A smartphone-integrated aptasensor for pesticide detection using gold-decorated microparticles.

The increasing incidence of environmental concerns related to excessive use of pesticides, such as imidacloprid and carbendazim, poses risks to pollinators, water bodies, and human health, prompting regulatory scrutiny and bans in developed countries. In this study, we propose a portable smartphone-based biosensor for rapid and label-free colorimetric detection by using the gold-decorated polystyrene microparticles (Ps-AuNP) functionalized with specific aptamers to imidacloprid and carbendazim on a microfluidic paper-based analytical device (µ-PAD). Four aptamers were selected for the detection of these pesticides and their sensitivity and selectivity performance was evaluated. The sensitivity results show a detection limit for imidacloprid of 3.12 ppm and 1.56 ppm for carbendazim. The aptamers also exhibited high selectivity performance against other pesticides, such as thiamethoxam, fenamiphos, isoproturon, and atrazine. However, the platform presented cross-selectivity when detecting imidacloprid, carbendazim, and linuron, which is discussed herein. Overall, we present a promising platform for simple, on-site, and rapid colorimetric screening of specific pesticides, while highlighting the challenges of aptasensors in achieving selectivity amidst diverse molecular structures.

Application of a dual-modality colorimetric analysis method to inkjet printing lateral flow detection of Salmonella typhimurium.

Lateral flow assay (LFA) color signal quantification methods were developed by utilizing both International Commission on Illumination (CIE) LAB (CIELAB) color space and grayscale intensity differences. The CIELAB image processing procedure included calibration, test, control band detection, and color difference calculation, which can minimize the noise from the background. The LFA platform showcases its ability to accurately discern relevant colorimetric signals. The rising occurrence of infectious outbreaks from foodborne pathogens like Salmonella typhimurium presents significant economic, healthcare, and public health risks. The study introduces an aptamer-based lateral flow (ABLF) platform by using inkjet printing for specially detecting S. typhimurium. The ABLF utilized gold-decorated polystyrene microparticles, functionalized with specific S. typhimurium aptamers (Ps-AuNPs-ssDNA). The platform demonstrates a detection limit of 102 CFU mL-1 in buffer solutions and 103 CFU mL-1 in romaine lettuce tests. Furthermore, it sustained performance for over 8 weeks at room temperature. The ABLF platform and analysis methods are expected to effectively resolve the low-sensitivity problems of the former LFA systems and to bridge the gap between lab-scale platforms to market-ready solutions by offering a simple, cost-effective, and consistent approach to detecting foodborne pathogens in real samples.

Recent Advances in Aptamer-Based Biosensors for Global Health Applications.

Since aptamers were first reported in the early 2000s, research on their use for the detection of health-relevant analytical targets has exploded. This review article provides a brief overview of the most recent developments in the field of aptamer-based biosensors for global health applications. The review provides a description of general aptasensing principles and follows up with examples of recent reports of diagnostics-related applications. These applications include detection of proteins and small molecules, circulating cancer cells, whole-cell pathogens, extracellular vesicles, and tissue diagnostics. The review also discusses the main challenges that this growing technology faces in the quest of bringing these new devices from the laboratory to the market.

Inkjet Printed Nanopatterned Aptamer-Based Sensors for Improved Optical Detection of Foodborne Pathogens.

The increasing incidence of infectious outbreaks from contaminated food and water supply continues imposing a global burden for food safety, creating a market demand for on-site, disposable, easy-to-use, and cost-efficient devices. Despite of the rapid growth of biosensors field and the generation of breakthrough technologies, more than 80% of the platforms developed at lab-scale never will get to meet the market. This work aims to provide a cost-efficient, reliable, and repeatable approach for the detection of foodborne pathogens in real samples. For the first time an optimized inkjet printing platform is proposed taking advantage of a carefully controlled nanopatterning of novel carboxyl-functionalized aptameric ink on a nitrocellulose substrate for the highly efficient detection of E. coli O157:H7 (25 colony forming units (CFU) mL-1 in pure culture and 233 CFU mL-1 in ground beef) demonstrating the ability to control the variation within ±1 SD for at least 75% of the data collected even at very low concentrations. From the best of the knowledge this work reports the lowest limit of detection of the state of the art for paper-based optical detection of E. coli O157:H7, with enough evidence (p > 0.05) to prove its high specificity at genus, species, strain, and serotype level.

