Aptamer-based CRISPR-Cas powered diagnostics of diverse biomarkers and small molecule targets.

CRISPR-Cas systems have been widely used in genome editing and transcriptional regulation. Recently, CRISPR-Cas effectors are adopted for biosensor construction due to its adjustable properties, such as simplicity of design, easy operation, collateral cleavage activity, and high biocompatibility. Aptamers' excellent sensitivity, specificity, in vitro synthesis, base-pairing, labeling, modification, and programmability has made them an attractive molecular recognition element for inclusion in CRISPR-Cas systems. Here, we review current advances in aptamer-based CRISPR-Cas sensors. We briefly discuss aptamers and the knowledge of Cas effector proteins, crRNA, reporter probes, analytes, and applications of target-specific aptamers. Next, we provide fabrication strategies, molecular binding, and detection using fluorescence, electrochemical, colorimetric, nanomaterials, Rayleigh, and Raman scattering. The application of CRISPR-Cas systems in aptamer-based sensing of a wide range of biomarkers (disease and pathogens) and toxic contaminants is growing. This review provides an update and offers novel insights into developing CRISPR-Cas-based sensors using ssDNA aptamers with high efficiency and specificity for point-of-care setting diagnostics.

Quercetin-Mediated Silver Nanoparticle Formation for the Colorimetric Detection of Infectious Pathogens Coupled with Loop-Mediated Isothermal Amplification.

Here, quercetin-mediated silver nanoparticle (AgNP) formation combined with loop-mediated isothermal amplification (LAMP) was introduced to colorimetrically detect two major infectious pathogens, SARS-CoV-2 and Enterococcus faecium, using a foldable PMMA microdevice. The nitrogenous bases of LAMP amplicons can readily form a complex with Ag+ ions, and the catechol moiety in quercetin, which acted as a reducing agent, could be chelated with Ag+ ions, resulting in the easy electron transfer from the oxidant to the reductant and producing brown-colored AgNPs within 5 min. The introduced method exhibited higher sensitivity than agarose gel electrophoresis due to more active redox centers in quercetin. The detection limit was attained at 101 copies µL-1 and 101 CFU mL-1 for SARS-CoV-2 RNA and E. faecium, respectively. A foldable microdevice made of two pieces of PMMA that fully integrates DNA extraction, amplification, and detection processes was fabricated to establish practical applicability. On one PMMA, DNA extraction was performed in a reaction chamber inserted with an FTA card, and then LAMP reagents were added for amplification. Silver nitrate was added to the reaction chamber after LAMP. On the other PMMA, quercetin-soaked paper discs loaded in the detection chamber were folded toward the reaction chamber for colorimetric detection. An intense brown color was produced within 5 min when heated at 65 °C. The introduced colorimetric assay, which is highly favorable for laboratory and on-site applications, could be a valuable alternative to conventional methods for detecting infectious diseases, given its unique principle, simplicity, and naked-eye detection.

Copper Sulfate-Induced Diphenylamine for Rapid Colorimetric Point-of-Care Detection of Contagious Pathogens Combined with Loop-Mediated Isothermal Amplification

Here, we developed a copper sulfate (CuSO4)-initiated diphenylamine (DPA)-based colorimetric strategy coupled with loop-mediated isothermal amplification (LAMP) for rapid detection of two critical contagious pathogens, SARS-CoV-2 and Enterococcus faecium. To detect the DNA, acid hydrolysis of LAMP amplicons was executed, enabling the development of a blue color. In the LAMP amplicons, the bond between the purines and deoxyribose is extremely labile. It can be broken using 70% sulfuric acid followed by phosphate group elimination, which generates a highly active keto aldehyde group. CuSO4 plays an imperative role inducing DPA to rapidly react with the keto aldehyde group, producing an intense blue color within 5 min. Moreover, low quantities such as 103 copies μL-1 of SARS-CoV-2 RNA and 102 CFU mL-1 of E. faecium were successfully detected, revealing the advantages of the introduced method. To confirm practical applicability, multiplex detection of pathogens was performed using a foldable microdevice comprising reaction and detection zones. Various reactions such as DNA extraction, LAMP, and acid hydrolysis occurred in the reaction zone. Then, colorimetric reagents (DPA, CuSO4, and ethylene glycol) contained in the detection zone were mixed with the keto aldehyde group by simply folding the microdevice, which was heated at 65 °C for 5 min for colorimetric detection. An intense blue color was developed where the target DNA was present. These results indicate that the method proposed in this study is highly suitable for point-of-care applications, especially in resource-limited settings for the rapid detection of harmful pathogens. © 2023 American Chemical Society.

