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In the past few years, the CRISPR (clustered regularly interspaced short palindromic repeats) applications in medicine and molecular biology have broadened. CRISPR has also been integrated with microfluidic-based biosensors to enhance the sensitivity and selectivity of medical diagnosis due to its great potentials. The CRISPR-powered microfluidics can help quantify DNAs and RNAs for different diseases such as cancer, and viral or bacterial diseases among others. Here in this review, we discussed the main applications of such tools along with their advantages and limitations.

Human African Trypanosomiasis (HAT) is caused by unicellular flagellated protozoan parasites of the genus Trypanosoma brucei. The subspecies T. b. gambiense is mainly responsible for mostly chronic anthroponotic infections in West- and Central Africa, accounting for roughly 95% of all HAT cases. Trypanosoma b. rhodesiense results in more acute zoonotic infections in East-Africa. Because HAT has a two-stage pathogenesis, treatment depends on clinical assessment of patients and the determination whether or not parasites have crossed the blood brain barrier. Today, ultimate confirmation of parasitemia is still done by microscopy analysis. However, the introduction of diagnostic lateral flow devices has been a major contributor to the recent dramatic drop in T. b. gambiense HAT. Other techniques such as loop mediated isothermal amplification (LAMP) and recombinant polymerase amplification (RPA)-based tests have been published but are still not widely used in the field. Most recently, CRISPR-Cas technology has been proposed to improve the intrinsic diagnostic characteristics of molecular approaches. This will become crucial in the near future, as preventing the resurgence of HAT will be a priority and will require tools with extreme high positive and negative predicted values, as well as excellent sensitivity and specificity. As for treatment, pentamidine and suramin have historically been the drugs of choice for the treatment of blood-stage gambiense-HAT and rhodesiense-HAT, respectively. For treatment of second-stage infections, drugs that pass the blood brain barrier are needed, and melarsoprol has been effectively used for both forms of HAT in the past. However, due to the high occurrence of post-treatment encephalopathy, the drug is not recommended for use in T. b. gambiense HAT. Here, a combination therapy of eflornithine and nifurtimox (NECT) has been the choice of treatment since 2009. As this treatment requires IV perfusion of eflornithine, efforts were launched in 2003 by the drugs for neglected disease initiative (DNDi) to find an oral-only therapy solution, suitable for rural sub-Saharan Africa treatment conditions. In 2019 this resulted in the introduction of fexinidazole, with a treatment regimen suitable for both the blood-stage and non-severe second-stage T. b. gambiense infections. Experimental treatment of T. b. rhodesiense HAT has now been initiated as well.

CoVID‐19 is a multi‐symptomatic disease which has made a global impact due to its ability to spread rapidly, and its relatively high mortality rate. Beyond the heroic efforts to develop vaccines, which we do not discuss herein, the response of scientists and clinicians to this complex problem has reflected the need to detect CoVID‐19 rapidly, to diagnose patients likely to show adverse symptoms, and to treat severe and critical CoVID‐19. Here we aim to encapsulate these varied and sometimes conflicting approaches and the resulting data in terms of chemistry and biology. In the process we highlight emerging concepts, and potential future applications that may arise out of this immense effort., The CoVID‐19 pandemic presented a significant technological challenge. Herein we review the role of interdisciplinary science and medicinal chemistry in detecting and directly combating the disease. We further suggest ways in which the fruits of these endeavours can be put to use in the future and how we can then hone such response efforts.

CRISPR-based diagnostics enable specific sensing of DNA and RNA biomarkers associated with human diseases. This is achieved through the binding of guide RNAs to a complementary sequence which activates Cas enzymes to cleave reporter molecules. Currently, most CRISPRbased diagnostics rely on target preamplification to reach sufficient sensitivity for clinical applications. This limits quantification capability and adds complexity to the reaction chemistry. Here, we show the combination of a CRISPR/Cas-based reaction with a Nanozyme-Linked ImmunoSorbent Assay which allows for the quantitative and colorimetric readout of Cas13- mediated RNA detection through catalytic metallic nanoparticles at room temperature (CrisprZyme). We demonstrate CrisprZyme is easily adaptable to a lateral-flow-based readout and different Cas enzymes, and enables the sensing of non-coding RNAs including microRNAs, long non-coding RNAs and circular RNAs. We utilise this platform to identify patients with acute myocardial infarction and to monitor cellular differentiation in vitro and in tissue biopsies from prostate cancer patients. We anticipate that CrisprZyme has significant potential as a universally applicable signal catalyst for CRISPR-based diagnostics which will expand the spectrum of targets for preamplification-free, quantitative detection.

