intact virions and an inexpensive smartphone-linked, pocket-size fluorimeter, called a "V-Pod” for its resemblance to an Apple AirPod™ headphone case. We characterize the V-Pod fluorimeter performance and the DDN-based assay to demonstrate a clinically relevant detection limit of 104 virus particles/mL for pseudo-typed WT SARS-CoV-2 and 105 virus particles/mL for real pathogenic variants, including Delta, Omicron, and D614g. © 2023 SPIE.

Ultra-sensitive Detection of Alzheimer's Biosmarkers Using Plasmonic Optical Fiber Sensors

The emergence of the Covid-19 pandemic has drawn great attention to vulnerable people affected by major diseases. Among them, Alzheimer's disease (AD) is the most prevalent disease. However, a long-standing challenge is to achieve early diagnosis of AD by detecting biomarkers such as amyloid beta (Aβ42), thus avoiding the labor of specialized hospital personnel and the high cost of imaging examinations using positron emission tomography. In this paper, we report a straightforward approach to realize a non-invasive lab-around fiber (LaF) optical sensor for AD biomarker detection, which is based on a tilted fiber Bragg grating (TFBG) combined with a nanoscale metallic thin film. We successfully demonstrated the detection of Aβ42 in complex biological matrices with a detection limit of 5 pg/mL. Therefore, our TFBG-SPR biosensor platform enables large-scale early disease screening and has great potential for clinical applications in early AD diagnosis. © 2022 IEEE.

Recent advances in cascade isothermal amplification techniques for ultra-sensitive nucleic acid detection.

Nucleic acid amplification techniques have always been one of the hot spots of research, especially in the outbreak of COVID-19. From the initial polymerase chain reaction (PCR) to the current popular isothermal amplification, each new amplification techniques provides new ideas and methods for nucleic acid detection. However, limited by thermostable DNA polymerase and expensive thermal cycler, PCR is difficult to achieve point of care testing (POCT). Although isothermal amplification techniques overcome the defects of temperature control, single isothermal amplification is also limited by false positives, nucleic acid sequence compatibility, and signal amplification capability to some extent. Fortunately, efforts to integrating different enzymes or amplification techniques that enable to achieve intercatalyst communication and cascaded biotransformations may overcome the corner of single isothermal amplification. In this review, we systematically summarized the design fundamentals, signal generation, evolution, and application of cascade amplification. More importantly, the challenges and trends of cascade amplification were discussed in depth.

A Review on the Application of Nanomaterials in Virus Detection

R apid, sensitive and specific detection of viruses is a key issue in the medical field. Since 2020, the global outbreak of COVID-19 requires more sensitive virus detection methods. With the development of new materials, especially nanomaterials, many materials have demonstrated great physical, chemical and mechanical properties, which present potential for virus detection. Nanomaterials can be divided into zero-dimensional materials, one-dimensional materials and two-dimensional materials by structure. In this paper, the classification and the latest progress of nanomaterials are reviewed, highlighting their applications in the field of virus detection. The future prospect of nanomaterials in virus detection is also presented and discussed. © 2023 Cailiao Daobaoshe/ Materials Review. All rights reserved.

3D interior hotspots embedded with viral lysates for rapid and label-free identification of infectious diseases

In recent decades, biomedical sensors based on surface-enhanced Raman spectroscopy (SERS), which reveals unique spectral features corresponding to individual molecular vibrational states, have attracted intensive attention. However, the lack of a system for precisely guiding biomolecules to active hotspot regions has impeded the broad application of SERS techniques. Herein, we demonstrate the irreversible active engineering of three-dimensional (3D) interior organo-hotspots via electrochemical (EC) deposition onto metal nanodimple (ECOMD) platforms with viral lysates. This approach enables organic seed-programmable Au growth and the spontaneous bottom-up formation of 3D interior organo-hotspots simultaneously. Because of the net charge effect on the participation rate of viral lysates, the number of interior organo-hotspots in the ECOMDs increases with increasingly positive polarity. The viral lysates embedded in the ECOMDs function as both a dielectric medium for field confinement and an analyte, enabling the highly specific and sensitive detection of SARS-CoV-2 lysates (SLs) at concentrations as low as 10-2 plaque forming unit/mL. The ECOMD platform was used to trace and detect the SLs in human saliva and diagnose of the delta-type SARS-CoV-2 in clinical environments;the results indicate that the proposed platform can provide point-of-care diagnoses of infectious diseases.

