Engineering CRISPR/Cas-based nanosystems for therapeutics,diagnosis and bioimaging

CRISPR/Cas system has been utilized to rationally manipulate intracellular genes, and it has been engineered as versatile and efficient gene editing tools with precise site-specificity and excellent targeting ability for therapeutics, diagnostics, and bioimaging. Here, the evolution and application of CRISPR/Cas systems were sketched chronologically. Landmark works were exemplified to illustrate the design principles of CRISPR/Cas systems. Furthermore, the delivery vectors of CRISPR/Cas system especially DNA nanomaterials-based vectors were categorized and illuminated. DNA nanomaterials are suitable for CRISPR/Cas system delivery via base pairing due to its sequence programmability and biocompatibility. Then the applications of CRISPR/Cas in diagnosis and genomic imaging were highlighted. At the end of the review, the challenges and opportunities of CRISPR/Cas systems were deeply discussed. We envision that the grant advances on CRISPR/Cas systems will promote the development of interdisciplinary fields in chemistry, biology and medicine.     

CRISPR-Cas Biochemistry and CRISPR-Based Molecular Diagnostics

Polymerase chain reaction (PCR)-based nucleic acid testing has played a critical role in disease diagnostics, pathogen surveillance, and many more. However, this method requires a long turnaround time, expensive equipment, and trained personnel, limiting its widespread availability and diagnostic capacity. On the other hand, the clustered regularly interspaced short palindromic repeats (CRISPR) technology has recently demonstrated capability for nucleic acid detection with high sensitivity and specificity. CRISPR-mediated biosensing holds great promise for revolutionizing nucleic acid testing procedures and developing point-of-care diagnostics. This review focuses on recent developments in both fundamental CRISPR biochemistry and CRISPR-based nucleic acid detection techniques. Four ongoing research hotspots in molecular diagnostics-target preamplification-free detection, microRNA (miRNA) testing, non-nucleic-acid detection, and SARS-CoV-2 detection-are also covered.     

CRISPR-Cas12a System for Biosensing and Gene Regulation

Clustered regularly interspaced short palindromic repeats (CRISPR) is a promising technology in the biological world. As one of the CRISPR-associated (Cas) proteins, Cas12a is an RNA-guided nuclease in the type V CRISPR-Cas system, which has been a robust tool for gene editing. In addition, due to the discovery of target-binding-induced indiscriminate single-stranded DNase activity of Cas12a, CRISPR-Cas12a also exhibits great promise in biosensing. This minireview not only gives a brief introduction to the mechanism of CRISPR-Cas12a but also highlights the recent developments and applications in biosensing and gene regulation. Finally, future prospects of the CRISPR-Cas12a system are also discussed. We expect this minireview will inspire innovative work on the CRISPR-Cas12a system by making full use of its features and advantages.     

Exploring the Trans‐Cleavage Activity of CRISPR‐Cas12a (cpf1) for the Development of a Universal Electrochemical Biosensor

An accurate, rapid, and cost‐effective biosensor for the quantification of disease biomarkers is vital for the development of early‐diagnostic point‐of‐care systems. The recent discovery of the trans‐cleavage property of CRISPR type V effectors makes CRISPR a potential high‐accuracy bio‐recognition tool. Herein, a CRISPR‐Cas12a (cpf1) based electrochemical biosensor (E‐CRISPR) is reported, which is more cost‐effective and portable than optical‐transduction‐based biosensors. Through optimizing the in vitro trans‐cleavage activity of Cas12a, E‐CRIPSR was used to detect viral nucleic acids, including human papillomavirus 16 (HPV‐16) and parvovirus B19 (PB‐19), with a picomolar sensitivity. An aptamer‐based E‐CRISPR cascade was further designed for the detection of transforming growth factor β1 (TGF‐β1) protein in clinical samples. As demonstrated, E‐CRISPR could enable the development of portable, accurate, and cost‐effective point‐of‐care diagnostic systems.     

