An Aptamer-Based Hydrogel Sensing Platform Enabling Low Micromolar Detection of Small Molecules Using Low Sample Volumes.

Poor fluorescence recovery at low analyte dosages and slow ligand binding kinetics are critical challenges currently limiting the use of aptamer-functionalized hydrogels for sensing small molecules. In this paper, we report an adenosine-responsive hydrogel sensor that integrates FRET-signaling aptamer switches into in situ-gelling thin-film hydrogels. The hydrogel sensor is able to entrap a high proportion of the sensing probes (>70% following vigorous washing), delay nucleolytic degradation, stabilize weak aptamer complexes to improve hybridization affinity and suppress fluorescence background, and provide high sensitivity in biological fluids (i.e., undiluted human serum). Furthermore, the developed hydrogel sensors were able to achieve low limits of detection (5.3 µM in buffer and 8.8 µM in serum) within 4 min of exposure to the sample, with signal generation requiring only 20 µL/well of analyte sample. The physical nature of the aptamer encapsulation allows this approach to accommodate virtually any small-molecule aptamer, avoiding the need for covalent anchoring and the complex modification of nucleic acid sequences typically required for effective aptamer-based molecular recognition.

Comprehensive fluorescence profiles of contamination-prone foods applied to the design of microcontact-printed in situ functional oligonucleotide sensors.

With both foodborne illness and food spoilage detrimentally impacting human health and the economy, there is growing interest in the development of in situ sensors that offer real-time monitoring of food quality within enclosed food packages. While oligonucleotide-based fluorescent sensors have illustrated significant promise, the development of such on-food sensors requires consideration towards sensing-relevant fluorescence properties of target food products-information that has not yet been reported. To address this need, comprehensive fluorescence profiles for various contamination-prone food products are established in this study across several wavelengths and timepoints. The intensity of these food backgrounds is further contextualized to biomolecule-mediated sensing using overlaid fluorescent oligonucleotide arrays, which offer perspective towards the viability of distinct wavelengths and fluorophores for in situ food monitoring. Results show that biosensing in the Cyanine3 range is optimal for all tested foods, with the Cyanine5 range offering comparable performance with meat products specifically. Moreover, recognizing that mass fabrication of on-food sensors requires rapid and simple deposition of sensing agents onto packaging substrates, RNA-cleaving fluorescent nucleic acid probes are successfully deposited via microcontact printing for the first time. Direct incorporation onto food packaging yields cost-effective sensors with performance comparable to ones produced using conventional deposition strategies.

Sub-second timing irregularities in a simple motor task in autism spectrum disorder: Preliminary effects of intermittent light stimulation.

Several authors have contributed extensively to the neurocognitive understanding of timing. In Autism Spectrum Disorder (ASD) on the contrary, internal timing and its functioning is not well understood. In this study, we have adapted a simple finger-tapping motor task, with a timing component, as we aim at understanding whether the processing of time is preserved in this population. We have tested a group of people on the autism spectrum without intellectual disabilities and a control sample recruited from the general population, matched for age, sex, schooling and general cognitive abilities on this task with a learning and testing phase. In the testing phase, we have added two exploratory conditions where participants were exposed to intermittent light stimulation of 4 and 8 Hz. Results show that both in the learning and testing phase, besides troubles in the motor component encountered by the people on the spectrum, their timing component performance was also problematic. This reveals to be especially true for time intervals below the 1 s range, as hypothesized, whereas performance in longer intervals is clearly preserved. It was also observed that the exposure to intermittent light stimulation specifically overcomes the difficulties observed in the autistic group, at the timing components at this millisecond time range. The observed timing difficulties in this group seem to be restricted to the system responsible for the processing of time intervals in the milliseconds range, which helps accommodate disparate findings in the literature.

Material Breakthroughs in Smart Food Monitoring: Intelligent Packaging and On-Site Testing Technologies for Spoilage and Contamination Detection.