Detection of SARS-CoV-2 receptor binding domain using fluorescence probe and DNA flowers enabled by rolling circle amplification.

Using rolling circle amplification (RCA) and two different ways of signal readout, we developed analytical methods to detect the receptor-binding domain (RBD) of SARS-CoV-2 spike protein (S protein). We modified streptavidin-coated magnetic beads with an aptamer of RBD through a biotin-tagged complementary DNA strand (biotin-cDNA). Binding of RBD caused the aptamer to dissociate from the biotin-cDNA, making the cDNA available to initiate RCA on the magnetic beads. Detection of RBD was achieved using a dual signal output. For fluorescence signaling, the RCA products were mixed with a dsDNA probe labeled with fluorophore and quencher. Hybridization of the RCA products caused the dsDNA to separate and to emit fluorescence (λex = 488 nm, λem = 520 nm). To generate easily detectable UV-vis absorbance signal, the RCA amplification was extended to produce DNA flower to encapsulate horseradish peroxidase (HRP). The HRP-encapsulated DNA flower catalyzed a colorimetric reaction between H2O2 and 3,3',5,5'-tetramethylbenzidine (TMB) to generate an optical signal (λabs = 450 nm). The fluorescence and colorimetric assays for RBD have low detection limits (0.11 pg mL-1 and 0.904 pg mL-1) and a wide linear range (0.001-100 ng mL-1). For detection of RBD in human saliva, the recovery was 93.0-100% for the fluorescence assay and 87.2-107% for the colorimetric assay. By combining fluorescence and colorimetric detection with RCA, detection of the target RBD in human saliva was achieved with high sensitivity and selectivity.

Nanomolecular Magnetic Probe for Colorimetric RT-LAMP of Viral RNA

Amplification of the RNA from the Covid-19 virus is considered the main objective of the molecular diagnosis of SARS-Cov-2. However, the use of target-based amplification methods such as polymerase chain reaction requires a step to convert the RNA of the Covid-19 virus into a DNA template to lead to amplification. In addition, isolating the RNA of the Covid-19 virus requires RNA purification kits, which will increase the time and costs of molecular detection of this virus. In this study, the magnetic nanoprobe is introduced that it could capture and amplify Covid-19 RNA through an isothermal amplification process called loop-mediated isothermal amplification without requiring a step to convert the viral RNA into a DNA template. By using the engineered sequences corresponding to the target nucleic acid attached to magnetic nanoparticles, it becomes possible to identify the target RNA of this virus through color changes due to pH changes that can be seen with the naked eye due to the presence of pH indicators in the reaction mix. According to the isothermal amplification of the viral RNA via LAMP assisted with the magnetic nanoprobe, the nanomolecular method eliminated the need for special equipment and the time for detecting Covid-19 in specimens. © 2022 by the authors.

Nanozyme-Based Colorimetric SARS-CoV-2 Nucleic Acid Detection by Naked Eye.

Fluorescence-based PCR and other amplification methods have been used for SARS-CoV-2 diagnostics, however, it requires costly fluorescence detectors and probes limiting deploying large-scale screening. Here, a cut-price colorimetric method for SARS-CoV-2 RNA detection by iron manganese silicate nanozyme (IMSN) is established. IMSN catalyzes the oxidation of chromogenic substrates by its peroxidase (POD)-like activity, which is effectively inhibited by pyrophosphate ions (PPi). Due to the large number of PPi generated by amplification processes, SARS-CoV-2 RNA can be detected by a colorimetric readout visible to the naked eye, with the detection limit of 240 copies mL-1 . This conceptually new method has been successfully applied to correctly distinguish positive and negative oropharyngeal swab samples of COVID-19. Colorimetric assay provides a low-cost and instrumental-free solution for nucleic acid detection, which holds great potential for facilitating virus surveillance.