Recent Advances on Electrochemical Biosensing Strategies toward Universal Point‐of‐Care Systems

A number of very recently developed electrochemical biosensing strategies are promoting electrochemical biosensing systems into practical point‐of‐care applications. The focus of research endeavors has transferred from detection of a specific analyte to the development of general biosensing strategies that can be applied for a single category of analytes, such as nucleic acids, proteins, and cells. In this Minireview, recent cutting‐edge research on electrochemical biosensing strategies are described. These developments resolved critical challenges regarding the application of electrochemical biosensors to practical point‐of‐care systems, such as rapid readout, simple biosensor fabrication method, ultra‐high detection sensitivity, direct analysis in a complex biological matrix, and multiplexed target analysis. This Minireview provides general guidelines both for scientists in the biosensing research community and for the biosensor industry on development of point‐of‐care system, benefiting global healthcare.     

Nucleic Acid Integrated Technologies for Electrochemical Point-of-Care Diagnostics: A Comprehensive Review

The need for routine and immediate healthcare monitoring has inspired “near-patient testing” or in other words “point-of-care testing (POCT)”. Therefore, POCT can be defined as laboratory tests that are performed at the patient's bedside or in the immediate vicinity of the incident. Among many POCTs, nucleic acid-based testing has attracted enormous attention for the diagnosis of important genetic, inherited and infectious diseases such as cancer and coronavirus. In this review, we outline the integration of nucleic acids into the remarkable electrochemical point-of-care diagnostics including microfluidic, paper and smartphone-based approaches, CRISPR/Cas and liquid biopsy related systems and DNA damage monitoring.     

Biosensors in Wine Production Monitoring

ABSTRACT

An overview of probe-type and flow-through biosensors, together with biosensing systems, both batch and continuous biosensing systems, for monitoring typical species in wine processes is presented with the aim of showing the advantages and disadvantages involved in the use of each type of devices. Thus, biosensors and biosensing systems for the determination of ethanol (individual or together with that of other compounds), organic acids, glycerol, reducing sugars, acetaldehyde and sulfur dioxide/sulfite anions are reviewed and critically compared. The versatility and capability of continuous biosensing systems as compared with biosensors, particularly with probe-type biosensors working in a discontinuous, batch manner, is demonstrated.     

纳米材料在电化学生物传感器中的应用

纳米材料因其具有独特的性质,被广泛应用于研制和发展具有超高灵敏度、超高选择性的电化学生物传感器.本文总结了纳米材料在电化学生物传感系统中的主要功能,介绍了近年来国内外基于纳米材料构建的电化学生物传感器的研究进展,并对该领域的发展前景做出了展望.     

Highly Efficient and Rapid Detection of the Cleavage Activity of Cas9/gRNA via a Fluorescent Reporter

The RNA-guided endonuclease clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) derived from CRISPR systems is a simple and efficient genome-editing technology applied to various cell types and organisms. So far, the extensive approach to detect the cleavage activity of customized Cas9/guide RNA (gRNA) is T7 endonuclease I (T7EI) assay, which is time and labor consuming. In this study, we developed a visualized fluorescent reporter system to detect the specificity and cleavage activity of gRNA. Two gRNAs were designed to target porcine immunoglobulin M and nephrosis 1 genes. The cleavage activity was measured by using the traditional homology-directed repair (HDR)-based fluorescent reporter and the single-strand annealing (SSA)-based fluorescent reporter we established in this study. Compared with the HDR assay, the SSA-based fluorescent reporter approach was a more efficient and dependable strategy for testing the cleavage activity of Cas9/gRNA, thereby providing a universal and efficient approach for the application of CRISPR/Cas9 in generating gene-modified cells and organisms.     

Microfluidic device architecture for electrochemical patterning and detection of multiple DNA sequences

Electrochemical biosensors pose an attractive solution for point-of-care diagnostics because they require minimal instrumentation and they are scalable and readily integrated with microelectronics. The integration of electrochemical biosensors with microscale devices has, however, proven to be challenging due to significant incompatibilities among biomolecular stability, operation conditions of electrochemical sensors, and microfabrication techniques. Toward a solution to this problem, we have demonstrated here an electrochemical array architecture that supports the following processes in situ, within a self-enclosed microfluidic device: (a) electrode cleaning and preparation, (b) electrochemical addressing, patterning, and immobilization of sensing biomolecules at selected sensor pixels, (c) sequence-specific electrochemical detection from multiple pixels, and (d) regeneration of the sensing pixels. The architecture we have developed is general, and it should be applicable to a wide range of biosensing schemes that utilize gold-thiol self-assembled monolayer chemistry. As a proof-of-principle, we demonstrate the detection and differentiation of polymerase chain reaction (PCR) amplicons diagnostic of human (H1N1) and avian (H5N1) influenza.     

