Ratoon Stunting Disease of Sugarcane: A Review Emphasizing Detection Strategies and Challenges.

Sugarcane (Saccharum hybrid) is an important cash crop grown in tropical and subtropical countries. Ratoon stunting disease (RSD), caused by a xylem-inhabiting bacterium, Leifsonia xyli subsp. xyli (Lxx) is one of the most economically significant diseases globally. RSD results in severe yield losses because its highly contagious nature and lack of visually identifiable symptoms make it harder to devise an effective management strategy. The efficacy of current management practices is hindered by implementation difficulties caused by lack of resources, high cost, and difficulties in monitoring. Rapid detection of the causal pathogen in vegetative planting material is crucial for sugarcane growers to manage this disease. Several microscopic, serological, and molecular-based methods have been developed and used for detecting the RSD pathogen. Although these methods have been used across the sugarcane industry worldwide to diagnose Lxx, some lack reliability or specificity, are expensive and time-consuming to apply, and most of all, are not suitable for on-farm diagnosis. In recent decades, there has been significant progress in the development of integrated isothermal amplification-based microdevices for accurate human and plant pathogen detection. There is a significant opportunity to develop a novel diagnostic method that integrates nanobiosensing with isothermal amplification within a microdevice format for accurate Lxx detection. In this review, we summarize (i) the historical background and current knowledge of sugarcane ratoon stunting disease, including some aspects related to transmission, pathosystem, and management practices; and (ii) the drawbacks of current diagnostic methods and the potential for application of advanced diagnostics to improve disease management.

Toward Personalized Nanomedicine: The Critical Evaluation of Micro and Nanodevices for Cancer Biomarker Analysis in Liquid Biopsy.

Liquid biopsy for the analysis of circulating cancer biomarkers (CBs) is a major advancement toward the early detection of cancer. In comparison to tissue biopsy techniques, liquid biopsy is relatively painless, offering multiple sampling opportunities across easily accessible bodily fluids such as blood, urine, and saliva. Liquid biopsy is also relatively inexpensive and simple, avoiding the requirement for specialized laboratory equipment or trained medical staff. Major advances in the field of liquid biopsy are attributed largely to developments in nanotechnology and microfabrication that enables the creation of highly precise chip-based platforms. These devices can overcome detection limitations of an individual biomarker by detecting multiple markers simultaneously on the same chip, or by featuring integrated and combined target separation techniques. In this review, the major advances in the field of portable and semi-portable micro, nano, and multiplexed platforms for CB detection for the early diagnosis of cancer are highlighted. A comparative discussion is also provided, noting merits and drawbacks of the platforms, especially in terms of portability. Finally, key challenges toward device portability and possible solutions, as well as discussing the future direction of the field are highlighted.

Low-cost electrochemical paper-based device for exosome detection.

Exosomes are vesicles released by healthy and cancer cells into the extracellular matrix and bodily fluid. Cancer cell-derived exosomes have attracted much attention in early-stage detection and prognostication of treatment response. Thus, detecting exosomes is of great interest to biology and medicine. However, many conventional detection methods require high-cost equipment and centralized laboratory facilities, making diagnostics inaccessible in limited-resource settings. This study reports a proof-of-concept low-cost electrochemical paper-based analytical device to quantify both the total bulk and cancer cell-derived exosomes in cell culture media. The device employs a sandwich immune assay design, where exosomes are initially captured using the electrode-bound generic antibodies (i.e. CD9) and subsequently detected via ovarian cancer-specific CA125 antibodies. Our proposed device quantifies the total bulk exosome concentration with a detection limit of 9.3 × 107 exosomes per mL and ovarian cancer cell-derived exosomes with a detection limit of 7.1 × 108 exosomes per mL, with a relative standard deviation of <10% (n = 3). We suggest that this low-cost and simple electrochemical paper-based device could be an alternative tool for detecting disease-specific exosomes in biological samples with the potential to be further developed for point-of-care diagnosis.

Detection of the SARS-CoV-2 humanized antibody with paper-based ELISA.