Versatile printed microheaters to enable low-power thermal control in paper diagnostics.

As the capabilities of low-resource field testing have begun to expand to incorporate more complex diagnostic technologies, many of these devices remain tethered to large heaters requiring relatively high-power inputs. Highly efficient microheaters would enable miniaturization of devices for more economic and effective heating with high temperatures and sustained incubation. This work reports the development and application of resistive microheaters printed with nanosilver ink for improved methods of automated sample heating in paper-based point-of-care (POC) and in-field diagnostics. Resistance is easily predicted, and shapes can be altered to fit space and heat-transfer needs, sustained and discrete heating of precise regions are possible. Here, we demonstrate both isothermal nucleic acid amplification at 65 °C and bacterial culture at 37 °C using our microheaters. Printed nanosilver microheaters are easily integrated into reactions that require low-power battery heating, can sustain heating for 16-hour incubations, and cost between 0.17 and 0.58 US dollars each. Further, the microheaters are reusable, stable over 6 months, and can be wetted without degradation or reduction in conductivity. These versatile printed microheaters enable thermal control for a variety of low power heating applications.

Aminolated and Thiolated PEG-Covered Gold Nanoparticles with High Stability and Antiaggregation for Lateral Flow Detection of Bisphenol A.

The few lateral flow assays (LFAs) established for detecting the endocrine disrupting chemical bisphenol A (BPA) have employed citrate-stabilized gold nanoparticles (GNPs), which have inevitable limitations and instability issues. To address these limitations, a more stable and more sensitive biosensor is developed by designing strategies for modifying the surfaces of GNPs with polyethylene glycol and then testing their effectiveness and sensitivity toward BPA in an LFA. Without the application of any enhancement strategy, this modified BPA LFA can achieve a naked-eye limit of detection (LOD) of 0.8 ng mL-1 , which is 12.5 times better than the LOD of regular BPA LFAs, and a quantitative LOD of 0.472 ng mL-1 . This modified LFA has the potential to be applied to the detection of various antigens.

Effects of impaired membrane interactions on α-synuclein aggregation and neurotoxicity.

The post-mortem brains of individuals with Parkinson's disease (PD) and other synucleinopathy disorders are characterized by the presence of aggregated forms of the presynaptic protein α-synuclein (aSyn). Understanding the molecular mechanism of aSyn aggregation is essential for the development of neuroprotective strategies to treat these diseases. In this study, we examined how interactions between aSyn and phospholipid vesicles influence the protein's aggregation and toxicity to dopaminergic neurons. Two-dimensional NMR data revealed that two familial aSyn mutants, A30P and G51D, populated an exposed, membrane-bound conformer in which the central hydrophobic region was dissociated from the bilayer to a greater extent than in the case of wild-type aSyn. A30P and G51D had a greater propensity to undergo membrane-induced aggregation and elicited greater toxicity to primary dopaminergic neurons compared to the wild-type protein. In contrast, the non-familial aSyn mutant A29E exhibited a weak propensity to aggregate in the presence of phospholipid vesicles or to elicit neurotoxicity, despite adopting a relatively exposed membrane-bound conformation. Our findings suggest that the aggregation of exposed, membrane-bound aSyn conformers plays a key role in the protein's neurotoxicity in PD and other synucleinopathy disorders.

Overexpression of alpha-synuclein at non-toxic levels increases dopaminergic cell death induced by copper exposure via modulation of protein degradation pathways.