Despite extensive commercial and regulatory interventions, food spoilage and contamination continue to impose massive ramifications on human health and the global economy. Recognizing that such issues will be significantly eliminated by the accurate and timely monitoring of food quality markers, smart food sensors have garnered significant interest as platforms for both real-time, in-package food monitoring and on-site commercial testing. In both cases, the sensitivity, stability, and efficiency of the developed sensors are largely informed by underlying material design, driving focus toward the creation of advanced materials optimized for such applications. Herein, a comprehensive review of emerging intelligent materials and sensors developed in this space is provided, through the lens of three key food quality markers - biogenic amines, pH, and pathogenic microbes. Each sensing platform is presented with targeted consideration toward the contributions of the underlying metallic or polymeric substrate to the sensing mechanism and detection performance. Further, the real-world applicability of presented works is considered with respect to their capabilities, regulatory adherence, and commercial potential. Finally, a situational assessment of the current state of intelligent food monitoring technologies is provided, discussing material-centric strategies to address their existing limitations, regulatory concerns, and commercial considerations.

Miniaturization of an enclosed electrospinning process to enhance reproducibility in the fabrication of rapidly dissolving cell-based biosensors.

There is broad interest in producing electrospun films embedded with biological materials. It is well known that electrospinning requires careful control of the process conditions, especially the environmental conditions such as relative humidity (RH). Given that commercial electrospinning systems are expensive (> $10,000) and are typically too large to be used in standard biological safety cabinets (BSC), we designed and built a miniaturized electrospinning box (E-Box) that will fit inside a BSC, and the RH can be easily controlled using simple instrumentation (gas cylinder, regulator, needle valve, rotameter). It uses an inexpensive computerized numerical control machine to control the spinneret positioning and collector rotational speed-all the parts for the device (except the syringe pump and voltage supply) can be purchased for approximately $1000. We demonstrate the usefulness of our design in optimizing the production of Escherichia coli-embedded pullulan-trehalose films to be used as rapidly dissolving biosensors for environmental monitoring. At a fixed electrospinning recipe, we showed that decreasing the RH from approximately 48% to 22% resulted in the average fiber diameter increasing from 240 (± 11) nm to 314 (± 8) nm. We also demonstrate the usefulness of our design in performing sequential electrospinning experiments to evaluate process performance reproducibility. For example, from just 1 mL of a polymer solution, we produced 16 electrospun films (approximately 3 cm by 8 cm each)-from those films we hole-punched approximately 80 biosensor discs which were then used in subsequent experiments to determine the amount of two different biocides (Grotan BK and triclosan) in aqueous samples. The technique developed in this study is ideal for creating electrospun materials in high quantities that are highly reproducible through the precise control of RH.

Advancing In Situ Food Monitoring through a Smart Lab-in-a-Package System Demonstrated by the Detection of Salmonella in Whole Chicken.

With food production shifting away from traditional farm-to-table approaches to efficient multistep supply chains, the incidence of food contamination has increased. Consequently, pathogen testing via inefficient culture-based methods has increased, despite its lack of real-time capabilities and need for centralized facilities. While in situ pathogen detection would address these limitations and enable individual product monitoring, accurate detection within unprocessed, packaged food products without user manipulation has proven elusive. Herein, "Lab-in-a-Package" is presented, a platform capable of sampling, concentrating, and detecting target pathogens within closed food packaging, without intervention. This system consists of a newly designed packaging tray and reagent-infused membrane that can be paired universally with diverse pathogen sensors. The inclined food packaging tray maximizes fluid localization onto the sensing interface, while the membrane acts as a reagent-immobilizing matrix and an antifouling barrier for the sensor. The platform is substantiated using a newly discovered Salmonella-responsive nucleic acid probe, which enables hands-free detection of 103 colony forming units (CFU) g-1 target pathogen in a packaged whole chicken. The platform remains effective when contamination is introduced with toolsand surfaces, ensuring widespread efficacy. Its real-world use for in situ detection is simulated using a handheld fluorescence scanner with smartphone connectivity.

