CRISPR/Cas13-Based Approaches for Ultrasensitive and Specific Detection of microRNAs

MicroRNAs (miRNAs) have a prominent role in virtually every aspect of cell biology. Due to the small size of mature miRNAs, the high degree of similarity between miRNA family members, and the low abundance of miRNAs in body fluids, miRNA expression profiling is technically challenging. Biosensors based on electrochemical detection for nucleic acids are a novel category of inexpensive and very sensitive diagnostic tools. On the other hand, after recognizing the target sequence, specific CRISPR-associated proteins, including orthologues of Cas12, Cas13, and Cas14, exhibit collateral nonspecific catalytic activities that can be employed for specific and ultrasensitive nucleic acid detection from clinically relevant samples. Recently, several platforms have been developed, connecting the benefits of enzyme-assisted signal amplification and enzyme-free amplification biosensing technologies with CRISPR-based approaches for miRNA detection. Together, they provide high sensitivity, precision, and fewer limitations in diagnosis through efficient sensors at a low cost and a simple miniaturized readout. This review provides an overview of several CRISPR-based biosensing platforms that have been developed and successfully applied for ultrasensitive and specific miRNA detection.

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Development of an Integrated Instrument for Nucleic Acid Pyrosequencing Detection

Key Engineering Materials ◽

10.4028/www.scientific.net/kem.315-316.469 ◽

2006 ◽

Vol 315-316 ◽

pp. 469-473

Author(s):

Ji Jun Zhu ◽

H.N. Shi ◽

J. Cheng ◽

X.Y. Wei ◽

Zu Hong Lu

Keyword(s):

Nucleic Acid ◽

Personal Computer ◽

Low Cost ◽

High Sensitivity ◽

Structure Design ◽

Nucleic Acid Detection ◽

Data Sampling ◽

Sampling Device ◽

Automatic Instrument ◽

Injection Process

This paper introduces a kind of new homemade and low cost multi-channel nucleic acid pyrosequence detector for at least ninety-six channels and presents the detail of related software and hardware development. We construct a kind of automatic instrument to fulfill the pyrosequencing processes. First we select the X-86 personal computer as host computer, the AT89C51 micro-controller as slave computer, the PMT (photoelectric multiply tube) as photoelectric transformation equipment, and the HY-6022 as data sampling device; Second we use the Visual C++ 6.0 as coding tools to design the measure and control system based on Windows 2000 operating system; Third we sample the fluorescent signal in all of the cuvettes during the reaction between nucleic acid and reagent; Last we analyze these data to realize the function of the multi-channel nucleic acid detection. In this paper the whole instrument design and key parts design are both introduced such as the liquid injection process and related structure design, the communication module between the host personal computer and the MCS51, the high sensitivity multi-channel detector (at least 96 channels, the sensitivity is 2.45×10-9w) etc. The result of the instrument for two channels data processing is also reported in this paper.

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Rapid detection of SARS-CoV-2 with CRISPR-Cas12a

PLoS Biology ◽

10.1371/journal.pbio.3000978 ◽

2020 ◽

Vol 18 (12) ◽

pp. e3000978

Author(s):

Dan Xiong ◽

Wenjun Dai ◽

Jiaojiao Gong ◽

Guande Li ◽

Nansong Liu ◽

...

Keyword(s):

Detection Method ◽

Point Of Care ◽

High Sensitivity ◽

Nucleic Acid Detection ◽

Global Pandemic ◽

Portable Detection ◽

Recent Outbreak ◽

Associated Proteins ◽

Specialized Equipment ◽

Viral Diagnosis

The recent outbreak of betacoronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which is responsible for the Coronavirus Disease 2019 (COVID-19) global pandemic, has created great challenges in viral diagnosis. The existing methods for nucleic acid detection are of high sensitivity and specificity, but the need for complex sample manipulation and expensive machinery slow down the disease detection. Thus, there is an urgent demand to develop a rapid, inexpensive, and sensitive diagnostic test to aid point-of-care viral detection for disease monitoring. In this study, we developed a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated proteins (Cas) 12a-based diagnostic method that allows the results to be visualized by the naked eye. We also introduced a rapid sample processing method, and when combined with recombinase polymerase amplification (RPA), the sample to result can be achieved in 50 minutes with high sensitivity (1–10 copies per reaction). This accurate and portable detection method holds a great potential for COVID-19 control, especially in areas where specialized equipment is not available.