Discovery and translation of functional nucleic acids for clinically diagnosing infectious diseases: Opportunities and challenges

Functional nucleic acids (FNAs) are short, single-stranded nucleic acids that can be derived from synthetic nucleic acid libraries using test-tube selection experiments. Due to their excellent chemical stability, high binding affinities and specificities, compatibility with a variety of signal-transduction mechanisms, and ease of synthesis and modification, FNAs have a great potential to overcome some of the limitations of current pathogen diagnostic methods by acting as molecular recognition elements (MREs) for point-of-care testing. This review summarizes the development of FNA-based biosensors for viral and bacterial detection in clinical samples. We first discuss examples of selecting FNAs for recognizing biomarkers of viral and bacterial pathogens. This is followed by discussion on integrating FNAs into fluorescent, colorimetric, and electrochemical biosensors and applying these sensors towards clinically diagnosing infectious diseases caused by many important bacterial and viral pathogens. Finally, the challenges of making FNA-based biosensors for infectious diseases are provided. (c) 2022 Elsevier B.V. All rights reserved.

Environmental routes of virus transmission and the application of nanomaterial-based sensors for virus detection

Many outbreaks of emerging disease (e.g., avian influenza, SARS, MERS, Ebola, COVID-19) are caused by viruses. In addition to direct person-to-person transfer, the movement of these viruses through environmental matrices (water, air, and food) can further disease transmission. There is a pressing need for rapid and sensitive virus detection in environmental matrices. Nanomaterial-based sensors (nanosensors), which take advantage of the unique optical, electrical, or magnetic properties of nanomaterials, exhibit significant potential for environmental virus detection. Interactions between viruses and nanomaterials (or recognition agents on the nanomaterials) can induce detectable signals and provide rapid response times, high sensitivity, and high specificity. Facile and field-deployable operations can be envisioned due to the small size of the sensing elements. In this frontier review, we summarize virus transmission via environmental pathways and then comprehensively discuss recent applications of nanosensors to detect various viruses. This review provides guidelines for virus detection in the environment through the use of nanosensors as a tool to decrease environmental transmission of current and emerging diseases.

Systematically investigating the fluorescent signal readout of CRISPR-Cas12a for highly sensitive SARS-CoV-2 detection.

The CRISPR/Cas system is widely used for molecular diagnostics after the discovery of trans-cleavage activity, especially now with the COVID-19 outbreak. However, the majority of contemporary trans-cleavage activity-based CRISPR/Cas biosensors exploited standard single-strand DNA (ssDNA) reporters, which were based on the FRET principle from pioneering research. An in-depth comparison and understanding of various fluorescent readout types are essential to facilitate the outstanding analytical performance of CRISPR probes. We investigated various types of fluorescent reporters of Cas12a comprehensively. Results show that trans-cleavage of Cas12a is not limited to ssDNA and dsDNA reporters, but can be extended to molecular beacons (MB). And MB reporters can achieve superior analytical performance compared with ssDNA and ds DNA reporters at the same conditions. Accordingly, we developed a highly-sensitive SARS-CoV-2 detection with the sensitivity as low as 100 fM were successfully achieved without amplification strategy. The model target of ORF1a could robustly identify the current widespread emerging SARS-CoV-2 variants. A real coronavirus GX/P2V instead of SARS-CoV-2 were chosen for practical application validation. And a minimum of 27 copies/mL was achieved successfully. This inspiration can also be applied to other Cas proteins with trans-cleavage activity, which provides new perspectives for simple, highly-sensitive and universal molecular diagnosis in various applications.

