scholarly journals Comparative Study of Gold and Carbon Nanoparticles in Nucleic Acid Lateral Flow Assay

Nanomaterials ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 741
Author(s):  
Juan Carlos Porras ◽  
Mireia Bernuz ◽  
Jennifer Marfa ◽  
Arnau Pallares-Rusiñol ◽  
Mercè Martí ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Comparative Study ◽  
Carbon Nanoparticles ◽  
Limit Of Detection ◽  
Early Stage ◽  
High Sensitivity ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Complex Samples ◽  
Increased Sensitivity

A lateral flow assay (LFA) is a paper-based, point-of-need test designed to detect a specific analyte in complex samples in low-resource settings. Although LFA has been successfully used in different applications, its use is still limited when high sensitivity is required, especially in the diagnosis of an early-stage condition. The limit of detection (LOD) is clearly related to the signal-generating system used to achieve the visual readout, in many cases involving nanoparticles coupled to a biomolecule, which, when combined, provides sensitivity and specificity, respectively. While colloidal gold is currently the most-used label, other detection systems are being developed. Carbon nanoparticles (CNPs) demonstrate outstanding features to improve the sensitivity of this technology by producing an increased contrast in the paper background. Based on the necessity of sensitivity improvement, the aim of this work is a comparative study, in terms of analytical performance, between commercial streptavidin gold nanoparticles (streptAv-AuNPs) and avidin carbon nanoparticles (Av-CNPs) in a nucleic acid lateral flow assay. The visual LOD of the method was calculated by serial dilution of the DNA template, ranging from 0.0 to 7 pg μL−1/1.5 × 104 CFU mL−1). The LFA achieved visual detection of as low as 2.2 × 10−2 pg μL−1 using Av-CNPs and 8.4 × 10−2 pg μL−1 using streptAv-AuNPs. These LODs could be obtained without the assistance of any instrumentation. The results demonstrate that CNPs showed an increased sensitivity, achieving the nanomolar range even by visual inspection. Furthermore, CNPs are the cheapest labels, and the suspensions are very stable and easy to modify.

scholarly journals One-Tube SARS-CoV-2 Detection Platform Based on RT-RPA and CRISPR/Cas12a

2020 ◽  
Author(s):  
Yangyang Sun ◽  
Lei Yu ◽  
Chengxi Liu ◽  
Wei Chen ◽  
Dechang Li ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Limit Of Detection ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
N Gene ◽  
Clinical Samples ◽  
Nucleic Acid Detection ◽  
Detection Process ◽  
Human Coronaviruses ◽  
Negative Controls

Abstract Background: COVID-19 has spread rapidly around the world, affecting almost every person. When lifting certain mandatory measures for an economic restart, robust surveillance must be established and implemented, with nucleic acid detection for SARS-CoV-2 as an essential component. Methods: We designed RT-RPA (Reverse Transcription and Recombinase Polymerase Isothermal Amplification) primers of RdRp gene and N gene according to the SARS-CoV-2 gene sequence. We optimized the components in the reaction so that the detection process could be carried out in one tube. The specificity was demonstrated through detecting nucleic acid samples from seven human coronaviruses. Clinical samples were used to validate the platform and all results were compared to rRT-PCR. RNA standards diluted by different gradients were used to demonstrate the limit of detection. Furthermore, we have developed a lateral flow assay based on OR-DETECTR for the detection of COVID-19. Results: We have developed a o ne-tube detection platform based on R T- R PA and DNA Endonuclease-Targeted CRISPR Trans Reporter ( DETECTR ) technology, termed OR-DETECTR, to detect SARS-CoV-2. The detection process is completed in one tube, and the time is 50min. The method can specifically detect SARS-CoV-2 from seven human coronaviruses with a low detection limit of 2.5 copies/µl input. Results from six SARS-CoV-2 patient samples, eight samples from patients with fever but no SARS-CoV-2 infection, and one mixed sample from 40 negative controls showed that OR-DETECTR is 100% consistent with rRT-PCR. Furthermore, we have developed a lateral flow assay based on OR-DETECTR for the detection of COVID-19. Conclusions: OR-DETECTR detection platform is rapid, accurate, tube closed, easy-to-operate, and free of large instruments for COVID-19 detection.