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NANOPOROUS MEMBRANE FOR BIOSENSING APPLICATIONS

Nano LIFE ◽

10.1142/s1793984411000323 ◽

2012 ◽

Vol 02 (01) ◽

pp. 1230003 ◽

Cited By ~ 10

Author(s):

YANG MO ◽

TAN FEI

Keyword(s):

Low Cost ◽

High Sensitivity ◽

Detection Methods ◽

Nucleic Acid Detection ◽

Nanoporous Membranes ◽

Inorganic Membranes ◽

Detection Techniques ◽

Nanoporous Membrane ◽

Surface Functionality ◽

Surface Modification Techniques

Synthetic nanoporous membranes have been used in numerous biosensing applications, such as glucose detection, nucleic acid detection, bacteria detection, and cell-based sensing. The increased surface affinity area and enhanced output sensing signals make the nanoporous membranes increasingly attractive as biosensing platforms. Surface modification techniques can be used to improve surface properties for realizable bioanalyte immobilization, conjugation, and detection. Combined with realizable detection techniques such as electrochemical and optical detection methods, nanoporous membrane–based biosensors have advantages, including rapid response, high sensitivity, and low cost. In this paper, an overview of nanoporous membranes for biosensing application is given. Types of nanoporous membranes including polymer membranes, inorganic membranes, membranes with nanopores fabricated using nanolithography, and nanotube-based membranes are introduced. The fabrication techniques of nanoporous membranes are also discussed. The key requirements of nanoporous membranes for biosensing applications include surface functionality for bioanalyte immobilization, biocompatibility, mechanical and chemical stability, and anti-biofouling capability. The recent advances and development of nanoporous membrane–based biosensors are discussed, especially for the sensing mechanism and surface functionalization strategies. Finally, the challenges and future development of nanoporous membrane for biosensing applications are discussed.

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Recent Advances in Cellulose-Based Biosensors for Medical Diagnosis

Biosensors ◽

10.3390/bios10060067 ◽

2020 ◽

Vol 10 (6) ◽

pp. 67 ◽

Cited By ~ 8

Author(s):

Samir Kamel ◽

Tawfik A. Khattab

Keyword(s):

Molecular Design ◽

Low Cost ◽

High Sensitivity ◽

Biologically Active ◽

Diagnostic Tools ◽

Cellulose Derivatives ◽

Label Free ◽

Cellulose Paper ◽

Recent Developments ◽

Nano Cellulose

Cellulose has attracted much interest, particularly in medical applications such as advanced biosensing devices. Cellulose could provide biosensors with enhanced biocompatibility, biodegradability and non-toxicity, which could be useful for biosensors. Thus, they play a significant role in environmental monitoring, medical diagnostic tools, forensic science, and foodstuff processing safety applications. This review summarizes the recent developments in cellulose-based biosensors targeting the molecular design principles toward medical detection purposes. The recognition/detection mechanisms of cellulose-based biosensors demonstrate two major classes of measurable signal generation, including optical and electrochemical cellulosic biosensors. As a result of their simplicity, high sensitivity, and low cost, cellulose-based optical biosensors are particularly of great interest for including label-free and label-driven (fluorescent and colorimetric) biosensors. There have been numerous types of cellulose substrates employed in biosensors, including several cellulose derivatives, nano-cellulose, bacterial cellulose, paper, gauzes, and hydrogels. These kinds of cellulose-based biosensors were discussed according to their preparation procedures and detection principle. Cellulose and its derivatives with their distinctive chemical structure have demonstrated to be versatile materials, affording a high-quality platform for accomplishing the immobilization process of biologically active molecules into biosensors. Cellulose-based biosensors exhibit a variety of desirable characteristics, such as sensitivity, accuracy, convenience, quick response, and low-cost. For instance, cellulose paper-based biosensors are characterized as being low-cost and easy to operate, while nano-cellulose biosensors are characterized as having a good dispersion, high absorbance capacity, and large surface area. Cellulose and its derivatives have been promising materials in biosensors which could be employed to monitor various bio-molecules, such as urea, glucose, cell, amino acid, protein, lactate, hydroquinone, gene, and cholesterol. The future interest will focus on the design and construction of multifunctional, miniaturized, low-cost, environmentally friendly, and integrated biosensors. Thus, the production of cellulose-based biosensors is very important.

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ASSESSMENT OF EFFICIENCY AND OFF-TARGET ACTIVITY OF CRISPR/CAS RIBONUCLEOPROTEIN COMPLEXES

Molecular Diagnostics and Biosafety – 2020. Russian national scientific and practical conference with international participation (October, 6–8, 2020): Conference Proceedings ◽

10.36233/978-5-9900432-9-9-98 ◽

2020 ◽

Author(s):

Y.V. Mikhaylova ◽

◽

M.A. Tyumentseva ◽

A.A. Shelenkov ◽

Y.G. Yanushevich ◽

...