Highly sensitive and rapid detection of SARS-CoV-2 via a portable CRISPR-Cas13a-based lateral flow assay.

To rapidly identify individuals infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and control the spread of coronavirus disease (COVID-19), there is an urgent need for highly sensitive on-site virus detection methods. A clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas)-based molecular diagnostic method was developed for this purpose. Here, a CRISPR system-mediated lateral flow assay (LFA) for SARS-CoV-2 was established based on multienzyme isothermal rapid amplification, CRISPR-Cas13a nuclease, and LFA. To improve the limit of detection (LoD), the crispr RNA, amplification primer, and probe were screened, in addition to concentrations of various components in the reaction system. The LoD of CRISPR detection was improved to 0.25 copy/µl in both fluorescence- and immunochromatography-based assays. To enhance the quality control of the CRISPR-based LFA method, glyceraldehyde-3-phosphate dehydrogenase was detected as a reference using a triple-line strip design in a lateral flow strip. In total, 52 COVID-19-positive and 101 COVID-19-negative clinical samples examined by reverse transcription polymerase chain reaction (RT-PCR) were tested using the CRISPR immunochromatographic detection technique. Results revealed 100% consistency, indicating the comparable effectiveness of our method to that of RT-PCR. In conclusion, this approach significantly improves the sensitivity and reliability of CRISPR-mediated LFA and provides a crucial tool for on-site detection of SARS-CoV-2.

RAPID DETECTION OF NOVEL CORONAVIRUS SARS-COV-2 BY SOLID-STATE NANOPORE

The COVID-19 pandemic spreads rapidly and globally. To quell the pandemic propagation, rapid and accurate detection of SARS-CoV-2 is urgently needed. Here, we present a nanopore coupled RT-LAMP method for SARSCoV-2 detection. After comparing all information from the nanopore experiment, we develop a method to use the event rate change of the amplicons translocation event to measure the amplification. As a result, our platform can distinguish positive from negative samples in 15 min with around 65 copies/reaction limit of detection and 100% specificity. We believe that the nanopore coupled RT-LAMP platform would provide a sensitive and specific detection for SARS-COV-2. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

Sequence-Specific Recognition of SARS COV-2 with Solid-State CRISPR-Cas12a-Assisted Nanopores (SCAN)

The current outbreak of the SARS-CoV-2 caused the COVID-19 disease to spread rapidly globally. Specific and sensitive detection of SARS-CoV-2 is needed to prevent the disease from spread. Here, we present a solid-state CRISPR-Cas12a-assisted nanopores (SCAN) system to detect SARS-CoV-2. We introduced a new scheme using current drops and dwell times of ssDNA reporter translocation events to estimate the cleavage activity. We validated this scheme by a statistical model approximating the reporter length distribution over the cleavage reaction. We believe that the SCAN would provide a sensitive and specific detection method for SARS-COV-2. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

COVID-19: Prevent Infection, Diagnosis and Vaccination using Nanotechnology

The world has been struggling with the major health crisis of the coronavirus disease 2019 (COVID-19) pandemic since December 2019. The highly virulent nature and the mutation rates of the novel virus resulted in less sensitive detection techniques. The main objective of this review paper is to present the possibility of using nanotechnology for COVID-19 prevention, diagnosis, and for developing vaccination. There are different research activities conducted using dissimilar types of masks to control COVID-19 transmission, and their results showed that using single fabric mask with holes was not effective for filtering coronavirus 2019, however, the charged polyvinylidene difluoride (PVDF) nanofiber prevent fast-spreading of COVID-19 and the filtration efficiency may reach 98%. Many researchers suggested that nanosorbents can be used to adsorb COVID-19 virus depending on size, surface area, and the presence of appropriate functional groups. The color of nanosorbents changes relatively by increasing the amount of sorbent virus from the air (virus concentration) and can be measured using different spectral techniques. Nanotechnology can also be an ideal solution to overcome the drawbacks of inactivated vaccines. The common traditional vaccines which depend on live-attenuated or inactivated pathogens present different risks from reflection to pathogenic virulence or guide to a weak immune response. So, the non-traditional vaccines based on nanoparticles (NPs) can enhance strong immunogenicity and antigen presentation by controlling the surface, shape, size and functional groups. © 2021 American Institute of Chemical Engineers. All rights reserved.