Gene multiplications or point mutations in alpha (α)-synuclein are associated with familial and sporadic Parkinson's disease (PD). An increase in copper (Cu) levels has been reported in the cerebrospinal fluid and blood of PD patients, while occupational exposure to Cu has been suggested to augment the risk to develop PD. We aimed to elucidate the mechanisms by which α-synuclein and Cu regulate dopaminergic cell death. Short-term overexpression of wild type (WT) or mutant A53T α-synuclein had no toxic effect in human dopaminergic cells and primary midbrain cultures, but it exerted a synergistic effect on Cu-induced cell death. Cell death induced by Cu was potentiated by overexpression of the Cu transporter protein 1 (Ctr1) and depletion of intracellular glutathione (GSH) indicating that the toxic effects of Cu are linked to alterations in its intracellular homeostasis. Using the redox sensor roGFP, we demonstrated that Cu-induced oxidative stress was primarily localized in the cytosol and not in the mitochondria. However, α-synuclein overexpression had no effect on Cu-induced oxidative stress. WT or A53T α-synuclein overexpression exacerbated Cu toxicity in dopaminergic and yeast cells in the absence of α-synuclein aggregation. Cu increased autophagic flux and protein ubiquitination. Impairment of autophagy by overexpression of a dominant negative Atg5 form or inhibition of the ubiquitin/proteasome system (UPS) with MG132 enhanced Cu-induced cell death. However, only inhibition of the UPS stimulated the synergistic toxic effects of Cu and α-synuclein overexpression. Our results demonstrate that α-synuclein stimulates Cu toxicity in dopaminergic cells independent from its aggregation via modulation of protein degradation pathways.

Cu(II) promotes amyloid pore formation.

The aggregation of α-synuclein is associated with dopamine neuron death in Parkinson's disease. There is controversy in the field over the question of which species of the aggregates, fibrils or protofibrils, are toxic. Moreover, compelling evidence suggested the exposure to heavy metals to be a risk of PD. Nevertheless, the mechanism of metal ions in promoting PD remains unclear. In this research, we investigated the structural basis of Cu(II) induced aggregation of α-synuclein. Using transmission electron microscopy experiments, Cu(II) was found to promote in vitro aggregation of α-synuclein by facilitating annular protofibril formation rather than fibril formation. Furthermore, neuroprotective baicalein disaggregated annular protofibrils accompanied by considerable decrease of ß-sheet content. These results strongly support the hypothesis that annular protofibrils are the toxic species, rather than fibrils, thereby inspiring us to search novel therapeutic strategies for the suppression of the toxic annular protofibril formation.

In vitro study of α-synuclein protofibrils by cryo-EM suggests a Cu(2+)-dependent aggregation pathway.

The aggregation of α-synuclein is thought to play a role in the death of dopamine neurons in Parkinson's disease (PD). Alpha-synuclein transitions itself through an aggregation pathway consisting of pathogenic species referred to as protofibrils (or oligomer), which ultimately convert to mature fibrils. The structural heterogeneity and instability of protofibrils has significantly impeded advance related to the understanding of their structural characteristics and the amyloid aggregation mystery. Here, we report, to our knowledge for the first time, on α-synuclein protofibril structural characteristics with cryo-electron microscopy. Statistical analysis of annular protofibrils revealed a constant wall thickness as a common feature. The visualization of the assembly steps enabled us to propose a novel, to our knowledge, mechanisms for α-synuclein aggregation involving ring-opening and protofibril-protofibril interaction events. The ion channel-like protofibrils and their membrane permeability have also been found in other amyloid diseases, suggesting a common molecular mechanism of pathological aggregation. Our direct visualization of the aggregation pathway of α-synuclein opens up fresh opportunities to advance the understanding of protein aggregation mechanisms relevant to many amyloid diseases. In turn, this information would enable the development of additional therapeutic strategies aimed at suppressing toxic protofibrils of amyloid proteins involved in neurological disorders.

Simultaneous colorimetric and electrochemical detection of trace mercury (Hg2+) using a portable and miniaturized aptasensor.

We report a novel aptasensor for the simultaneous colorimetric and electrochemical detection of mercury (Hg2+). This device consists of a paper-based microfluidic component (µ-PAD) incorporated into a miniaturized three-electrode system fabricated through printed circuit board (PCB) technology. This biosensor is portable, rapid, versatile, and can detect Hg2+ down to 0.01 ppm based on 3σ of the blank/slope criteria. Moreover, it is highly selective against As2+, Cu2+, Zn2+, Pb2+, Cd2+, Mg2+, and Fe2+, reaching up to 13 times more of the input signal than the other heavy metals. The colorimetric detection mechanism uses aptamer functionalized polystyrene (PS)-AgNPs and Ps-AuNPs microparticles' specific aggregation. The Ps-AuNPs-based system allows qualitative detection (LOD 5 ppm) and stability over seven days (up to 97.59% signal retention). For the Ps-AgNPs-based system, the detection limit is 0.5 ppm with a linear range from 0.5 to 20 ppm (adjusted R2= 0.986) and stability over 30 days (up to 94.95% signal retention). The electrochemical component measures changes in charge transfer resistance upon target-aptamer hybridization using a [Ru (NH3)6]3+Cl3] redox probe. The latest component presents a linear range from 0.01 to 1 ppm (adjusted R2= 0.935) with a LOD of 0.01 ppm and performance stability over seven days (up to 102.52 ± 11.7 signal retention). This device offers a universal dual detection platform with multiplexing, multi-replication, quantitative color analysis, and minimization of false results. Furthermore, detection results in river samples showed recoveries up to 91.12% (RSD 0.85) and 105.61% (RSD 1.62) for the electrochemical and colorimetric components, respectively. The proposed system is highly selective with no false-positive or false-negative results in an overall wide linear range and can safeguard the accuracy of detection results in aptasensing platforms in general.