Universal and high-fidelity DNA single nucleotide polymorphism detection based on a CRISPR/Cas12a biochip

Single nucleotide polymorphisms (SNPs) are associated with many human diseases, so accurate and efficient SNP detection is of great significance for early diagnosis and clinical prognosis. This report proposes a universal and high-fidelity genotyping method in microfluidic point-of-care equipment based on the clustered regularly interspaced short palindromic repeat (CRISPR) system. Briefly, by systematically inserting the protospacer-adjacent-motif (PAM) sequence, we improved the universality of the CRISPR/Cas12a based SNP detection; by removing the complementary ssDNA and introducing an additional nucleotide mismatch, we improved the sensitivity and specificity. We preloaded the CRISPR/Cas12a reagents into the point-of-care biochip for automating the process, increasing the stability and long-term storage. This biochip enables us to rapidly and conveniently detect the genotypes within 20 min. In a practical application, the CRISPR/Cas12a biochip successfully distinguished three genotypes (homozygous wild type; the homozygous mutant type; and the heterozygous mutant type) of the CYP1A1*2 (A4889G, rs1048943), CYP2C19*2 (G681A, rs4244285), CYP2C9*3 (A1075C, rs1057910), and CYP2C19*3 (G636A, rs4986893) genes related to multiple cancers from 17 clinical blood samples. This CRISPR/Cas12a-based SNP genotyping method, being universal, accurate, and sensitive, will have broad applications in molecular diagnostics and clinical research.

A universal and high-fidelity genotyping method based on the clustered regularly interspaced short palindromic repeat (CRISPR) system was performed on the microfluidic point-of-care equipment.     

Novel Electrochemical Biosensing for Detection of Neglected Tropical Parasites of Animal Origin: Recent Advances

Parasitic diseases are among neglected disease of human and animals, especially in tropical and sub tropical regions. In the era of artificial intelligence, the novel biosensing diagnostic platform is needed for an early control measure implementation. This goal can be successfully achieved by onsite application of electrochemical biosensors. They are being developed towards point of care diagnostics; however commercial availability is scanty. The recent developments during last one decade in terms of the electrode surface modification for rapid diagnosis of important emerging/re-emerging parasites is presented. The information would help future improvement in the electrochemical biosensing of parasites.     

Current progresses and trends in carbon nanomaterials-based electrochemical and electrochemiluminescence biosensors

The enormous potential of biosensors in medical diagnostics has motivated scientists to develop newer innovative tools and advance biosensing technologies. The use of cell, organelles, nucleotides, aptamers, antibodies, affibodies, proteins, peptides, molecules, and printed polymers, merged with nanotechnology, offers excellent tools to prepare highly sensitive and advanced biosensors. Therefore, the current decade has witnessed a rapid surge in the fabrication of different nanomaterial-based biosensors. Among them, carbon nanomaterials (CNMs) have emerged highly attractive in the fabrication of both electrochemical and electrochemiluminescence (ECL) biosensors. On one hand, CNMs bear prominent electrical conductivity, large surface area to immobilize adequate amount of biomolecules, an enhanced loading capacity, improved biocompatibility, and active site for electrochemical reaction. Additionally, CNMs could be chemically modified for the covalent coupling with the biomolecules. On the other hand, both electrochemical and ECL biosensors allow for cost-effective, rapid, and real-time detection with excellent sensitivity and selectivity, with the capability of integrating different biomolecules and CNMs on the same chip. However, currently there is not a single review, which includes CNM-based electrochemical and ECL biosensors' current progress and trends. Therefore, this review intends to survey the current progress and future trends in CNM-based electrochemical and ECL biosensors.     

CRISPR Mediated Biosensing Toward Understanding Cellular Biology and Point-of-Care Diagnosis

Recent advances in CRISPR based biotechnologies have greatly expanded our capabilities to repurpose CRISPR for the development of biomolecular sensors for diagnosing diseases and understanding cellular pathways. The key attribute that allows CRISPR to be widely utilized is the programmable and highly selective mechanism. In this Minireview, we first illustrate the molecular principle of CRISPR functioning process from sensing to actuating. Next, the CRISPR based biosensing strategies for nucleic acids, proteins and small molecules are summarized. We highlight some of recent advances in applications for in vitro detection of biomolecules and in vivo imaging of cellular networks. Finally, the challenges with, and exciting prospects of, CRISPR based biosensing developments are discussed.     