Recent advances on gold and silver nanoparticle-based colorimetric strategies for the detection of different substances and SARS-CoV-2: a comprehensive review

Nanoparticle (NP)-based colorimetric methods are extensively used for the rapid detection of environmental contaminants, different substances and SARS-CoV-2 in various fields such as environmental science, virology, pollution research, and the food industry, as well as biomedicine. Colorimetric sensors exhibit high sensitivity and selectivity, are easy to handle, portable, safe for screening purposes and can be visualized by the naked eye. Herein, the colorimetric sensing approaches of the two most commonly used metallic NPs, i.e., gold (Au) and silver (Ag), and their physicochemical methods are discussed, as metallic NPs show good efficiency due to their unique optical and chemical properties. This review summarizes the progress on colorimetric sensors based on metallic NPs as sensors and their applications, elucidating the utility and superior features of metallic-NP-based colorimetric assay for the detection of different environmental contaminants, biomolecules and SARS-CoV-2 in the environmental as well as human biological samples. An outlook with respect to the trends and future development of the proposed sensors is also provided.

A Novel, Cleaved Probe-Based Reverse Transcription Loop-Mediated Isothermal Amplification Method for Specific and Sensitive Detection of Porcine Deltacoronavirus.

Porcine deltacoronavirus (PDCoV) causes watery diarrhea, vomiting, and 30-40% mortality in newborn piglets. A simple, rapid, and sensitive method for PDCoV detection is valuable in its surveillance and control. Here, we developed a novel, cleaved probe-based reverse transcription loop-mediated isothermal amplification (CP-RT-LAMP) method for PDCoV detection. A cleaved probe with a ribonucleotide insertion that targeted the N gene of PDCoV was designed. During the reaction, the enzyme ribonuclease H2 is activated only when the cleaved probe is perfectly complementary to the template, leading to the hydrolytic release of a quencher moiety and signal output. This method can be easily used on a real-time fluorescence quantitative equipment or an on-site isothermal instrument combined with a smartphone. The specificity assay showed no cross-reactivity with other porcine enteric pathogens. This method had a detection limit of 25 copies/µL, suggesting comparable sensitivity with reverse transcription quantitative PCR (RT-qPCR). In detecting 100 clinical samples (48 fecal swab specimens and 52 intestinal specimens), the detection rate of the CP-RT-LAMP method (26%) was higher than that of RT-qPCR (17%). Thus, it is a highly specific and sensitive diagnostic method for PDCoV, with a great application potential for monitoring PDCoV in the laboratory or point-of-care testing in the field.

Terahertz Impedance Spectroscopy of Biological Nanoparticles by a Resonant Metamaterial Chip for Breathalyzer-Based COVID-19 Prompt Tests

We propose a tested, sensitive, and prompt COVID-19 breath screening method that takes less than 1 min. The method is nonbiological and is based on the detection of a shift in the resonance frequency of a nanoengineered inductor-capacitor (LC) resonant metamaterial chip, caused by viruses and mainly related exhaled particles, when performing terahertz spectroscopy. The chip consists of thousands of microantennas arranged in an array and enclosed in a plastic breathalyzer-like disposable capsule kit. After an appreciable agreement between numerical simulations (COMSOL and CST) and experimental results was reached using our metamaterial design, low-scale clinical trials were conducted with asymptomatic and symptomatic coronavirus patients and healthy individuals. It is shown that coronavirus-positive individuals are effectively screened upon observation of a shift in the transmission resonance frequency of about 1.5-9 GHz, which is diagnostically different from the resonance shift of healthy individuals who display a 0-1.5 GHz shift. The initial results of screening coronavirus patients yielded 88% agreement with the realtime quantitative polymerase chain reaction (RT-qPCR) results (performed concurrently with the breath test) with an outcome of a positive predicted value of 87% and a negative predicted value of 88%.