A Point of Care Lateral Flow Assay for Rapid and Colorimetric Detection of Interleukin 6 and Perspectives in Bedside Diagnostics

2020 ◽  
Author(s):  
Carla Eiras
Keyword(s):  
Interleukin 6 ◽  
Limit Of Detection ◽  
Inflammatory Diseases ◽  
Point Of Care ◽  
Colorimetric Detection ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Aggregation Assay ◽  
Before And After ◽  
Bedside Diagnosis

Interleukin-6 (IL-6) is a multifunctional cytokine and high bloodstream levels of which have been associated with severe inflammatory diseases, such as dengue fever, sepsis, various cancers, and visceral leishmaniasis (VL). Rapid tests for the quantification of IL-6 would be of great assistance for the bedside diagnosis and treatment of diseases such as VL. We have developed a lateral flow assay (LFA) for rapid and colorimetric IL-6 detection, consisting of anti-IL-6 antibodies conjugated to gold nanoparticles (AuNPs). The optimal concentration of anti-IL-6 used in the conjugate was determined to be 800.0 μg/mL, based on an aggregation assay using LFA. A linear relationship between IL-6 standard concentration and color intensity was observed after 20 min, with a linear range between 1.25 ng/mL and 9,000 ng/mL. The limit of detection for this method was estimated a t0.38 ng/mL. The concentration of IL-6 in five patients with severe VL was measured using LFA, and the results were consistent with those obtained using the cytometric bead array (CBA) method. A thorough analysis of the LFA membranes’ surface morphology, before and after sample contact, was performed using atomic force microscopy (AFM).The prototype described here is still being tested and improved, but this LFA will undoubtedly be of great help in the clinical quantification of IL-6.

A Point of Care Lateral Flow Assay for Rapid and Colorimetric Detection of Interleukin 6 and Perspectives in Bedside Diagnostics

2020 ◽  
Author(s):  
Carla Eiras
Keyword(s):  
Interleukin 6 ◽  
Limit Of Detection ◽  
Inflammatory Diseases ◽  
Point Of Care ◽  
Colorimetric Detection ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Aggregation Assay ◽  
Before And After ◽  
Bedside Diagnosis

Interleukin-6 (IL-6) is a multifunctional cytokine and high bloodstream levels of which have been associated with severe inflammatory diseases, such as dengue fever, sepsis, various cancers, and visceral leishmaniasis (VL). Rapid tests for the quantification of IL-6 would be of great assistance for the bedside diagnosis and treatment of diseases such as VL. We have developed a lateral flow assay (LFA) for rapid and colorimetric IL-6 detection, consisting of anti-IL-6 antibodies conjugated to gold nanoparticles (AuNPs). The optimal concentration of anti-IL-6 used in the conjugate was determined to be 800.0 μg/mL, based on an aggregation assay using LFA. A linear relationship between IL-6 standard concentration and color intensity was observed after 20 min, with a linear range between 1.25 ng/mL and 9,000 ng/mL. The limit of detection for this method was estimated at a t0.38 ng/mL. The concentration of IL-6 in five patients with severe VL was measured using LFA, and the results were consistent with those obtained using the cytometric bead array (CBA) method. A thorough analysis of the LFA membranes’ surface morphology, before and after sample contact, was performed using atomic force microscopy (AFM). The prototype described here is still being tested and improved, but this LFA will undoubtedly be of great help in the clinical quantification of IL-6.

scholarly journals Rational Programming of Cas12a for Early-Stage Detection of COVID-19 by Lateral Flow Assay and Portable Real-Time Fluorescence Readout Facilities

Biosensors ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 11
Author(s):  
Zhijian Yi ◽  
Jean de Dieu Habimana ◽  
Omar Mukama ◽  
Zhiyuan Li ◽  
Nelson Odiwuor ◽  
...  
Keyword(s):  
Real Time ◽  
Early Stage ◽  
Limited Resource ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Capture Probe ◽  
Fluorescence Detector ◽  
Lateral Flow Strip ◽  
Global Pandemic ◽  
Novel Approach

Coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus has led to a global pandemic with a high spread rate and pathogenicity. Thus, with limited testing solutions, it is imperative to develop early-stage diagnostics for rapid and accurate detection of SARS-CoV-2 to contain the rapid transmission of the ongoing COVID-19 pandemic. In this regard, there remains little knowledge about the integration of the CRISPR collateral cleavage mechanism in the lateral flow assay and fluorophotometer. In the current study, we demonstrate a CRISPR/Cas12a-based collateral cleavage method for COVID-19 diagnosis using the Cas12a/crRNA complex for target recognition, reverse transcription loop-mediated isothermal amplification (RT-LAMP) for sensitivity enhancement, and a novel DNA capture probe-based lateral flow strip (LFS) or real-time fluorescence detector as the parallel system readout facility, termed CRICOLAP. Our novel approach uses a customized reporter that hybridizes an optimized complementary capture probe fixed at the test line for naked-eye result readout. The CRICOLAP system achieved ultra-sensitivity of 1 copy/µL in ~32 min by portable real-time fluorescence detection and ~60 min by LFS. Furthermore, CRICOLAP validation using 60 clinical nasopharyngeal samples previously verified with a commercial RT-PCR kit showed 97.5% and 100% sensitivity for S and N genes, respectively, and 100% specificity for both genes of SARS-CoV-2. CRICOLAP advances the CRISPR/Cas12a collateral cleavage result readout in the lateral flow assay and fluorophotometer, and it can be an alternative method for the decentralized field-deployable diagnosis of COVID-19 in remote and limited-resource locations.

scholarly journals 1832. Development of an Ultrasensitive Field-Applicable Plasmodium falciparum Assay for Malaria Diagnosis and Eradication

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S42-S43
Author(s):  
Rose Lee ◽  
Helena De Puig Guixe ◽  
Jeffrey Dvorin ◽  
James Collins
Keyword(s):  
Plasmodium Falciparum ◽  
Nucleic Acid ◽  
Genomic Dna ◽  
Limit Of Detection ◽  
Malaria Diagnosis ◽  
High Sensitivity ◽  
Nucleic Acid Detection ◽  
One Pot ◽  
Minimal Sample ◽  
Isothermal Assay

Abstract Background Malaria control and eradication have been hampered by asymptomatic carriage which serves as a parasite reservoir. Low-density infections (< 100 parasites/microliter) frequently fall below the limit of detection (LOD) of microscopy and rapid diagnostic tests (RDT) which are antigen-based tests. Molecular methods such as polymerase chain reaction are capable of higher sensitivity yet remain impractical for resource-limited settings. We describe development of an isothermal assay using the nucleic acid detection platform SHERLOCK (Specific High-Sensitivity Enzymatic Reporter UnLOCKing), which may also be increasingly important as there has been rising detection of histidine-rich protein 2 (HRP2) gene deletions in Plasmodium spp. HRP2 is the most commonly used antigen in RDTs and deletion of this gene would render many RDTs obsolete. Methods SHERLOCK leverages the endonucleases of CRISPR-associated microbial adaptive immunity. Cas12a is an RNA-guided, DNA-cleaving enzyme, which can be programmed with guide RNAs to cleave nontarget reporter ssDNA. We exploit the nonspecific degradation of labeled ssDNA to detect the presence of the dsDNA target that activated Cas12a (Figure 1). Recombinase polymerase amplification (RPA) coupled with Cas12a detection enables a lower LOD. Plasmodium falciparum whole genomic DNA was compared with parasites cultured in red blood cells (RBCs) with known parasitemia and boiled at 95°C for 5 minutes for lysis of RBCs/parasites then diluted 1:2.5 to prevent solidification. Results This SHERLOCK assay detected simulated Plasmodium falciparum infection at attomolar LODs when applied to whole genomic DNA and simulated samples of infected RBCs spiked into whole blood. The genomic assay detected down to 0.2 parasites/microliter and the simulated sample detected to 10 parasites/microliter in the final reaction volume. In comparison, LODs from the initial input volume was 5aM and 250aM, respectively (Figure 2). Conclusion We demonstrate an isothermal nucleic acid detection platform capable of diagnosis in 60 minutes in a one-pot assay requiring minimal sample preparation and reaching an LOD recommended by the WHO for malaria eradication. In summary, we illustrate the utility of the SHERLOCK platform in application to malaria and global health. Disclosures All Authors: No reported Disclosures.