Tuning the sensitivity of cell-based biosensors for the detection of biocides.

The presence of biocides in wastewater can negatively impact the efficiency of wastewater treatment processes, particularly the process of nitrification. In this paper, we describe the development of cell-based biosensors (CBBs) with tunable levels of sensitivity for rapidly detecting the presence and predicting the type and concentration of biocides. The CBB assay developed is performed by first exposing a panel of bacterial strains (E. coli, B. subtilis, B. cereus) to the sample being tested and to the control sample without biocide, and then adding a fluorescent dye (LIVE/DEAD BacLight). We then compare the fluorescence signals generated by the two samples, and the differences in the signals indicate the presence of a biocide, as previously reported in the literature. We found that the sensitivity of the CBB assay can be improved by 'tuning' the type/salinity of the buffer used to suspend the cells, and by changing the number of cells used in the assay. These changes improved the level of detection (LOD) of the biocide Cetyltrimethylammonium bromide (CTAB) from 10 ppm to 0.625 ppm and the biocide Grotan® BK from 500 ppm to 7.8 ppm. With the optimized conditions for each strain, we also establish that the combined response from the panel of bacterial strains can be used to predict the type and concentration of biocide sample tested. Additionally, we provide evidence that the CBB assay can be performed using a compact, commercially available fluorometer. Overall, the significance of this work will improve point-of-use testing and enable the discrimination between biocide-containing samples of similar toxicity and detection of lower toxicity samples, thereby improving the accuracy of the CBB assay.

A Simple Colorimetric Au-on-Au Tip Sensor with a New Functional Nucleic Acid Probe for Food-borne Pathogen Salmonella typhimurium.

An Au-on-Au tip sensor is developed for the detection of Salmonella typhimurium (Salmonella), using a new synthetic nucleic acid probe (NAP) as a linker for the immobilization of a DNA-conjugated Au nanoparticle (AuNP) onto a DNA-attached thin Au layer inside a pipette tip. In the presence of Salmonella, RNase H2 from Salmonella (STH2) cleaves the NAP and the freed DNA-conjugated AuNP can be visually detected by a paper strip. This portable biosensor does not require any electronic, electrochemical or optical equipment. It delivers a detection limit of 3.2×103  CFU mL-1 for Salmonella in 1 h without cell-culturing or signal amplification and does not show cross-reactivity with several control bacteria. Further, the sensor reliably detects Salmonella spiked in food samples, such as ground beef and chicken, milk, and eggs. The sensor can be reused and is stable at ambient temperature, showing its potential as a point-of-need device for the prevention of food poisoning by Salmonella.

Detection of Large Genomic RNA via DNAzyme-Mediated RNA Cleavage and Rolling Circle Amplification: SARS-CoV-2 as a Model.

A new method for the detection of genomic RNA combines RNA cleavage by the 10-23 DNAzyme and use of the cleavage fragments as primers to initiate rolling circle amplification (RCA). 230 different 10-23 DNAzyme variants were screened to identify those that target accessible RNA sites within the highly structured RNA transcripts of SARS-CoV-2. A total of 28 DNAzymes were identified with >20 % cleavage, 5 with >40 % cleavage and one with >60 % in 10 min. The cleavage fragments from these reactions were then screened for coupling to an RCA reaction, leading to the identification of several cleavage fragments that could efficiently initiate RCA. Using a newly developed quasi-exponential RCA method with a detection limit of 500 aM of RNA, 14 RT-PCR positive and 15 RT-PCR negative patient saliva samples were evaluated for SARS-CoV-2 genomic RNA, achieving a clinical sensitivity of 86 % and specificity of 100 % for detection of the virus in <2.5 h.

Accidental guide wire migration and late percutaneous externalization after central venous catheterization.