This work reports the development of a rapid, simple and inexpensive colorimetric paper-based assay for the detection of the severe acute respiratory symptom coronavirus 2 (SARS-CoV-2) humanized antibody. The paper device was prepared with lamination for easy sample handling and coated with the recombinant SARS-CoV-2 nucleocapsid antigen. This assay employed a colorimetric reaction, which is followed by horseradish peroxidase (HRP) conjugated detecting antibody in the presence of the 3,3',5,5'-tetramethylbenzidine (TMB) substrate. The colorimetric readout was evaluated and quantified for specificity and sensitivity. The characterization of this assay includes determining the linear regression curve, the limit of detection (LOD), the repeatability, and testing complex biological samples. We found that the LOD of the assay was 9.00 ng µL-1 (0.112 IU mL-1). The relative standard deviation was approximately 10% for a sample number of n = 3. We believe that our proof-of-concept assay has the potential to be developed for clinical screening of the SARS-CoV-2 humanized antibody as a tool to confirm infected active cases or to confirm SARS-CoV-2 immune cases during the process of vaccine development.

Correction: Soda et al. Electrochemical Detection of Global DNA Methylation Using Biologically Assembled Polymer Beads. Cancers 2021, 13, 3787.

In the original publication [...].

CRISPR/Cas-Based Diagnostics in Agricultural Applications.

Pests and disease-causing pathogens frequently impede agricultural production. An early and efficient diagnostic tool is crucial for effective disease management. Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated protein (Cas) have recently been harnessed to develop diagnostic tools. The CRISPR/Cas system, composed of the Cas endonuclease and guide RNA, enables precise identification and cleavage of the target nucleic acids. The inherent sensitivity, high specificity, and rapid assay time of the CRISPR/Cas system make it an effective alternative for diagnosing plant pathogens and identifying genetically modified crops. Furthermore, its potential for multiplexing and suitability for point-of-care testing at the field level provide advantages over traditional diagnostic systems such as RT-PCR, LAMP, and NGS. In this review, we discuss the recent developments in CRISPR/Cas based diagnostics and their implications in various agricultural applications. We have also emphasized the major challenges with possible solutions and provided insights into future perspectives and potential applications of the CRISPR/Cas system in agriculture.

Placental Exosomes as Biomarkers for Maternal Diseases: Current Advances in Isolation, Characterization, and Detection.

Serving as the interface between fetal and maternal circulation, the placenta plays a critical role in fetal growth and development. Placental exosomes are small membrane-bound extracellular vesicles released by the placenta during pregnancy. They contain a variety of biomolecules, including lipids, proteins, and nucleic acids, which can potentially be biomarkers of maternal diseases. An increasing number of studies have demonstrated the utility of placental exosomes for the diagnosis and monitoring of pathological conditions such as pre-eclampsia and gestational diabetes. This suggests that placental exosomes may serve as new biomarkers in liquid biopsy analysis. This review provides an overview of the current understanding of the biological function of placental exosomes and their potential as biomarkers of maternal diseases. Additionally, this review highlights current barriers and the way forward for standardization and validation of known techniques for exosome isolation, characterization, and detection. Finally, microfluidic devices for exosome research are discussed.

Potential Avenues for Exosomal Isolation and Detection Methods to Enhance Small-Cell Lung Cancer Analysis.

Around the world, lung cancer has long been the main factor in cancer-related deaths, with small-cell lung cancer (SCLC) being the deadliest form of lung cancer. Cancer cell-derived exosomes and exosomal miRNAs are considered promising biomarkers for diagnosing and prognosis of various diseases, including SCLC. Due to the rapidity of SCLC metastasis, early detection and diagnosis can offer better diagnosis and prognosis and therefore increase the patient's chances of survival. Over the past several years, many methodologies have been developed for analyzing non-SCLC-derived exosomes. However, minimal advances have been made in SCLC-derived exosome analysis methodologies. This Review discusses the epidemiology and prominent biomarkers of SCLC. Followed by a discussion about the effective strategies for isolating and detecting SCLC-derived exosomes and exosomal miRNA, highlighting the critical challenges and limitations of current methodologies. Finally, an overview is provided detailing future perspectives for exosome-based SCLC research.