Progression in sensing cardiac troponin biomarker charge transductions on semiconducting nanomaterials

A real-time ability to interpret the interaction between targeted biomolecules and the surface of semiconductors (metal transducers) into readable electrical signals, without biomolecular modification involving fluorescence dyes, redox enzymes, and radioactive labels, created by label-free biosensors has been extensively researched. Field-effect transistor (FET)- and capacitor-based biosensors are among the diverse electrical charge biosensing architectures that have drawn much attention for having charge transduction; thus, enabling the early and rapid diagnosis of the appropriate cardiac biomarkers at lower concentrations. These semiconducting material-based transducers are very suitable to be integrated with portable electronic devices for future online collection, transmission, reception, analysis, and reporting. This overview elucidates and clarifies two major electrical label-free systems (FET- and capacitor-based biosensors) with cardiac troponin (cTn) biomarker-mediated charge transduction for acute myocardial infarction (AMI) diagnosis. Advances in these systems are highlighted by their progression in bridging the laboratory and industry; the foremost technologies have made the transition from benchtop to bedside and beyond.     

Digital biosensors with built-in logic for biomedical applications—biosensors based on a biocomputing concept

This article reviews biomolecular logic systems for bioanalytical applications, specifically concentrating on the prospects and fundamental and practical challenges of designing digitally operating biosensors logically processing multiple biochemical signals. Such digitally processed information produces a final output in the form of a yes/no response through Boolean logic networks composed of biomolecular systems, and hence leads to a high-fidelity biosensing compared with traditional single (or parallel) sensing devices. It also allows direct coupling of the signal processing with chemical actuators to produce integrated “smart” “sense/act” (biosensor-bioactuator) systems. Unlike common biosensing devices based on a single input (analyte), devices based on biochemical logic systems require a fundamentally new approach for the sensor design and operation and careful attention to the interface of biocomputing systems and electronic transducers. As common in conventional biosensors, the success of the enzyme logic biosensor would depend, in part, on the immobilization of the biocomputing reagent layer. Such surface confinement provides a contact between the biocomputing layer and the transducing surface and combines efficiently the individual logic-gate elements. Particular attention should thus be given to the composition, preparation, and immobilization of the biocomputing surface layer, to the role of the system scalability, and to the efficient transduction of the output signals. By processing complex patterns of multiple physiological markers, such multisignal digital biosensors should have a profound impact upon the rapid diagnosis and treatment of diseases, and particularly upon the timely detection and alert of medical emergencies (along with immediate therapeutic intervention). Other fields ranging from biotechnology to homeland security would benefit from these advances in new biocomputing biosensors and the corresponding closed-loop “add/act” operation.     

Recent Progress on the Development of Biofuel Cells for Self‐Powered Electrochemical Biosensing and Logic Biosensing: A Review

Biofuel cells (BFCs) based on enzymes and microorganisms have been recently received considerable attention because they are recognized as an attractive type of energy conversion technology. In addition to the research activities related to the application of BFCs as power source, we have witnessed recently a growing interest in using BFCs for self‐powered electrochemical biosensing and electrochemical logic biosensing applications. Compared with traditional biosensors, one of the most significant advantages of the BFCs‐based self‐powered electrochemical biosensors and logic biosensors is their ability to detect targets integrated with chemical‐to‐electrochemical energy transformation, thus obviating the requirement of external power sources. Following my previous review (Electroanalysis­ 2012 , 24, 197–209), the present review summarizes, discusses and updates the most recent progress and latest advances on the design and construction of BFCs‐based self‐powered electrochemical biosensors and logic biosensors. In addition to the traditional approaches based on substrate effect, inhibition effect, blocking effect and gene regulation effect for BFCs‐based self‐powered electrochemical biosensors and logic biosensors design, some new principles including enzyme effect, co‐stabilization effect, competition effect and hybrid effect are summarized and discussed by me in details. The outlook and recommendation of future directions of BFCs‐based self‐powered electrochemical biosensors and logic biosensors are discussed in the end.