A 70-year-old man was admitted to the emergency department with recent spontaneous externalization of a metallic device from his right inner thigh. He had been experiencing mild local pain for 2 weeks and had a recent hospitalization due to cardiogenic hemodynamic instability, requiring a central venous catheter placement in his right internal jugular vein 3 months earlier. Doppler ultrasound confirmed the intravascular foreign body hypothesis as a guidewire was identified inside the right femoral vein, associated with femoropopliteal venous thrombosis. The guidewire was successfully removed percutaneously through simple manual traction guided by radioscopy. The patient was discharged the following day on oral anticoagulation with rivaroxaban. On outpatient follow-up 4 weeks post discharge, he had no complaints in the right lower limb except for slight swelling. Central venous catheterization is a common invasive procedure that, although unquestionably safe and well stablished in medical practice, can lead to serious complications when performed without proper technique.

Three on Three: Universal and High-Affinity Molecular Recognition of the Symmetric Homotrimeric Spike Protein of SARS-CoV-2 with a Symmetric Homotrimeric Aptamer.

Our previously discovered monomeric aptamer for SARS-CoV-2 (MSA52) possesses a universal affinity for COVID-19 spike protein variants but is ultimately limited by its ability to bind only one subunit of the spike protein. The symmetrical shape of the homotrimeric SARS-CoV-2 spike protein presents the opportunity to create a matching homotrimeric molecular recognition element that is perfectly complementary to its structural scaffold, causing enhanced binding affinity. Here, we describe a branched homotrimeric aptamer with three-fold rotational symmetry, named TMSA52, that not only possesses excellent binding affinity but is also capable of binding several SARS-CoV-2 spike protein variants with picomolar affinity, as well as pseudotyped lentiviruses expressing SARS-CoV-2 spike protein variants with femtomolar affinity. Using Pd-Ir nanocubes as nanozymes in an enzyme-linked aptamer binding assay (ELABA), TMSA52 was capable of sensitively detecting diverse pseudotyped lentiviruses in pooled human saliva with a limit of detection as low as 6.3 × 103 copies/mL. The ELABA was also used to test 50 SARS-CoV-2-positive and 60 SARS-CoV-2-negative patient saliva samples, providing sensitivity and specificity values of 84.0 and 98.3%, respectively, thus highlighting the potential of TMSA52 for the development of future rapid tests.

Disentangling response initiation difficulties from response inhibition in autism spectrum disorder: A sentence-completion task.

It has been proposed that individuals with autism spectrum disorder (ASD) struggle both with response initiation and with response inhibition, both of which are functions of the executive system. Experimental tasks are unlikely pure measures of a single cognitive domain, and in this study we aim at understanding the contributions of response initiation difficulties to possible deficits in inhibitory control in autism. A sample of adults diagnosed with ASD and a control sample participated in this study. To participants it was asked to perform a sentence-completion task with two different condition: Part A-targeting response initiation and Part B-engaging inhibitory processes. Importantly, we have analyzed the B-A latencies that have been proposed for the removal of the response initiation confound effect. Results show that no differences between the groups were found in accuracy measures, either in Part A (ASD: M = 0.78; Controls: M = 0.90) nor Part B (ASD: M = 0.03; Controls: M = 0.02). However, in both conditions autistic participants were significantly slower to respond than the group of participants with typical development (Part A-ASD: M = 2432.5 ms; Controls M = 1078.5 ms; Part B-ASD M = 6758.3 ms; Controls M = 3283.9 ms). Critically, we show that when subtracting the response times of Part A from Part B (B-A latencies) no group differences attributable to inhibitory processes remained (ASD: M = 4325.76; Controls: M = 2205.46). With this study we corroborate the existence of difficulties with response initiation in autism and we question the existence of troubles in inhibition per se.

Microwave versus Conventional Sintering of NiTi Alloys Processed by Mechanical Alloying.