scholarly journals CRISPR/Cas13a powered electrochemical microfluidic biosensor for nucleic acid amplification-free miRNA diagnostics

2019 ◽  
Author(s):  
Richard Bruch ◽  
Julia Baaske ◽  
Claire Chatelle ◽  
Mailin Meirich ◽  
Sibylle Madlener ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Limit Of Detection ◽  
Early Stage ◽  
Low Cost ◽  
Clinical Diagnostics ◽  
Nucleic Acid Amplification ◽  
Process Time ◽  
Serum Samples ◽  
Microfluidic Biosensor ◽  
Pcr Method

Non-coding small RNAs, such as microRNAs, are becoming the biomarkers of choice for multiple diseases in clinical diagnostics. A dysregulation of these microRNAs can be associated to many different diseases, such as cancer, dementia or cardiovascular conditions. The key for an effective treatment is an accurate initial diagnosis at an early stage, improving the patient’s survival chances. Here, we introduce a CRISPR/Cas13a powered microfluidic, integrated electrochemical biosensor for the on-site detection of microRNAs. Through this unique combination, the quantification of the potential tumor markers microRNA miR-19b and miR-20a has been realized without any nucleic acid amplification. With a readout time of 9 minutes and an overall process time of less than 4 hours, a limit of detection of 10 pM was achieved, using a measuring volume of less than 0.6 µl. Furthermore, we demonstrate the feasibility of our versatile sensor platform to detect miR-19b in serum samples of children, suffering from brain cancer. The validation of our results with a standard qRT-PCR method shows the ability of our system to be a low-cost and target amplification-free tool for nucleic acid based diagnostics.

scholarly journals Engineered CRISPR/Cas12a Enables Rapid SARS-CoV-2 Detection

2020 ◽  
Author(s):  
Long T. Nguyen ◽  
Santosh R. Rananaware ◽  
Brianna L.M. Pizzano ◽  
Brandon T. Stone ◽  
Piyush K. Jain
Keyword(s):  
High Sensitivity ◽  
High Specificity ◽  
Single Copy ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Magnesium Concentration ◽  
Clinical Validation ◽  
Respiratory Pathogens ◽  
Reporter Assay ◽  
Wide Range

ABSTRACTThe coronavirus disease (COVID-19) caused by SARS-CoV-2 has swept through the globe at an unprecedented rate. CRISPR-based detection technologies such as DETECTR, SHERLOCK, and STOPCovid have emerged as a rapid and affordable platform that can shape the future of diagnostics. Recently, we reported engineered crRNAs for Cas12a, called ENHANCE, that enables enhanced detection of nucleic acids. Here we report development, clinical validation, and advancement of ENHANCE platform for detecting SARS-CoV-2. With an RT-LAMP pre-amplification step, ENHANCE detects samples down to a single copy with 95% accuracy and shows high specificity towards various isolates of SARS-CoV-2 against 31 highly similar and common respiratory pathogens. Utilizing LbCas12a-mediated trans-cleavage activity, ENHANCE works robustly in a wide range of magnesium concentration (3 mM-13 mM), allowing for further assay optimization. Additionally, ENHANCEv2 is developed to further improve the previously reported ENHANCE. ENHANCEv2 employs mutated LbCas12aD156R, engineered chimeric DNA-extended crRNA, and a dual reporter for both fluorescence-based reporter assay and lateral flow assay. Both ENHANCE and ENHANCEv2 are validated in 62 clinical nasopharyngeal swabs, showing 60/62 (96.7%) agreement with RT-qPCR results, and using only 5 μL of sample and 20 minutes of CRISPR reaction. Lateral flow assay on paper strips displays 100% agreement with fluorescence-based reporter assay in the clinical validation. Following a 30-minute pre-amplification RT-LAMP step, the lyophilized ENHANCEv2 is shown to achieve high sensitivity and specificity while reducing CRISPR reaction time to as low as 3 minutes and maintaining its detection capability upon storage at room temperature for several weeks.

scholarly journals A Novel Lateral Flow Assay for Rapid and Sensitive Nucleic Acid Detection of Avibacterium paragallinarum