Smartphone-based colorimetric detection of cardiac troponin T via label-free aptasensing.

We report an aptasensing platform for the detection of cardiac troponin T (cTnT) in the immediate and early phases of acute myocardial infarction (AMI). High-flow filter paper was used to fabricate a microfluidic paper-based analytical device (µ-PAD), which was further modified with gold-decorated polystyrene microparticles functionalized with a highly specific cTnT aptamer. Herein, cTnT detection is presented in two linear ranges (0.01-0.8 µg/ml and 6.25-50 µg/ml) with an LoD of 3.9X10-4 µg/ml, which is in agreement with reference values determined by the American Heart Association. The proposed platform showed remarkable selectivity against AMI-associated cardiac biomarkers such as TNF-alpha, interleukin-6, cardiac troponin I, and reactive protein-C. This aptasensor is a label-free assay that relies only on smartphone-based image analysis and takes less processing time in comparison with traditional methods like ELISA. Furthermore, it exhibits outstanding stability over 23 days when devices are stored at 4 °C. The reported platform is a stable and cost-effective method for the on-site and user-friendly detection of cTnT in normal saline buffer and diluted human serum.

Cold Atmospheric Plasma-Assisted Direct Deposition of Polypyrrole-Ag Nanocomposites for Flexible Electronic Sensors.

Conductive polymers and their composite materials have attracted considerable interest due to their potential applications in sensors, actuators, drug delivery systems, and energy storage devices. Despite their wide range of applications, many challenges remain primarily with respect to the complex synthesis and time-consuming manufacturing steps that are often required in the fabrication process of various devices with conductive polymers. Here, we demonstrate the novel use of cold atmospheric plasma (CAP)-assisted deposition technologies as a solvent-free and scalable approach for in situ polymerization and direct deposition of conductive polypyrrole-silver (PPy-Ag) nanocomposites onto the desired substrates under atmospheric conditions. In this study, a systematic approach with different precursor composition mixtures containing pyrrole as the monomer and AgNO3 as the photoinitiator was investigated to assess the effect of precursor composition on the final chemical, electrical, and mechanical properties of the PPy-Ag nanocomposite thin-film coatings which were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, and cyclic bending tests. The characterizations indicated the possibility of fabricating PPy-Ag nanocomposite films with tunable degrees of polymerization and Ag nanoparticle loading by simply varying the percentage of AgNO3 in precursor composition mixtures. Finally, as a proof of concept, the potential use of the PPy-Ag nanocomposite films with different Ag nanoparticle loading percentages was assessed for humidity sensing by measuring their level of change in electrical resistance in the relative humidity range of 12-60%. It is envisioned that the developed CAP-assisted deposition technology can provide a new stepping stone toward scalable additive manufacturing of various functional nanocomposite films for different low-cost and flexible electronic applications.

Cu2O and Au/Cu2O particles: surface properties and applications in glucose sensing.

In this work we investigated the surface and facet-dependent catalytic properties of metal oxide particles as well as noble metal/metal oxide heterogeneous structures, with cuprous oxide (Cu(2)O) and Au/Cu(2)O being selected as model systems. As an example of application, we explored the potential of these materials in developing electrocatalytic devices. Cu(2)O particles were synthesized in various shapes, then used for testing their morphology-dependent electrochemical properties applied to the detection of glucose. While we did not attempt to obtain the best detection limit reported to date, the octahedral and hexapod Cu(2)O particles showed reasonable detection limits of 0.51 and 0.60 mM, respectively, which are physiologically relevant concentrations. However, detection limit seems to be less affected by particle shapes than sensitivity. Heterogeneous systems where Au NPs were deposited on the surface of Cu(2)O particles were also tested with similar results in terms of the effect of surface orientation.