Zearalenone (ZEN) is harmful to animals and human beings, so it is very important to develop a rapid and sensitive method for the detection of ZEN. In this paper, we proposed a novel ZEN-monitoring method using two aptamers as recognition elements and EnGen LbaCas12a and Nt.AlwI nicking endonuclease as signal amplifiers. When ZEN was present, it bound to the aptamer Z0 and, Z1 was released into solution. The solution was then separated and the Nt.AlwI enzyme was added in order to form a nicking-enzyme cycle, thereby producing large amounts of the ssDNA Z3 for 30 min. The Z3 formed a CRISPR-Cas12a-Z3 complex with CRISPR-Cas12a, activated the trans-cleavage ability of Cas12a, cleaved the Quenched Reporter for 20 min, and underwent fluorescence recovery. The aptasensor was able to sensitively detect ZEN in the linear range of 1–1000 pg/mL, with a detection limit as low as 0.213 pg/mL. The detection time lasted for 2 h. Additionally, this detection technology can also be used to monitor other hazards.

Recently emerged novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the resulting corona virus disease 2019 (COVID-19) led to urgent search for methods to prevent and treat COVID-19. Among important disciplines that were mobilized is the biomaterials science and engineering. Biomaterials offer a range of possibilities to develop disease models, protective, diagnostic, therapeutic, monitoring measures, and vaccines. Among the most important contributions made so far from this field are tissue engineering, organoids, and organ-on-a-chip systems, which have been the important frontiers in developing tissue models for viral infection studies. Also, due to low bioavailability and limited circulation time of conventional antiviral drugs, controlled and targeted drug delivery could be applied alternatively. Fortunately, at the time of writing this paper, we have two successful vaccines and new at-home detection platforms. In this paper, we aim to review recent advances of biomaterial-based platforms for protection, diagnosis, vaccination, therapeutics, and monitoring of SARS-CoV-2 and discuss challenges and possible future research directions in this field. American Heart AssociationAmerican Heart Association [442611-NU-80922]; 2232 International Fellowship for Outstanding Researchers Program of TUBITAKTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [118C346] The authors acknowledge funding from the American Heart Association (442611-NU-80922). Y. N. Ertas acknowledges funding support from 2232 International Fellowship for Outstanding Researchers Program of TUBITAK (Project No: 118C346). Thanks are due to Fatemeh Rastegar-Adib for helping with drawing parts of Fig. 1 and Fig. 5.

Viruses are ubiquitous in the environment. While many impart no deleterious effects on their hosts, several are major pathogens. This risk of pathogenicity, alongside the fact that many viruses can rapidly mutate highlights the need for suitable, rapid diagnostic measures. This review provides a critical analysis of widely used methods and examines their advantages and limitations. Currently, nucleic-acid detection and immunoassay methods are among the most popular means for quickly identifying viral infection directly from source. Nucleic acid-based detection generally offers high sensitivity, but can be time-consuming, costly, and require trained staff. The use of isothermal-based amplification systems for detection could aid in the reduction of results turnaround and equipment-associated costs, making them appealing for point-of-use applications, or when high volume/fast turnaround testing is required. Alternatively, immunoassays offer robustness and reduced costs. Furthermore, some immunoassay formats, such as those using lateral-flow technology, can generate results very rapidly. However, immunoassays typically cannot achieve comparable sensitivity to nucleic acid-based detection methods. Alongside these methods, the application of next-generation sequencing can provide highly specific results. In addition, the ability to sequence large numbers of viral genomes would provide researchers with enhanced information and assist in tracing infections.

The field of genome editing started with the discovery of meganucleases (e.g., the LAGLIDADG family of homing endonucleases) in yeast. After the discovery of transcription activator-like effector nucleases and zinc finger nucleases, the recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated proteins (Cas) system has opened a new window of applications in the field of gene editing. Here, we review different Cas proteins and their corresponding features including advantages and disadvantages, and we provide an overview of the different endonuclease-deficient Cas protein (dCas) derivatives. These dCas derivatives consist of an endonuclease-deficient Cas9 which can be fused to different effector domains to perform distinct in vitro applications such as tracking, transcriptional activation and repression, as well as base editing. Finally, we review the in vivo applications of these dCas derivatives and discuss their potential to perform gene activation and repression in vivo, as well as their potential future use in human therapy.