2021 ◽  
Vol 8 ◽  
Author(s):  
Caiyun Huo ◽  
Donghai Li ◽  
Zhenguo Hu ◽  
Guiping Li ◽  
Yanxin Hu ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Gel Electrophoresis ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Nucleic Acid Hybridization ◽  
Nucleic Acid Detection ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Infectious Coryza ◽  
Avibacterium Paragallinarum

Avibacterium paragallinarum, the pathogen of infectious coryza, caused a highly contagious respiratory disease that poses a serious threat to chickens. Hence, it is necessary to do diagnostic screening for Av. paragallinarum. Existing technologies have been used for Av. paragallinarum testing, which, however, have some drawbacks such as time consuming and expensive that require well-trained personnel and sophisticated infrastructure, especially when they are limitedly feasible in some places for lack of resources. Nucleic acid hybridization-based lateral flow assay (LFA) is capable of dealing with these drawbacks, which is attributed to the advantages, such low cost, rapid, and simple. However, nucleic acid determination of Av. paragallinarum through LFA method has not been reported so far. In this study, we developed a novel LFA method that employed gold nanoparticle probes to detect amplified Av. paragallinarum dsDNA. Compared with agarose gel electrophoresis, this LFA strip was inexpensive, simple- to- use, and time- saving, which displayed the visual results within 5–8 min. This LFA strip had higher sensitivity that achieved the detection limit of 101 CFU/ml compared with 102 CFU/ml in agarose gel electrophoresis. Besides, great sensitivity was also shown in the LFA strip, and no cross reaction existed for other bacteria. Furthermore, Av. paragallinarum in clinical chickens with infectious coryza were perfectly detected by our established LFA strip. Our study is the first to develop the LFA integrated with amplification and sample preparation techniques for better nucleic acid detection of Av. paragallinarum, which holds great potential for rapid, accurate, and on-site determination methods for early diagnosis of Av. paragallinarum to control further spreading.

scholarly journals Engineered CRISPR/Cas12a Enables Rapid SARS-CoV-2 Detection

2021 ◽  
Author(s):  
Long Nguyen ◽  
Santosh Rananaware ◽  
Brianna Pizzano ◽  
Brandon Stone ◽  
Piyush Jain
Keyword(s):  
High Sensitivity ◽  
High Specificity ◽  
Single Copy ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Magnesium Concentration ◽  
Clinical Validation ◽  
Respiratory Pathogens ◽  
Reporter Assay ◽  
Wide Range

Abstract The coronavirus disease (COVID-19) caused by SARS-CoV-2 has swept through the globe at an unprecedented rate. CRISPR-based detection technologies such as DETECTR, SHERLOCK, and others have emerged as a rapid and affordable platform that can shape the future of diagnostics. Recently, we reported engineered crRNAs for Cas12a, called ENHANCE, that enables enhanced detection of nucleic acids. Here we report development, clinical validation, and advancement of ENHANCE platform for detecting SARS-CoV-2. With an RT-LAMP pre-amplification step, ENHANCE detects samples down to a single copy with 95% accuracy and shows high specificity towards various isolates of SARS-CoV-2 against 31 highly similar and common respiratory pathogens. Utilizing LbCas12a-mediated trans-cleavage activity, ENHANCE works robustly in a wide range of magnesium concentration (3 mM-13 mM), allowing for further assay optimization. Additionally, ENHANCEv2 is developed to further improve the previously reported ENHANCE. ENHANCEv2 employs mutated LbCas12aD156R, engineered chimeric DNA-extended crRNA, and a dual reporter for both fluorescence-based reporter assay and lateral flow assay. Both ENHANCE and ENHANCEv2 are validated in 62 clinical nasopharyngeal swabs, showing 60/62 (96.7%) agreement with RT-qPCR results, and using only 5 µL of sample and 20 minutes of CRISPR reaction. Lateral flow assay on paper strips displays 100% agreement with fluorescence-based reporter assay in the clinical validation. Following a 30-minute pre-amplification RT-LAMP step, the lyophilized ENHANCEv2 is shown to achieve high sensitivity and specificity while reducing CRISPR reaction time to as low as 3 minutes and maintaining its detection capability upon storage at room temperature for several weeks.

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