Rapid Disease Diagnostics Research Articles

First report of Colletotrichum truncatum causing anthracnose of ashwagandha (Withania somnifera) in India.

Ashwagandha (Withania somnifera), enriched in alkaloids, steroidal lactones and saponins, is a valuable herb in Indian Ayurvedic medicine. During December 2023, Va-1 (Vallabh Ashwagandha-1) plants at ICAR -Central Tobacco Research Institute, Vedasandur, Tamil Nadu (10.53717ºN, 77.9507°E) were noticed with anthracnose-like disease symptoms, characterized by irregular necrotic lesions on stem, leaves and pods. Infected plants dried entirely in the advanced stage and had pin-head-shaped fruiting bodies. In the assessment of 185 plants, 42 were symptomatic, while five were dried, and the disease severity varied between 17 to 22%. Pathogen was isolated from infected leaf and stem tissues (3 to 5 mm2) using a potato dextrose agar (PDA) medium amended with streptomycin sulphate (20 mg/l). Infected tissues were surface sterilized using 1% sodium hypochlorite, and the PDA plates were incubated at 27°C for three days. The pathogen was purified, and two cultures, AVCT01 and AVCT02, were used for the downstream characterization. Colonies on the PDA medium were initially pale white and later turned to dark grey, with an average growth of 42.6 mm (diameter) on the 7th day after inoculation (DAI). Conidia were smooth, hyaline, aseptate and curved, with a tapering end and truncate base. The conidial dimensions (n=30) were 22.8 ± 1.32 × 3.4 ± 0.29 µm (20.6 to 25.8 × 2.9 to 4.2). Acervuli were scattered, containing dark brown conidiomata with septate, pointed setae and measured 119 ± 16.6 × 4.2 ± 0.5 µm (81 to 148 × 3.1 to 5.3) in dimension. Morphological and cultural characteristics were identical to Colletotrichum truncatum described by Damm et al. (2009). Further, the molecular characterization was executed with primers targeting ITS (ITS1/ITS4) and three housekeeping genes; act (ACT-512F/ACT-783R), tub2 (Bt2a/Bt2b) and gapdh (GDF1/GDFR1) (Kim et al. 2020). The amplified products were sequenced bidirectionally and in BLASTn analysis, sequences revealed 100% identity with C. truncatum [(NCBI GenBank OM327672 (ITS), OR147336 (act), MG214130 (tub2) and MK675260 (gapdh)]. In the maximum likelihood phylogeny of concatenated alignments, these cultures clustered with CBS119189. All sequences were deposited in NCBI GenBank (ITS: PP919605 and PP919606; act: PP921525 and PP921526; tub2: PP921527 and PP921528; gapdh: PP921529 and PP921530). Pathogenicity was done by spraying conidial suspension of AVCT01 (1 × 106/ml) on leaves of three plants (two months old). Non-injury technique of Gupta et al. (2020) was conducted for stem inoculation. The inoculated and control plants (sprayed with sterile distilled water) were maintained separately at 25°C with 90% relative humidity. Necrotic lesions were observed on leaves and stems of inoculated plants on 8th DAI confirming Koch's postulates and the pathogen re-isolation was also done. Disease severity was assessed using the scale of Yang and Hartman, 2015. Previously, leaf spots caused by C. gloeosporioides and C. dematium were reported in ashwagandha from West Bengal (Sarkar and Dasgupta, 2017) and Uttar Pradesh (Wijeskara et al. 2005) states of India. However, our study marks the first report of C. truncatum in ashwagandha, alerting the adaptation of multiple Colletotrichum species to this highly valued medicinal herb. Elucidating the etiology of newly adapted pathogen species paves the way for rapid disease diagnostics to implement effective disease management strategies.

Evaporation Dynamics on a Lithium Niobate Surface.

Manipulating the water evaporation dynamics is a prerequisite in various modern-day applications like DNA stretching, rapid disease diagnostics, and inkjet printing. One method to affect the evaporation dynamics of droplets is to externally apply electric fields. However, surfaces that bear an intrinsic surface charge have not yet been investigated with respect to their evaporation behavior. In this study, we investigate water droplet evaporation on lithium niobate (LN), a ferroelectric material with a very high spontaneous polarization of 0.7 . Our results show that a droplet deposited on an LN surface evaporates in three stages: (i) constant contact radius (ii) mixed phase (iii) stick-slip, which is likely originating from the intrinsic surface charge. The influence of the polarization direction of the LN surface as well as the relative humidity of the environment on various evaporation characteristics were studied. The results suggest that the specific adsorption layers forming on charged surfaces, e. g. from the humidity of the surrounding air, play a key role in the evaporation process. Furthermore, compared to other materials with similar contact angles, LN demonstrated a significantly large evaporation rate. This property might also be attributed to the intrinsic surface charge and could be exploited in heat transfer applications.

Drop-On-Demand Microdroplet Generation under Charge Injection by Corona Discharge.