Trends in nanomaterial-based biosensors for viral detection

Pandemics such as COVID-19 have highlighted the importance of point-of-care sensors for testing, tracing, and treatment to minimize and manage infection. Biosensors have been widely deployed in portable devices such as glucose sensors and pregnancy tests. Their development for point-of-exposure virus detection or point-of-care devices is anticipated but their reliability for the accurate detection of viruses is critical. Nanomaterials, such as metal nanoparticles (NPs), magnetic NPs, quantum dots, carbon-based nanomaterials, and molecularly imprinted polymer (MIP) NPs, have been utilized in biosensors to enhance sensitivity. Molecular imprinting is a cost-effective method to synthesize polymers for selective binding, which have excellent properties as biosensors. More research on MIP NPs can be expected in the near future. The utilization of nanomaterials in several types of transducers for biosensor devices is also illustrated to give an overview of their use. Finally, a summary is given together with a future perspective on how biosensors can be further developed as reliable, portable viral biosensors.

Exploring the entropy-driven amplification reaction and trans-cleavage activity of CRISPR-Cas12a for the development of an electrochemiluminescence biosensor for the detection of the SARS-CoV-2 RdRp gene in real samples and environmental surveillance

In this paper, we present the first idea of using a DNA triple helix structure to inhibit CRISPR-Cas12a activity and apply it to the design of an electrochemiluminescent biosensor for the detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) gene in real samples and environmental surveillance. We employed a segment from the RdRp gene of SARS-CoV-2 by an entropy-driven reaction, which was paired with double-stranded DNA that can activate CRISPR-Cas12a activity by Hoogsteen pairing to form triple-stranded DNA, thereby inhibiting the binding interaction of the double-stranded DNA with CRISPR-Cas12a, which in turn inhibits the trans cleavage activity of CRISPR-Cas12a. The inhibited CRISPR-Cas12a is unable to cut the nucleic acid modified on the electrode surface, resulting in the inability of the ferrocene (Fc) modified on the other end of the nucleic acid to move away from the electrode surface, and thus failing to cause electrochemiluminescence changes in GOAu-Ru modified on the electrode surface. The extent of the electrogenic chemiluminescence change can reflect the concentration of the gene to be tested. Using this system, we achieved the detection of the SARS-CoV-2 RdRp gene with a detection limit of 32.80 aM.

Rapid field determination of SARS-CoV-2 by a colorimetric and fluorescent dual-functional lateral flow immunoassay biosensor.

The rapid and accurate diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the early stage of virus infection can effectively prevent the spread of the virus and control the epidemic. Here, a colorimetric and fluorescent dual-functional lateral flow immunoassay (LFIA) biosensor was developed for the rapid and sensitive detection of spike 1 (S1) protein of SARS-CoV-2. A novel dual-functional immune label was fabricated by coating a single-layer shell formed by mixing 20 nm Au nanoparticles (Au NPs) and quantum dots (QDs) on SiO2 core to produce strong colorimetric and fluorescence signals and ensure good monodispersity and high stability. The colorimetric signal was used for visual detection and rapid screening of suspected SARS-CoV-2 infection on sites. The fluorescence signal was utilized for sensitive and quantitative detection of virus infection at the early stage. The detection limits of detecting S1 protein via colorimetric and fluorescence functions of the biosensor were 1 and 0.033 ng/mL, respectively. Furthermore, we evaluated the performance of the biosensor for analyzing real samples. The novel biosensor developed herein had good repeatability, specificity and accuracy, which showed great potential as a tool for rapidly detecting SARS-CoV-2.