The field of genome editing started with the discovery of meganucleases (e.g., the LAGLIDADG family of homing endonucleases) in yeast. After the discovery of transcription activator-like effector nucleases and zinc finger nucleases, the recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated proteins (Cas) system has opened a new window of applications in the field of gene editing. Here, we review different Cas proteins and their corresponding features including advantages and disadvantages, and we provide an overview of the different endonuclease-deficient Cas protein (dCas) derivatives. These dCas derivatives consist of an endonuclease-deficient Cas9 which can be fused to different effector domains to perform distinct in vitro applications such as tracking, transcriptional activation and repression, as well as base editing. Finally, we review the in vivo applications of these dCas derivatives and discuss their potential to perform gene activation and repression in vivo, as well as their potential future use in human therapy.

[EN] Food allergy is reported as the commonest adverse reaction to food components, whose prevalence has increased in recent years. As food avoidance is mainly in practice the only way to prevent hypersensitive consumers from ingesting allergenic substances, it is imperative to provide complete and accurate in-formation on food ingredients. In this scenario, there is a need for precise, fast and cost-effective methods for the high-throughput screening of specific allergen content in food products. This work reviews recent approaches, existing kits for food-borne allergen detection and cutting-edge applications by focusing on the sensitivity, selectivity and applicability of current methods in food samples. In addition, the advantages, benefits and limitations of each approach are discussed to establish the most suitable methods and which challenges are to be addressed in forthcoming years from an analytical viewpoint. (C) 2020 Elsevier B.V. All rights reserved., This work has been funded by the Agencia Estatal de Investigacion, Spain (CTQ2016-75749-R, FEDER)

The excessive use of antibiotics in human and veterinary medicine causes the emergence of multidrug resistant bacteria. In this context, the surveillance of many different antibiotics provokes a worldwide challenge. Hence, fast and versatile multianalyte single-use biosensors are of increasing interest for many fields such as medical analysis or environmental and food control. Here we present a microfluidic platform enabling the electrochemical readout of up to eight enzyme-linked assays (ELAs), simultaneously. To demonstrate the applicability of this platform for the surveillance and monitoring of antibiotics, we used highly sensitive biomolecular sensor systems for the simultaneous detection of two commonly employed antibiotic classes tetracycline and streptogramin. Thus, microfluidic channel networks are designed, comprising distinct numbers of immobilization sections with a very low volume of 680 nL each. These passively metered sections can be actuated separately for an individual assay procedure. The limits of detection (LOD) are determined, with high precision, to 6.33 and 9.22 ng mL–1 for tetracycline and pristinamycin, respectively. The employed channel material, dry film photoresist (DFR), allows an easy storage of preimmobilized assays with a shelf life of at least 3 months. Multianalyte measurements in a complex medium are demonstrated by the simultaneous detection of both antibiotics in spiked human plasma within a sample-to-result time of less than 15 min., Analytical Chemistry, 88 (20), ISSN:1520-6882, ISSN:0003-2700

Although polymerase chain reaction (PCR) is the most widely used method for DNA amplification, the requirement of thermocycling limits its non-laboratory applications. Isothermal DNA amplification techniques are hence valuable for on-site diagnostic applications in place of traditional PCR. Here we describe a true isothermal approach for amplifying and detecting double-stranded DNA based on a CRISPR–Cas9-triggered nicking endonuclease-mediated Strand Displacement Amplification method (namely CRISDA). CRISDA takes advantage of the high sensitivity/specificity and unique conformational rearrangements of CRISPR effectors in recognizing the target DNA. In combination with a peptide nucleic acid (PNA) invasion-mediated endpoint measurement, the method exhibits attomolar sensitivity and single-nucleotide specificity in detection of various DNA targets under a complex sample background. Additionally, by integrating the technique with a Cas9-mediated target enrichment approach, CRISDA exhibits sub-attomolar sensitivity. In summary, CRISDA is a powerful isothermal tool for ultrasensitive and specific detection of nucleic acids in point-of-care diagnostics and field analyses., Isothermal DNA amplification techniques are useful for diagnostic applications in place of traditional PCR. Here the authors describe CRISDA, which combines CRISPR–Cas9 with strand displacement amplification and exhibits attomolar sensitivity and single-nucleotide specificity in DNA detection.