An Interfacial Affinity Interaction-Based Method for Detecting HOTAIR lncRNA in Cancer Plasma Samples.

Long non-coding RNA Homeobox transcript antisense intergenic RNA (HOTAIR) is recognized as a participant in different processes of normal cell development. Aberrant overexpression of HOTAIR contributes to the initiation, growth, and invasiveness of ovarian cancer. Using the affinity interaction of target HOTAIR lncRNA sequences towards a screen-printed gold electrode (SPE-Au), herein we report on a novel, rapid and simple method to detect HOTAIR sequences. HOTAIR lncRNA sequences were first extracted from ovarian cancer cell lines and patient plasma samples and were magnetically captured and purified by complimentary capture probe-functionalized magnetic beads. Isolated target HOTAIR lncRNAs were directly adsorbed onto unmodified screen-printed gold electrodes (SPE-Au) for direct quantification with [Fe(CN)6]3-/4- redox couple. Our assay achieved a linear dynamic range of 100 nM and 1 pM for detecting pre-clinical model HOTAIR lncRNA samples (%RSD ≤ 5%, for n = 3) and was highly specific, showing clear distinction between HOTAIR lncRNA targets and non-specific miR-891 and miR-486 (100 nM) (%RSD ≤ 5%, for n = 3). The method was tested using ovarian cancer-specific cell lines (SKOV3 and OVCAR3) and mesothelial cell line (MeT-5A)-derived lncRNAs. The analytical performance of our method was validated using RT-qPCR. Finally, the method was tested using clinical samples from ovarian cancer patients and the resulting electrochemical responses show a clear distinction between the ovarian carcinoma and benign samples.

Current and future strategies for diagnostic and management of obstructive sleep apnea.

INTRODUCTION: Obstructive sleep apnea (OSA) is a common sleep disorder with multiple comorbidities including hypertension, diabetes, and cardiovascular disorders. Detected based on an overnight sleep study is called polysomnography (PSG); OSA still remains undiagnosed in majority of the population mainly attributed to lack of awareness. To overcome the limitations posed by PSG such as patient discomfort and overnight hospitalization, newer technologies are being explored. In addition, challenges associated with current management of OSA using continuous positive airway pressure (CPAP), etc. presents several pitfalls. AREAS COVERED: Conventional and modern detection/management techniques including PSG, CPAP, smart wearable/pillows, bio-motion sensors, etc., have both pros and cons. To fulfill the limitations in OSA diagnostics, there is an imperative need for new technology for screening of symptomatic and more importantly asymptomatic OSA patients to reduce the risk of several associated life-threatening comorbidities. In this line, molecular marker-based diagnostics have shown great promises. EXPERT OPINION: A detailed overview is presented on the OSA management and diagnostic approaches and recent advances in the molecular screening methods. The potentials of biomarker-based detection and its limitations are also portrayed and a comparison between the standard, current modern approaches, and promising futuristic technologies for OSA diagnostics and management is set forth.ABBREVIATIONS AHI: Apnea hypopnea index; AI: artificial intelligence; CAM: Cell adhesion molecules; CPAP: Continuous Positive Airway Pressure; COVID-19: Coronavirus Disease 2019; CVD: Cardiovascular disease; ELISA: Enzyme linked immunosorbent assay; HSAT: Home sleep apnea testing; IR-UWB: Impulse radio-ultra wideband; MMA: maxillomandibular advancement; PSG: Polysomnography; OSA: Obstructive sleep apnea; SOD: Superoxide dismutase; QD: Quantum dot.

Bioengineered Polymer Nanobeads for Isolation and Electrochemical Detection of Cancer Biomarkers.