In printing, microreactors, and bioassays, the precise control of micrometer-scale droplet generation is essential but challenging, often restricted by the equipment and nozzles used in traditional methods. We introduce a needle-plate electrode corona discharge technique that injects charges into an oil layer, enabling the precise manipulation of droplet polarization and splitting. This method allows for meticulous adjustment of microdroplet formation regarding location, size, and quantity by modulating the discharge voltage, discharge time, and electrode positioning. It enables the immediate initiation and cessation of droplet production, thereby facilitating on-demand droplet generation. Our study on the voltage-dependent droplet stretch coefficient shows that as the voltage increases, the droplets transition from controlled splitting to regular Taylor cone-like ejections, eventually reaching the Rayleigh limit and fully breaking apart. These advancements significantly improve microfluidic droplet manipulation, offering considerable benefits for applications in targeted drug delivery, rapid disease diagnostics, and precise environmental monitoring.

A portable droplet microfluidic device for cortisol measurements using a competitive heterogeneous assay.

Point-of-care monitoring of chemical biomarkers in real-time holds great potential in rapid disease diagnostics and precision medicine. However, monitoring is still rare in practice, as the measurement of biomarkers often requires time consuming and labour intensive assay procedures such as enzyme linked immunosorbent assay (ELISA), which pose a challenge to an autonomous point-of-care device. This paper describes a prototype device capable of performing ELISA autonomously and repeatedly in a high frequency using droplet microfluidics. Driven by a specially designed peristaltic pump, the device can collect liquid samples from a reservoir, produce trains of droplets, complete magnetic bead based ELISA protocols and provide readouts with colourimetric measurement. Here, cortisol was chosen as a target analyte as its concentration in the human body varies on a circadian rhythm which may be perturbed by disease. The prototype device draws in and analyses 350 nL of the sample containing free bioactive cortisol every 10 seconds, with a sample-to-signal time of 10 minutes, and measures favourably in the analytical range of 3.175-100 ng ml-1, with reliably lower variability compared with the well plate based assay. As most ELISA type assays share similar procedures, we envisage that this approach could form a platform technology for measurement or even continuous monitoring of biomarkers in biological fluids at the point-of-care.

A novel plant pathogen management tool for vector management.

Decision support systems often focus on insect control due to direct damage. However, when insects vector plant pathogens, these decision support systems must be tailored to disease management. However, a decision system that links diagnosticians to vector management is lacking and complicated by patterns of insect abundance over space and time. Here, we describe an approach that integrated monitoring of an insect pest (aster leafhopper; Macrosteles quadrilineatus, Forbes) that vectors aster yellows phytoplasma (Candidatus Phytoplasma spp.), with rapid disease diagnostics and web-based text messaging in two crops, carrots and celery. From 2014-2019, a total of 8,343 aster leafhoppers were collected, 99 of these were infected with phytoplasma. Text messaging reduced the number of infected leafhoppers. When we compared infected leafhopper density across crops, their temporal patterns were most similar at a 2-week delay. Comparisons within crop indicated that in celery uninfected and infected leafhopper density was most similar at a 2-week delay, but there was no similar pattern in carrots. Leafhopper density and infectivity were not similar beyond individual farms. Our results suggest that farmers should account for these temporal and spatial patterns when managing leafhoppers infected with aster yellows phytoplasma to improve pest management. By combining extensive monitoring, with rapid disease diagnostics, and text messaging, we demonstrate the value of our decision support tool. © 2020 Society of Chemical Industry.

Sensitive DNA detection by polymerase chain reaction with gold nanoparticles

We developed a novel strategy for rapid colorimetric detection of specific DNA sequence based on gold nanoparticles assemblies induced by polymerase chain reaction (PCR) product. In the presence of target DNA, the two DNA-functionalized AuNP probes selectively hybridized with the prohibited nucleic acid segments of two primers owing to the zipping off of the hairpin structures during PCR process, resulted in the aggregation of AuNPs with a concomitant color change from red to blue-purple. It is a convenient and universal method for sensitive DNA detection with no need for any further post-treatment of the PCR products. Most importantly, our method showed a low limit of detection (LOD) of 4.3 fM with a wide range of target DNA from 16 fM to 1.6 nM. Owing to the versatility and low cost, the proposed strategy could be extremely useful for a wide range of applications, providing a promising tool for rapid disease diagnostics and gene sequencing.

Soft Material-Enabled, Flexible Hybrid Electronics for Medicine, Healthcare, and Human-Machine Interfaces.

Flexible hybrid electronics (FHE), designed in wearable and implantable configurations, have enormous applications in advanced healthcare, rapid disease diagnostics, and persistent human-machine interfaces. Soft, contoured geometries and time-dynamic deformation of the targeted tissues require high flexibility and stretchability of the integrated bioelectronics. Recent progress in developing and engineering soft materials has provided a unique opportunity to design various types of mechanically compliant and deformable systems. Here, we summarize the required properties of soft materials and their characteristics for configuring sensing and substrate components in wearable and implantable devices and systems. Details of functionality and sensitivity of the recently developed FHE are discussed with the application areas in medicine, healthcare, and machine interactions. This review concludes with a discussion on limitations of current materials, key requirements for next generation materials, and new application areas.