Noble-Nanoparticle-Decorated Ti3C2T x MXenes for Highly Sensitive Volatile Organic Compound Detection.

Two-dimensional transition-metal carbides and nitrides (MXenes) have been regarded as promising sensing materials because of their high surface-to-volume ratios and outstanding electronic, optical, and mechanical properties with versatile transition-metal and surface chemistries. However, weak gas-molecule adsorption of MXenes poses a serious limitation to their sensitivity and selectivity, particularly for trace amounts of volatile organic compounds (VOCs) at room temperature. To deal with these issues, Au-decorated MXenes are synthesized by a facile solution mixing method for room-temperature sensing of a wide variety of oxygen-based and hydrocarbon-based VOCs. Dynamic sensing experiments reveal that optimal decoration of Au nanoparticles (NPs) on Ti3C2T x MXene significantly elevates the response and selectivity of the flexible sensors, especially in detecting formaldehyde. Au-Ti3C2T x gas sensors exhibited an extremely low limit of detection of 92 ppb for formaldehyde at room temperature. Au-Ti3C2T x provides reliable gas response, low noise level, ultrahigh signal-to-noise ratio, high selectivity, as well as parts per billion level of formaldehyde detection. The prominent mechanism for Au-Ti3C2T x in sensing formaldehyde is elucidated theoretically from density functional theory simulations. The results presented here strongly suggest that decorating noble-metal NPs on MXenes is a feasible strategy for the development of next-generation ultrasensitive sensors for Internet of Things.

DNA-Functionalized Ti3C2Tx MXenes for Selective and Rapid Detection of SARS-CoV-2 Nucleocapsid Gene.

Coronavirus disease 2019 (COVID-19) is an emerging human infectious disease caused by severe acute respiratory syndrome 2 (SARS-CoV-2, initially called novel coronavirus 2019-nCoV) virus. Thus, an accurate and specific diagnosis of COVID-19 is urgently needed for effective point-of-care detection and disease management. The reported promise of two-dimensional (2D) transition-metal carbides (Ti3C2Tx MXene) for biosensing owing to a very high surface area, high electrical conductivity, and hydrophilicity informed their selection for inclusion in functional electrodes for SARS-CoV-2 detection. Here, we demonstrate a new and facile functionalization strategy for Ti3C2Tx with probe DNA molecules through noncovalent adsorption, which eliminates expensive labeling steps and achieves sequence-specific recognition. The 2D Ti3C2Tx functionalized with complementary DNA probes shows a sensitive and selective detection of nucleocapsid (N) gene from SARS-CoV-2 through nucleic acid hybridization and chemoresistive transduction. The fabricated sensors are able to detect the SARS-CoV-2 N gene with sensitive and rapid response, a detection limit below 105 copies/mL in saliva, and high specificity when tested against SARS-CoV-1 and MERS. We hypothesize that the MXenes' interlayer spacing can serve as molecular sieving channels for hosting organic molecules and ions, which is a key advantage to their use in biomolecular sensing.

Microfluidic paper-based aptasensor devices for multiplexed detection of pathogenic bacteria.

Foodborne pathogens are major public health concerns worldwide. Paper-based microfluidic devices are versatile, user friendly and low cost. We report a novel paper-based single input channel microfluidic device that can detect more than one whole-cell foodborne bacteria at the same time, and comes with quantitative reading via image analysis. This microfluidic paper-based multiplexed aptasensor simultaneously detects E. coli O157:H7 and S. Typhimurium. Custom designed particles provide colorimetric signal enhancement and false results prevention. Several aptamers were screened and the highest-affinity aptamers were optimized and employed for detection of these bacteria in solution, both in a buffer as well as pear juice. Image analysis was used to read and quantify the colorimetric signal and measure bacteria concentration, thus rendering this paper based microfluidic device quantitative. The colorimetric results show linearity over a wide concentration range (102CFU/mL to 108CFU/mL) and a limit of detection (LOD) of 103CFU/mL and 102CFU/mL for E. coli O157:H7 and S. Typhimurium, respectively. An insignificant change in colorimetric response for non-target bacteria indicates the aptasesnors are specific. The reported multiplexed colorimetric paper-based microfluidic devices is likely to perform well for on-site rapid screening of pathogenic bacteria in water and food products.