The present study shows a comparison between two sintering processes, microwave and conventional sintering, for the manufacture of NiTi porous specimens starting from powder mixtures of nickel and titanium hydrogenation-dehydrogenation (HDH) milled by mechanical alloying for a short time (25 min). The samples were sintered at 850 °C for 15 min and 120 min, respectively. Both samples exhibited porosity, and the pore size results are within the range of the human bone. The NiTi intermetallic compound (B2, R-phase, and B19') was detected in both sintered samples through X-ray diffraction (XRD) and electron backscattering diffraction (EBSD) on scanning electron microscopic (SEM). Two-step phase transformation occurred in both sintering processes with cooling and heating, the latter occurring with an overlap of the peaks, according to the differential scanning calorimetry (DSC) results. From scanning electron microscopy/electron backscatter diffraction, the R-phase and B2/B19' were detected in microwave and conventional sintering, respectively. The instrumented ultramicrohardness results show the highest elastic work values for the conventionally sintered sample. It was observed throughout this investigation that using mechanical alloying (MA) powders enabled, in both sintering processes, good results, such as intermetallic formation and densification in the range for biomedical applications.

Polyvinylamine with moderate binding affinity as a highly effective vehicle for RNA delivery.

Polymeric carriers for RNA therapy offer potential advantages in terms of low immunogenicity, promoting modifiability and accelerating intracellular transport. However, balancing high transfection efficacy with low toxicity remains challenging with polymer-based vehicles; indeed, polyethyleneimine (PEI) remains the "gold standard" polymer for this purpose despite its significant toxicity limitations. Herein, we demonstrate the potential of polyvinylamine (PVAm), a commodity high-charge cationic polymer used in the papermaking industry and has similar structure with PEI, as an alternative carrier for RNA delivery. High levels of transfection of normal, tumor, and stem cells with a variety of RNA cargoes including small interfering RNA (siRNA), microRNA (miRNA), and recombinant RNA can be achieved in vitro under the proper complex conditions. While, both the anti-tumor effect achieved in a xenograft osteosarcoma model and lipid-lowering activity observed in a hyperlipidemia mice indicate the potential for highly effective in vivo activity. Of note, both the transfection efficiency and the cytotoxicity of PVAm compare more favorably with those of PEI, with PVAm offering the additional advantages of simpler purification and significantly lower cost. In addition, the mechanism for the difference in transfection efficiency between PVAm and PEI is explored by molecular docking as well as analyzing the process of association and dissociation between polymers (PVAm and PEI) and nucleic acids. Our research provides a novel, non-toxic, and cost-effective carrier candidate for next generation RNA therapy, and elucidates the potential mechanism of PVAm for its efficient delivery of RNA.

Investigation of discordant SARS-CoV-2 RT-PCR results using minimally processed saliva.

Saliva is an attractive sample for coronavirus disease 2019 testing due its ease of collection and amenability to detect viral RNA with minimal processing. Using a direct-to-RT-PCR method with saliva self-collected from confirmed COVID-19 positive volunteers, we observed 32% false negative results. Confirmed negative and healthy volunteer samples spiked with 106 genome copies/mL of heat-inactivated severe acute respiratory syndrome coronavirus 2 showed false negative results of 10% and 13%, respectively. Additional sample heating or dilution of the false negative samples conferred only modest improvements. These results highlight the potential to significantly underdiagnose COVID-19 infections when testing directly from minimally processed heterogeneous saliva samples.

A Universal DNA Aptamer that Recognizes Spike Proteins of Diverse SARS-CoV-2 Variants of Concern.

Invited for the cover of this issue are John Brennan, Yingfu Li, and co-workers at McMaster University. The image depicts MSA52 as a universal DNA aptamer that recognizes spike proteins of diverse SARS-CoV-2 variants of concern. Read the full text of the article at 10.1002/chem.202200078.