Keyword(s):

High Sensitivity ◽

Correct Choice ◽

Target Sequence ◽

Dna Breaks ◽

Gene Encoding ◽

Guide Rna ◽

Guide Rnas ◽

Ribonucleoprotein Complexes ◽

Chemokine Receptor Ccr5 ◽

Target Activity

In this study, we assessed the efficiency and off-target activity of the CRISPR/CAS complex with one of the selected guide RNAs using the CIRCLE-seq technology. The gene encoding the human chemokine receptor CCR5 was used as a target sequence for genome editing. The results of this experiment indicate the correct choice of the guide RNA and efficient work of the CRISPR- CAS ribonucleoprotein complex used. CIRCLE-seq technology has shown high sensitivity compared to bioinformatic methods for predicting off-target activity of CRISPR/CAS complexes. We plan to evaluate the efficiency and off-target activity of CRISPR/CAS ribonucleoprotein complexes with other guide RNAs by slightly adjusting the CIRCLE-seq-technology protocol in order to reduce nonspecific DNA breaks and increase the number of reliable reads.

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A rapid, accurate, scalable, and portable testing system for COVID-19 diagnosis

Nature Communications ◽

10.1038/s41467-021-23185-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Guanhua Xun ◽

Stephan Thomas Lane ◽

Vassily Andrew Petrov ◽

Brandon Elliott Pepa ◽

Huimin Zhao

Keyword(s):

Limit Of Detection ◽

Low Cost ◽

High Volume ◽

N Gene ◽

Testing System ◽

Target Sequence ◽

One Pot ◽

Sequence Detection ◽

Highly Sensitive ◽

Speed Accuracy

AbstractThe need for rapid, accurate, and scalable testing systems for COVID-19 diagnosis is clear and urgent. Here, we report a rapid Scalable and Portable Testing (SPOT) system consisting of a rapid, highly sensitive, and accurate assay and a battery-powered portable device for COVID-19 diagnosis. The SPOT assay comprises a one-pot reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP) followed by PfAgo-based target sequence detection. It is capable of detecting the N gene and E gene in a multiplexed reaction with the limit of detection (LoD) of 0.44 copies/μL and 1.09 copies/μL, respectively, in SARS-CoV-2 virus-spiked saliva samples within 30 min. Moreover, the SPOT system is used to analyze 104 clinical saliva samples and identified 28/30 (93.3% sensitivity) SARS-CoV-2 positive samples (100% sensitivity if LoD is considered) and 73/74 (98.6% specificity) SARS-CoV-2 negative samples. This combination of speed, accuracy, sensitivity, and portability will enable high-volume, low-cost access to areas in need of urgent COVID-19 testing capabilities.

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Rapid and Sensitive Detection of miRNA Based on AC Electrokinetic Capacitive Sensing for Point-of-Care Applications

Sensors ◽

10.3390/s21123985 ◽

2021 ◽

Vol 21 (12) ◽

pp. 3985

Author(s):

Nan Wan ◽

Yu Jiang ◽

Jiamei Huang ◽

Rania Oueslati ◽

Shigetoshi Eda ◽

...

Keyword(s):

Limit Of Detection ◽

Point Of Care ◽

Low Cost ◽

Clinical Diagnostics ◽

Detection Methods ◽

Capacitive Sensing ◽

Application Potential ◽

Mirna Detection ◽

Mirna 25 ◽

Low Sensitivity

A sensitive and efficient method for microRNAs (miRNAs) detection is strongly desired by clinicians and, in recent years, the search for such a method has drawn much attention. There has been significant interest in using miRNA as biomarkers for multiple diseases and conditions in clinical diagnostics. Presently, most miRNA detection methods suffer from drawbacks, e.g., low sensitivity, long assay time, expensive equipment, trained personnel, or unsuitability for point-of-care. New methodologies are needed to overcome these limitations to allow rapid, sensitive, low-cost, easy-to-use, and portable methods for miRNA detection at the point of care. In this work, to overcome these shortcomings, we integrated capacitive sensing and alternating current electrokinetic effects to detect specific miRNA-16b molecules, as a model, with the limit of detection reaching 1.0 femto molar (fM) levels. The specificity of the sensor was verified by testing miRNA-25, which has the same length as miRNA-16b. The sensor we developed demonstrated significant improvements in sensitivity, response time and cost over other miRNA detection methods, and has application potential at point-of-care.