Folding dynamics of phenylalanine hydroxylase depends on the enzyme's metallation state: the native metal, iron, protects against aggregate intermediates.

Phenylalanine hydroxylase (PAH), a non-heme iron enzyme, is responsible for the phenylalanine conversion to tyrosine. Its malfunction causes phenylketonuria (PKU). To better understand how protein structure and folding profiles are affected by the metal cofactor, we investigated the chemical (un)folding of apo- and holo-PAH from Chromobacterium violaceum (cPAH) using circular dichroism (CD) and analytical ultracentrifugation (AUC). Holo-cPAH shows a two-state unfolding transition. In contrast, the unfolding profile for apo-cPAH reveals a three-state (un)folding pathway and accumulation of an intermediate (apo-cPAH(I)). This intermediate is also observed in refolding experiments. Fluorescence studies are consistent with the CD findings. The intermediate apo-cPAH(I) and unfolded state(s) of apo- and holo-cPAH(U) have been characterized by analytical ultracentrifugation (AUC). At 2.4 and 2.8 M GuHCl, 90% of the signal for apo-cPAH has a weight average sedimentation coefficient in water at 20°C (s20,w) of about 48 S, representing multiple aggregate species made of multiple monomers of cPAH. Aggregate formation for apo-cPAH is also confirmed by dynamic light scattering and electron microscopy giving a hydrodynamic radius (R(H)) of 41 nm for apo-cPAH(I) versus 3.5 nm for the native protein.

Structure of TRPV1 channel revealed by electron cryomicroscopy.

The transient receptor potential (TRP) family of ion channels participate in many signaling pathways. TRPV1 functions as a molecular integrator of noxious stimuli, including heat, low pH, and chemical ligands. Here, we report the 3D structure of full-length rat TRPV1 channel expressed in the yeast Saccharomyces cerevisiae and purified by immunoaffinity chromatography. We demonstrate that the recombinant purified TRPV1 channel retains its structural and functional integrity and is suitable for structural analysis. The 19-A structure of TRPV1 determined by using single-particle electron cryomicroscopy exhibits fourfold symmetry and comprises two distinct regions: a large open basket-like domain, likely corresponding to the cytoplasmic N- and C-terminal portions, and a more compact domain, corresponding to the transmembrane portion. The assignment of transmembrane and cytoplasmic regions was supported by fitting crystal structures of the structurally homologous Kv1.2 channel and isolated TRPV1 ankyrin repeats into the TRPV1 structure.

Roll-to-Roll Manufactured Sensors for Nitroaromatic Organophosphorus Pesticides Detection.

A fully roll-to-roll manufactured electrochemical sensor with high sensing and manufacturing reproducibility has been developed for the detection of nitroaromatic organophosphorus pesticides (NOPPs). This sensor is based on a flexible, screen-printed silver electrode modified with a graphene nanoplatelet (GNP) coating and a zirconia (ZrO2) coating. The combination of the metal oxide and the 2-D material provided advantageous electrocatalytic activity toward NOPPs. Manufacturing, scanning electron microscopy-scanning transmission electron microscopy image analysis, electrochemical surface characterization, and detection studies illustrated high sensitivity, selectivity, and stability (∼89% current signal retention after 30 days) of the platform. The enzymeless sensor enabled rapid response time (10 min) and noncomplex detection of NOPPs through voltammetry methods. Furthermore, the proposed platform was highly group-sensitive toward NOPPs (e.g., methyl parathion (MP) and fenitrothion) with a detection limit as low as 1 µM (0.2 ppm). The sensor exhibited a linear correlation between MP concentration and current response in a range from 1 µM (0.2 ppm) to 20 µM (4.2 ppm) and from 20 to 50 µM with an R2 of 0.992 and 0.991, respectively. Broadly, this work showcases the first application of GNPs/ZrO2 complex on flexible silver screen-printed electrodes fabricated by entirely roll-to-roll manufacturing for the detection of NOPPs.