Early sensitive diagnosis of cancer is critical for enhancing treatment success. We previously bioengineered multifunctional core-shell structures composed of a poly-3-hydroxybutyrate (PHB) core densely coated with protein functions for uses in bioseparation and immunodiagnostic applications. Here, we report bioengineering of Escherichia coli to self-assemble PHB inclusions that codisplay a ferritin-derived iron-binding peptide and the protein A-derived antibody-binding Z domain. The iron-binding peptide mediated surface coating with a ferrofluid imparting superparamagnetic properties, while the Z domain remained accessible for binding of cancer biomarker-specific antibodies. We demonstrated that these nanobeads can specifically bind biomarkers in complex mixtures, enabling efficient magnetic separation toward enhanced electrochemical detection of cancer biomarkers such as methylated DNA and exosomes from cancer cells. Our study revealed that superparamagnetic core-shell structures can be derived from biological self-assembly systems for uses in sensitive and specific electrochemical detection of cancer biomarkers, laying the foundation for engineering advanced nanomaterials for diverse diagnostic approaches.

A Portable Device for LAMP Based Detection of SARS-CoV-2.

This paper reports the design, development, and testing of a novel, yet simple and low-cost portable device for the rapid detection of SARS-CoV-2. The device performs loop mediated isothermal amplification (LAMP) and provides visually distinguishable images of the fluorescence emitted from the samples. The device utilises an aluminium block embedded with a cartridge heater for isothermal heating of the sample and a single-board computer and camera for fluorescence detection. The device demonstrates promising results within 20 min using clinically relevant starting concentrations of the synthetic template. Time-to-signal data for this device are considerably lower compared to standard quantitative Polymerase Chain Reaction(qPCR) machine (~10-20 min vs. >38 min) for 1 × 102 starting template copy number. The device in its fully optimized and characterized state can potentially be used as simple to operate, rapid, sensitive, and inexpensive platform for population screening as well as point-of-need severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) detection and patient management.

Electrochemical Detection of Global DNA Methylation Using Biologically Assembled Polymer Beads.

DNA methylation is a cell-type-specific epigenetic marker that is essential for transcriptional regulation, silencing of repetitive DNA and genomic imprinting. It is also responsible for the pathogenesis of many diseases, including cancers. Herein, we present a simple approach for quantifying global DNA methylation in ovarian cancer patient plasma samples based on a new class of biopolymer nanobeads. Our approach utilises the immune capture of target DNA and electrochemical quantification of global DNA methylation level within the targets in a three-step strategy that involves (i) initial preparation of target single-stranded DNA (ss-DNA) from the plasma of the patients' samples, (ii) direct adsorption of polymer nanobeads on the surface of a bare screen-printed gold electrode (SPE-Au) followed by the immobilisation of 5-methylcytosine (5mC)-horseradish peroxidase (HRP) antibody, and (iii) immune capture of target ss-DNA onto the electrode-bound PHB/5mC-HRP antibody conjugates and their subsequent qualification using the hydrogen peroxide/horseradish peroxidase/hydroquinone (H2O2/HRP/HQ) redox cycling system. In the presence of methylated DNA, the enzymatically produced (in situ) metabolites, i.e., benzoquinone (BQ), binds irreversibly to cellular DNA resulting in the unstable formation of DNA adducts and induced oxidative DNA strand breakage. These events reduce the available BQ in the system to support the redox cycling process and sequel DNA saturation on the platform, subsequently causing high Coulombic repulsion between BQ and negatively charged nucleotide strands. Thus, the increase in methylation levels on the electrode surface is inversely proportional to the current response. The method could successfully detect as low as 5% methylation level. In addition, the assay showed good reproducibility (% RSD ≤ 5%) and specificity by analysing various levels of methylation in cell lines and plasma DNA samples from patients with ovarian cancer. We envision that our bioengineered polymer nanobeads with high surface modification versatility could be a useful alternative platform for the electrochemical detection of varying molecular biomarkers.

An amplification-free method for the detection of HOTAIR long non-coding RNA.