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Membrane-associated phase separation: organization and function emerge from a two-dimensional milieu

Journal of Molecular Cell Biology ◽

10.1093/jmcb/mjab010 ◽

2021 ◽

Author(s):

Jonathon A Ditlev

Keyword(s):

Phase Separation ◽

Cell Biology ◽

Cellular Environment ◽

Condensate Formation ◽

Associated Proteins ◽

Available Technology ◽

And Function ◽

Surrounding Membrane

Abstract Liquid‒liquid phase separation (LLPS) of biomolecules has emerged as an important mechanism that contributes to cellular organization. Phase separated biomolecular condensates, or membrane-less organelles, are compartments composed of specific biomolecules without a surrounding membrane in the nucleus and cytoplasm. LLPS also occurs at membranes, where both lipids and membrane-associated proteins can de-mix to form phase separated compartments. Investigation of these membrane-associated condensates using in vitro biochemical reconstitution and cell biology has provided key insights into the role of phase separation in membrane domain formation and function. However, these studies have generally been limited by available technology to study LLPS on model membranes and the complex cellular environment that regulates condensate formation, composition, and function. Here, I briefly review our current understanding of membrane-associated condensates, establish why LLPS can be advantageous for certain membrane-associated condensates, and offer a perspective for how these condensates may be studied in the future.

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Integrating high-performing electrochemical transducers in lateral flow assay

Analytical and Bioanalytical Chemistry ◽

10.1007/s00216-021-03301-y ◽

2021 ◽

Author(s):

Antonia Perju ◽

Nongnoot Wongkaew

Keyword(s):

High Performance ◽

Point Of Care ◽

Low Cost ◽

High Sensitivity ◽

Lateral Flow ◽

Analytical Performance ◽

Label Free ◽

High Performing ◽

Lateral Flow Assays ◽

Electrochemical Transducers

AbstractLateral flow assays (LFAs) are the best-performing and best-known point-of-care tests worldwide. Over the last decade, they have experienced an increasing interest by researchers towards improving their analytical performance while maintaining their robust assay platform. Commercially, visual and optical detection strategies dominate, but it is especially the research on integrating electrochemical (EC) approaches that may have a chance to significantly improve an LFA’s performance that is needed in order to detect analytes reliably at lower concentrations than currently possible. In fact, EC-LFAs offer advantages in terms of quantitative determination, low-cost, high sensitivity, and even simple, label-free strategies. Here, the various configurations of EC-LFAs published are summarized and critically evaluated. In short, most of them rely on applying conventional transducers, e.g., screen-printed electrode, to ensure reliability of the assay, and additional advances are afforded by the beneficial features of nanomaterials. It is predicted that these will be further implemented in EC-LFAs as high-performance transducers. Considering the low cost of point-of-care devices, it becomes even more important to also identify strategies that efficiently integrate nanomaterials into EC-LFAs in a high-throughput manner while maintaining their favorable analytical performance.

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Microfluidic Based Physical Approaches towards Single-Cell Intracellular Delivery and Analysis

Micromachines ◽

10.3390/mi12060631 ◽

2021 ◽

Vol 12 (6) ◽

pp. 631

Author(s):

Kiran Kaladharan ◽

Ashish Kumar ◽

Pallavi Gupta ◽

Kavitha Illath ◽

Tuhin Subhra Santra ◽

...

Keyword(s):

Single Cell ◽

Cell Biology ◽

Transfection Efficiency ◽

Intracellular Delivery ◽

Diagnostic Tools ◽

Delivery Methods ◽

Physical Methods ◽

Cell Measurement ◽

Therapeutic Development ◽

Single Living

The ability to deliver foreign molecules into a single living cell with high transfection efficiency and high cell viability is of great interest in cell biology for applications in therapeutic development, diagnostics, and drug delivery towards personalized medicine. Various physical delivery methods have long demonstrated the ability to deliver cargo molecules directly to the cytoplasm or nucleus and the mechanisms underlying most of the approaches have been extensively investigated. However, most of these techniques are bulk approaches that are cell-specific and have low throughput delivery. In comparison to bulk measurements, single-cell measurement technologies can provide a better understanding of the interactions among molecules, organelles, cells, and the microenvironment, which can aid in the development of therapeutics and diagnostic tools. To elucidate distinct responses during cell genetic modification, methods to achieve transfection at the single-cell level are of great interest. In recent years, single-cell technologies have become increasingly robust and accessible, although limitations exist. This review article aims to cover various microfluidic-based physical methods for single-cell intracellular delivery such as electroporation, mechanoporation, microinjection, sonoporation, optoporation, magnetoporation, and thermoporation and their analysis. The mechanisms of various physical methods, their applications, limitations, and prospects are also elaborated.

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