Copper Sulfate-Induced Diphenylamine for Rapid Colorimetric Point-of-Care Detection of Contagious Pathogens Combined with Loop-Mediated Isothermal Amplification

Here, we developed a copper sulfate (CuSO4)-initiated diphenylamine (DPA)-based colorimetric strategy coupled with loop-mediated isothermal amplification (LAMP) for rapid detection of two critical contagious pathogens, SARS-CoV-2 and Enterococcus faecium. To detect the DNA, acid hydrolysis of LAMP amplicons was executed, enabling the development of a blue color. In the LAMP amplicons, the bond between the purines and deoxyribose is extremely labile. It can be broken using 70% sulfuric acid followed by phosphate group elimination, which generates a highly active keto aldehyde group. CuSO4 plays an imperative role inducing DPA to rapidly react with the keto aldehyde group, producing an intense blue color within 5 min. Moreover, low quantities such as 103 copies μL-1 of SARS-CoV-2 RNA and 102 CFU mL-1 of E. faecium were successfully detected, revealing the advantages of the introduced method. To confirm practical applicability, multiplex detection of pathogens was performed using a foldable microdevice comprising reaction and detection zones. Various reactions such as DNA extraction, LAMP, and acid hydrolysis occurred in the reaction zone. Then, colorimetric reagents (DPA, CuSO4, and ethylene glycol) contained in the detection zone were mixed with the keto aldehyde group by simply folding the microdevice, which was heated at 65 °C for 5 min for colorimetric detection. An intense blue color was developed where the target DNA was present. These results indicate that the method proposed in this study is highly suitable for point-of-care applications, especially in resource-limited settings for the rapid detection of harmful pathogens. © 2023 American Chemical Society

An achromatic colorimetric nanosensor for sensitive multiple pathogen detection by coupling plasmonic nanoparticles with magnetic separation.

Rapid screening of multiple pathogens will greatly improve the efficiency of pandemic prevention and control. Colorimetric methods exhibit the advantages of convenience, portability, low cost, time efficiency, and free of sophisticated instruments, yet usually have difficulties in simultaneous detection and suffer from monotonous color changes with low visual resolution and sensitivity. Hence, coupled three kinds of plasmonic nanoparticles (NPs) with magnetic separation, we developed an achromatic colorimetric nanosensor with highly enhanced visual resolution for simultaneous detection of SARS-CoV-2, Staphylococcus aureus, and Salmonella typhimurium. The achromatic nanosensor was composed of SARS-CoV-2-targeting red gold NPs, S. aureus-targeting yellow silver NPs and S. typhimurium-targeting blue silver triangle NPs mixed as black color. In the detection, three corresponding magnetic probes were added into the above mixture. In the presence of a target pathogen, it would be recognized and combined with corresponding colored reporters and magnetic probes to form sandwich complexes, which were removed by magnetic separation, and the sensor changed from black to a chromatic color (the color of the reporters remained in supernatant). Consequently, different target pathogen induced different color. For example, SARS-CoV-2, S. aureus, and S. typhimurium respectively produced green, purple, and orange. While coexistence of S. aureus and S. typhimurium produced red, and coexistence of S. aureus and SARS-CoV-2 produced blue, etc. Therefore, by observing the color change or measuring the absorption spectra, multiple pathogen detection was achieved conveniently. Compared with most colorimetric sensors, this achromatic nanosensor involved rich color change, thus significantly enhancing visual resolution and inspection sensitivity. Therefore, this sensor opened a promising avenue for efficient monitoring and early warning of food safety and quality.

Understanding Oligonucleotide Hybridization and the Role of Anchoring on the Single-Walled Carbon Nanotube Corona Phase for Viral Sensing Applications

Semiconducting single-walled carbon nanotubes (SWCNTs) with tailored corona phases (CPs), or surface adsorbed molecules, have emerged as a promising interface for sensing applications. The adsorption of an analyte can be specifically transduced as a modulation of their band-gap near infrared (nIR) photoluminescence (PL). One such CP ideal for this purpose is single-stranded DNA (ssDNA), where subsequent sequence-dependent hybridization can result in PL emission wavelength shifts. Due to ssDNA adsorption to the SWCNT surface, the resultant noncanonical hybridization and its effect on SWCNT photophysical properties are not well understood. In this work, we study 20-and 21-mer DNA and RNA hybridization on the complementary ssDNA-SWCNT CP in the context of nucleic acid sensing for SARS-CoV-2 sequences as model analytes. We found that the van't Hoff transition enthalpy of hybridization on SWCNT CP was -11.9 kJ mol-1, much lower than that of hybridization in solution (-707 kJ mol-1). We used SWCNT solvatochromism to calculate the solvent-exposed surface area to indicate successful hybridization. We found that having a 30-mer anchor region in addition to the complementary region significantly improved PL response sensitivity and selectivity, with a (GT)15 anchor preferred for RNA targets. Coincubation of ssDNA-SWCNTs with an analyte at 37 degrees C resulted in faster hybridization kinetics without sacrificing specificity. Other methods aimed to improve CP rearrangement kinetics such as bath sonication and surfactant additions were ineffective. We also determined that the target sequence choice is important as secondary structure formation in the target is negatively correlated with hybridization. Best performing CPs showed detection limits of 11 and 13 nM for DNA and RNA targets, respectively. Finally, we simulated sensing conditions using the saliva environment, showing sensor compatibility in biofluids. In total, this work elucidates key design features and processing to enable sequence-specific hybridization on ssDNA-SWCNT CPs.