The discovery of large transcripts of long RNAs that have limited protein coding capacity, known as long non-coding RNAs (lncRNAs) present new concepts on RNA-mediated gene regulation. Increasing evidence suggests that large intervening ncRNAs regulate key pathways in cancer genesis and metastasis. Among the most characterized lncRNAs, homeobox (HOX) transcript antisense intergenic RNA (HOTAIR) acts as an oncogenic molecule in different cancer cells, and thus its expression level serves as a potential biomarker for diagnostic and therapeutic purposes in several human cancers, such as breast, prostate, liver and ovarian cancer. This paper reports a simple and sensitive sensor platform for the detection of HOTAIR. Extracted HOTAIR sequences from ovarian cancer cells and plasma samples derived from ovarian cancer patients were magnetically isolated and purified, followed by a sandwich hybridization event at a screen-printed gold electrode. This event was monitored by amperometry using the hydrogen peroxide/horseradish peroxidase/hydroquinone (H2O2/HRP/HQ) system. The catalytic enhancement of the amperometric signal enabled our assay to achieve a detection limit of 1.0 fM with a good inter-assay reproducibility (relative standard deviation (%RSD) = < 5.0%, n = 3). The method was used for the analysis of specific HOTAIR in cell line and a small cohort of plasma samples derived from patients with ovarian cancer. The analytical performance of the method was also demonstrated using a standard RT-qPCR. We believe that the proof of the concept assay demonstrated here could be a cost-effective alternative platform for screening cancer-related lncRNAs in routine clinical settings.

PCR-Free Detection of Long Non-Coding HOTAIR RNA in Ovarian Cancer Cell Lines and Plasma Samples.

Long non-coding RNA HOX transcript antisense intergenic RNA (HOTAIR) is one of the promising biomarkers that has widely been used in determining the stages of many cancers, including ovarian cancer. In cancer diagnostics, the two key analytical challenges for detecting long non-coding RNA biomarkers are i) the low concentration levels (nM to fM range) in which they are found and ii) the analytical method where broad dynamic range is required (four to six orders of magnitude) due to the large variation in expression levels for different HOTAIR RNAs. To meet these challenges, we report on a biosensing platform for the visual (colorimetric) estimation and subsequent electrochemical quantification of ovarian-cancer-specific HOTAIR using a screen-printed gold electrode (SPE-Au). Our assay utilizes a two-step strategy that involves (i) magnetic isolation and purification of target HOTAIR sequences and (ii) subsequent detection of isolated sequences using a sandwich hybridization coupled with horseradish peroxidase (HRP)-catalyzed reaction of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide. The assay achieved a detection limit of 1.0 fM HOTAIR in spiked buffer samples with excellent reproducibility (% RSD ≤ 5%, for n = 3). It was successfully applied to detect HOTAIR in cancer cell lines and a panel of plasma samples derived from patients with ovarian cancer. The analytical performance of the method was validated with standard RT-qPCR. We believe that the proof of concept assay reported here may find potential use in routine clinical settings for the screening of cancer-related lncRNAs.

Advanced liquid biopsy technologies for circulating biomarker detection.

Liquid biopsy is a new diagnostic concept that provides important information for monitoring and identifying tumor genomes in body fluid samples. Detection of tumor origin biomolecules like circulating tumor cells (CTCs), circulating tumor specific nucleic acids (circulating tumor DNA (ctDNA), circulating tumor RNA (ctRNA), microRNAs (miRNAs), long non-coding RNAs (lnRNAs)), exosomes, autoantibodies in blood, saliva, stool, urine, etc. enables cancer screening, early stage diagnosis and evaluation of therapy response through minimally invasive means. From reliance on painful and hazardous tissue biopsies or imaging depending on sophisticated equipment, cancer management schemes are witnessing a rapid evolution towards minimally invasive yet highly sensitive liquid biopsy-based tools. Clinical application of liquid biopsy is already paving the way for precision theranostics and personalized medicine. This is achieved especially by enabling repeated sampling, which in turn provides a more comprehensive molecular profile of tumors. On the other hand, integration with novel miniaturized platforms, engineered nanomaterials, as well as electrochemical detection has led to the development of low-cost and simple platforms suited for point-of-care applications. Herein, we provide a comprehensive overview of the biogenesis, significance and potential role of four widely known biomarkers (CTCs, ctDNA, miRNA and exosomes) in cancer diagnostics and therapeutics. Furthermore, we provide a detailed discussion of the inherent biological and technical challenges associated with currently available methods and the possible pathways to overcome these challenges. The recent advances in the application of a wide range of nanomaterials in detecting these biomarkers are also highlighted.