Quantum dots and carbon dots based fluorescent sensors for TB biomarkers detection

The clinical use of volatile organic compounds (VOCs) detection for improved and rapid diagnoses of various diseases possesses a significant potential for the correct and timely screening of such diseases. Tuberculosis (TB) is a major pulmonary disease of concern, and can potentially be diagnosed using breath analysis techniques. Various sensing methods have been demonstrated and utilized to detect TB-VOCs. Some of the methods are based on electrical signal or change in resistance of device due to VOCs while some methods utilize mass spectroscopy. A low cost and quick method is very essential at this stage. Hence, in this paper, we report the synthesis of stable colloidal suspensions of CdSe QDs as well as carbon dots as a viable photoluminescent platform for detection of TB biomarkers. Such novel CdSe/carbon dots based sensing solution with tunable excitation and emission properties acts as a fluorescent probe for selective detection of TB biomarkers. The development of a smart, tunable fluorescent sensor system can provide an economically feasible solution and pave the way for rapid disease diagnostics.

Fabrication of Smart Au/TiO2 Nanotubes/Si Based Schottky-Tunneling Diode Sensors for Electrochemical Detection of Biomarkers

Rapid, point-of-care (POC) disease diagnostics presents several challenges including cost, detection time, equipment portability, and performance. Detection of volatile organic compounds (VOCs) based disease biomarkers from the breath provides a potential solution to the aforementioned problems. One such tuberculosis (TB) biomarker identified from breath is methyl nicotinate. In this regard, development of smart sensor systems can provide an economically feasible solution and pave the way for rapid disease diagnostics. In this paper, we present the fabrication of a smart sensor developed by synthesis of self-organized TiO2 nanotube arrays formed by anodization of thin Ti film deposited on Si wafers by direct current (D.C.) magnetron sputtering. Site-specific interdigitated patterned growth of gold electrode was achieved using photolithography techniques. The gold interdigitated electrodes behave as the working electrode while platinum deposited on the back surface of the Si wafer acts as the back contact. Au on TiO2 presents a Schottky junction due to differences in their work function and can be overcome by light irradiation. On the other hand, an ultra-thin, inherent SiO2 layer on Si aids in tunneling electrons across the TiO2 – Si junction. Thus, Au/TiO2/SiO2/Si behaves as a Schottky tunneling diode junction that electronically couples a VOC oxidation catalyst (Au) to a photovoltaic absorber (Si). A sweep of the bias voltage (-5V to 5V) and associated sensor response at room temperature upon exposure to both air and biomarker vapors (~1000 ppm) was carried out. From the current-voltage plot (I-V), the operating voltage was chosen overcoming the negative differential resistance such that the sensor current response to the biomarker vapor would be maximized under forward bias condition. The sensor current when exposed to the biomarker vapor was higher than that in air. All sensor measurements were carried out under illuminated conditions. The variation in the output current of the sensor biased at the chosen operating voltage (~ 0.7V) at room temperature was observed for the range of 0.01 ppm to 1000 ppm biomarker concentration. The sensor demonstrated a strong response and the results suggest that the sensor is capable of detecting extremely small concentrations of the biomarker in the vapor phase. This smart sensor presents significant enhancement in sensor response over previously reported metal functionalized TiO2 nanotube array based sensing platform developed in our group for electrochemical detection of similar disease biomarkers.

Developing a Broadband Amplifier for Analysis of DNA Structural and Molecular Characteristics

The recent emergence of DNA-based diagnostics increases the need for rapid DNA sequencing technologies. One method to achieve this is to pass DNA through a nanopore, recording the trans-membrane current with a low-noise current amplifier. The challenge presented in this method is that the bandwidth of commercially available current amplifiers is limited to 100kHz, which is not fast enough to resolve the signals present in DNA sequencing. To overcome this problem, researchers have developed a custom amplifier that has reached a bandwidth of approximately 1MHz, being able to reveal additional information during the DNA translocation events through a nanopore.We will demonstrate a design of a custom amplifier that offers a wider bandwidth than the current designs, enabling the study of DNA translocation without the need to limit the speed of translocation. In addition, an amplifier with a bandwidth larger than 1MHz allows discoveries to be made about information that might be present in a higher range of frequencies, enabling measurements at a higher time resolution than what was previously possible. The amplifier will be designed to allow direct integration of a micro- or nanopore sensing area on the same physical substrate, eliminating the need for external electrode wiring. The outcomes from this research open up the possibility of an integrated high-speed DNA sequencer chip enabling rapid disease diagnostics.

One-pot polymerase chain reaction with gold nanoparticles for rapid and ultrasensitive DNA detection

We report a novel method for rapid, colorimetric detection of a specific deoxyribonucleic acid (DNA) sequence by carrying out a polymerase chain reaction in the presence of gold nanoparticles functionalized with two primers. Extension of the primers when the target DNA is present as a template during the polymerase chain reaction process affords the complementary sequences on the gold nanoparticle surfaces and results in the formation of gold nanoparticle aggregates with a concomitant color change from red to pinkish/purple. This method provides a convenient and straightforward solution for ultrasensitive DNA detection without any further post-treatment of the polymerase chain reaction products being necessary, and is a promising tool for rapid disease diagnostics and gene sequencing.