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.

Electromechanical RT-LAMP device for portable SARS-CoV-2 detection.

Rapid point-of-care tests for infectious diseases are essential, especially in pandemic conditions. We have developed a point-of-care electromechanical device to detect SARS-CoV-2 viral RNA using the reverse-transcription loop-mediated isothermal amplification (RT-LAMP) principle. The developed device can detect SARS-CoV-2 viral RNA down to 103 copies/mL and from a low amount of sample volumes (2 µL) in less than an hour of standalone operation without the need for professional labor and equipment. Integrated Peltier elements in the device keep the sample at a constant temperature, and an integrated camera allows automated monitoring of LAMP reaction in a stirring sample by using colorimetric analysis of unfocused sample images in the hue/saturation/value color space. This palm-fitting, portable and low-cost device does not require a fully focused sample image for analysis, and the operation could be stopped automatically through image analysis when the positive test results are obtained. Hence, viral infections can be detected with the portable device produced without the need for long, expensive, and labor-intensive tests and equipment, which can make the viral tests disseminated at the point-of-care.

Nucleic acid amplification with specific signal filtration and magnification for ultrasensitive colorimetric detection.

Recently, sensitive, fast and low cost nucleic acid isothermal amplification technologies (such as loop-mediated isothermal amplification, LAMP) have attracted great attention in the urgent needs of point-of-care testing (POCT) and regular epidemic prevention and control. However, unlike PCR which usually employs TaqMan probe to report specific signals, specific-signal-output strategies in isothermal amplification are immature and visual detection even rare, which limits their popularity in POCT. We hypothesize to address this issue by designing a visual-signal-report system to both filtrate and magnify the target information in isothermal amplification. In this work, we developed a specific signal filtration and magnification colorimetric isothermal sensing platform (SFMC for short) for ultrasensitive detection of DNA and RNA. SFMC consists of two processes: an isothermal amplification with specific signal filtration and a self-replication catalyzed hairpin assembly (SRCHA) for rapid target-specific signal magnification and outputting. With these unique properties, this biosensing platform could detect target DNA as low as 5 copies per reaction and target RNA as low as 10 copies per reaction by naked eyes. Benefited from the excellent colorimetric detection performance, this biosensing platform has been successfully used for African swine fever virus (ASFV) and SARS-CoV-2 detection.

Cysteine-Encapsulated Liposome for Investigating Biomolecular Interactions at Lipid Membranes.

The development of a strategy to investigate interfacial phenomena at lipid membranes is practically useful because most essential biomolecular interactions occur at cell membranes. In this study, a colorimetric method based on cysteine-encapsulated liposomes was examined using gold nanoparticles as a probe to provide a platform to report an enzymatic activity at lipid membranes. The cysteine-encapsulated liposomes were prepared with varying ratios of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol through the hydration of lipid films and extrusions in the presence of cysteine. The size, composition, and stability of resulting liposomes were analyzed by scanning electron microscopy (SEM), dynamic light scattering (DLS), nuclear magnetic resonance (NMR) spectroscopy, and UV-vis spectrophotometry. The results showed that the increased cholesterol content improved the stability of liposomes, and the liposomes were formulated with 60 mol % cholesterol for the subsequent experiments. Triton X-100 was tested to disrupt the lipid membranes to release the encapsulated cysteine from the liposomes. Cysteine can induce the aggregation of gold nanoparticles accompanying a color change, and the colorimetric response of gold nanoparticles to the released cysteine was investigated in various media. Except in buffer solutions at around pH 5, the cysteine-encapsulated liposomes showed the color change of gold nanoparticles only after being incubated with Triton X-100. Finally, the cysteine-encapsulated liposomal platform was tested to report the enzymatic activity of phospholipase A2 that hydrolyzes phospholipids in the membrane. The hydrolysis of phospholipids triggered the release of cysteine from the liposomes, and the released cysteine was successfully detected by monitoring the distinct red-to-blue color change of gold nanoparticles. The presence of phospholipase A2 was also confirmed by the appearance of a peak around 690 nm in the UV-vis spectra, which is caused by the cysteine-induced aggregation of gold nanoparticles. The results demonstrated that the cysteine-encapsulated liposome has the potential to be used to investigate biological interactions occurring at lipid membranes.

A novel delayed lateral flow immunoassay for enhanced detection of SARS-CoV-2 spike antigen.

A new detection strategy was developed to improve the sensitivity of a lateral flow immunoassay platform utilizing a delayed hydrophobic barrier fabricated with trimethylsilyl cellulose (TMSC). The SARS-CoV-2 spike receptor-binding domain (SARS-CoV-2 SP RBD) antigen was chosen as a model analyte to demonstrate the superior detectability of this scheme. The novel device consists of 2 separate layers, so-called delayed lateral flow immunoassay (d-LFIA). The upper layer is intended for the analyte or sample flow path, where the test solution flows freely straight to the detection zone to bind with the primary antibody. The lower layer, located just underneath, is designed for the SARS-CoV-2 spike receptor-binding domain-conjugated gold nanoparticles (SARS-CoV-2 SP RBD-AuNPs) used for producing a colorimetric signal. This layer is fabricated with a TMSC barrier to time-delay the movement of SARS-CoV-2 SP RBD-AuNPs, thus allowing the antigen to bind with the primary antibody more efficiently. This platform exhibited a 2.6-fold enhancement in the sensitivity and 9.1-fold improvement in the limit of detection (LOD) as compared with the conventional LFIA. In addition, this d-LFIA device was satisfactorily applied to accurate screening of COVID-19 patients.

Colorimetric detection of viral RNA fragments based on an integrated logic-operated three-dimensional DNA walker.

Timely and accurate detection of virus is crucial for preventing spread of disease and early treatment of the infected cases. Herein we design an integrated logic-operated three-dimensional DNA walker for colorimetric detection of viral RNA fragments, by taking SARS-CoV-2 as an example. The DNA walker is composed of small amounts of dually-blocked walking strands and large amounts of dual-stem-loop track strands on gold nanoparticles. The walking strand contains a swing arm domain and a DNAzyme domain blocked at both sides of catalytic core, while the track strand contains a substrate domain located at the peripheral larger loop. Only the presence of both ORF1ab and N RNA fragments can fully de-block the walking strand, which then continuously hybridizes with track strands and cleaves them by DNAzyme-catalyzed hydrolysis. As the cleavage of track strands from long-stranded, double stem-loop structure to short-stranded, linear sequence, the DNA walker shows much lowered stability due to decreased negative charge density and diminished steric repulsion, which then gets aggregated at high salt concentration, accompanied by a visible color change. The colorimetric DNA walker detects RNA fragments down to 1 nM, responds dual viral genes in a "AND" logic way, and shows high specificity to target sequence. It can further detect large nucleic acids containing ORF1ab and N sequences, and reach 200 copies/mL detection limit by coupling a simple upstream amplification of sample. The method may provide a convenient way for reliable detection of viral RNA.

VALVE-ENABLED SEQUENTIAL REAGENT DELIVERY AND PAPER-BASED ENRICHMENT FOR SIMULTANEOUS DETECTION OF SARS-COV-2 AND INFLUENZA VIRUSES

We report a point-of-care (POC) testing platform for simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus. The POC device integrates sample preparation using ball-based valves for sequential delivery of reagents, viral RNA isolation and enrichment by paper-based filtration, with reverse transcription loop-mediated isothermal amplification (RT-LAMP) and colorimetric detection. The device is capable of detecting both viruses, showing high sensitivity and specificity. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

Visual diagnosis of COVID-19 disease based on serum metabolites using a paper-based electronic tongue.

This study aims to use a paper-based sensor array for point-of-care detection of COVID-19 diseases. Various chemical compounds such as nanoparticles, organic dyes and metal ion complexes were employed as sensing elements in the array fabrication, capturing the metabolites of human serum samples. The viral infection caused the type and concentration of serum compositions to change, resulting in different color responses for the infected and control samples. For this purpose, 118 serum samples of COVID-19 patients and non-COVID controls both men and women with the age range of 14-88 years were collected. The serum samples were initially subjected to the sensor, followed by monitoring the variation in the color of sensing elements for 5 min using a scanner. By taking into consideration the statistical information, this method was capable of discriminating COVID-19 patients and control samples with 83.0% accuracy. The variation of age did not influence the colorimetric patterns. The desirable correlation was observed between the sensor responses and viral load values calculated by the PCR test, proposing a rapid and facile way to estimate the disease severity. Compared to other rapid detection methods, the developed assay is cost-effective and user-friendly, allowing for screening COVID-19 diseases reliably.

Smartphone-Based Point-of- Care Testing

The outbreak of the COVID-19 has increased the demand for point-of-care testing ( POCT), and as the most indispensable tools for human beings at present, smartphones have great application potential in POCT. Smartphone-based POCT has the following unique advantages: ( 1) easy to operate and without the need for professional training;( 2) shorter wait times and quicker test results;( 3) low fabrication cost and convenient to use in limited-resource areas. Therefore, smartphone-based POCT is rapidly emerging as a potential alternative to traditional laboratory testing. Herein, we perform a comprehensive review of recent progress and applications of smartphone-based sensors in POCT for the past three years, which uses the tested objects ( body fluids, volatile organic compounds, vital signs) by POCT as the basis for classification, and combines with the current mainstream sensing strategies, including colorimetric, fluorescent, electrochemical technology, piezoelectric, pyroelectric, ultrasonic and photoelectric sensor, etc. We evaluate the performance and development potential of these sensors, in addition, the emerging technologies used in POCT are introduced, such as nanotechnology, flexible electronic devices, microfluidic technology, biodegradable technology, self- powered technology, multichannel detection and so on. Finally, current problems are summarized and the future development of the smartphone-based POCT is discussed.

Non-invasive detection of COVID-19 using a microfluidic-based colorimetric sensor array sensitive to urinary metabolites.

A colorimetric sensor array designed on a paper substrate with a microfluidic structure has been developed. This array is capable of detecting COVID-19 disease by tracking metabolites of urine samples. In order to determine minor metabolic changes, various colorimetric receptors consisting of gold and silver nanoparticles, metalloporphyrins, metal ion complexes, and pH-sensitive indicators are used in the array structure. By injecting a small volume of the urine sample, the color pattern of the sensor changes after 7 min, which can be observed visually. The color changes of the receptors (recorded by a scanner) are subsequently calculated by image analysis software and displayed as a color difference map. This study has been performed on 130 volunteers, including 60 patients infected by COVID-19, 55 healthy controls, and 15 cured individuals. The resulting array provides a fingerprint response for each category due to the differences in the metabolic profile of the urine sample. The principal component analysis-discriminant analysis confirms that the assay sensitivity to the correctly detected patient, healthy, and cured participants is equal to 73.3%, 74.5%, and 66.6%, respectively. Apart from COVID-19, other diseases such as chronic kidney disease, liver disorder, and diabetes may be detectable by the proposed sensor. However, this performance of the sensor must be tested in the studies with a larger sample size. These results show the possible feasibility of the sensor as a suitable alternative to costly and time-consuming standard methods for rapid detection and control of viral and bacterial infectious diseases and metabolic disorders.

Nanomolecular Magnetic Probe for Colorimetric RT-LAMP of Viral RNA

Amplification of the RNA from the Covid-19 virus is considered the main objective of the molecular diagnosis of SARS-Cov-2. However, the use of target-based amplification methods such as polymerase chain reaction requires a step to convert the RNA of the Covid-19 virus into a DNA template to lead to amplification. In addition, isolating the RNA of the Covid-19 virus requires RNA purification kits, which will increase the time and costs of molecular detection of this virus. In this study, the magnetic nanoprobe is introduced that it could capture and amplify Covid-19 RNA through an isothermal amplification process called loop-mediated isothermal amplification without requiring a step to convert the viral RNA into a DNA template. By using the engineered sequences corresponding to the target nucleic acid attached to magnetic nanoparticles, it becomes possible to identify the target RNA of this virus through color changes due to pH changes that can be seen with the naked eye due to the presence of pH indicators in the reaction mix. According to the isothermal amplification of the viral RNA via LAMP assisted with the magnetic nanoprobe, the nanomolecular method eliminated the need for special equipment and the time for detecting Covid-19 in specimens. © 2022 by the authors.