A Universal DNA Aptamer that Recognizes Spike Proteins of Diverse SARS-CoV-2 Variants of Concern.

We report on a unique DNA aptamer, denoted MSA52, that displays universally high affinity for the spike proteins of wildtype SARS-CoV-2 as well as the Alpha, Beta, Gamma, Epsilon, Kappa, Delta and Omicron variants. Using an aptamer pool produced from round 13 of selection against the S1 domain of the wildtype spike protein, we carried out one-round SELEX experiments using five different trimeric spike proteins from variants, followed by high-throughput sequencing and sequence alignment analysis of aptamers that formed complexes with all proteins. A previously unidentified aptamer, MSA52, showed Kd values ranging from 2 to 10 nM for all variant spike proteins, and also bound similarly to variants not present in the reselection experiments. This aptamer also recognized pseudotyped lentiviruses (PL) expressing eight different spike proteins of SARS-CoV-2 with Kd values between 20 and 50 pM, and was integrated into a simple colorimetric assay for detection of multiple PL variants. This discovery provides evidence that aptamers can be generated with high affinity to multiple variants of a single protein, including emerging variants, making it well-suited for molecular recognition of rapidly evolving targets such as those found in SARS-CoV-2.

LISzyme Biosensors: DNAzymes Embedded in an Anti-biofouling Platform for Hands-free Real-Time Detection of Bacterial Contamination in Milk.

Nonspecific binding is a significant challenge associated with biosensors in complex food textures. To overcome this, we have developed LISzymes, which are DNAzymes incorporated in lubricant-infused surfaces (LISs). Using milk as a complex background matrix, we show that LISzyme biosensors are significantly more effective in preventing nonspecific binding compared to other commonly used "blocking" methods. The use of lubricant infusion to treat sensing surfaces results in a 4-fold increase in the signal-to-noise ratio obtained with the DNAzyme with respect to untreated surfaces, when detecting the presence of specific bacteria in milk. This is a striking improvement upon previous DNAzyme sensors. We also show that the use of LISs does not affect the DNAzyme's ability to effectively and specifically detect its target─a protein specifically produced by Escherichia coli (E. coli), in a complex sample matrix such as milk. LISzymes drastically improve DNAzyme performance, resulting in target detection associated with E. coli at concentrations as low as 250 CFU/mL in milk in less than an hour, which is currently not possible using other optical platforms. LISzymes are promising tools for the real-time monitoring of food contamination and may prove valuable within many other biosensing applications.

Stress Exposure of Evolved Bacteriophages under Laboratory versus Food Processing Conditions Highlights Challenges in Translatability.

Bacterial viruses, or bacteriophages, are highly potent, target-specific antimicrobials. Bacteriophages can be safely applied along the food production chain to aid control of foodborne pathogens. However, bacteriophages are often sensitive to the environments encountered in food matrices and under processing conditions, thus limiting their applicability. We sought to address this challenge by exposing commercially available Listeria monocytogenes bacteriophage, P100, to three stress conditions: desiccation, elevated temperature, and low pH, to select for stress-resistant bacteriophages. The stressed bacteriophage populations lost up to 5.1 log10 in infectivity; however, the surviving subpopulation retained their stress-resistant phenotype through five passages with a maximum of 2.0 log10 loss in infectivity when exposed to the same stressor. Sequencing identified key mutation regions but did not reveal a clear mechanism of resistance. The stress-selected bacteriophage populations effectively suppressed L. monocytogenes growth at a modest multiplicity of infection of 0.35-0.43, indicating no trade-off in lytic ability in return for improved survivability. The stressed subpopulations were tested for survival on food grade stainless steel, during milk pasteurization, and within acidic beverages. Interestingly, air drying on stainless steel and pasteurization in milk led to significantly less stress and titer loss in bacteriophage compared to similar stress under model lab conditions. This led to a diminished benefit for stress-selection, thus highlighting a major challenge in real-life translatability of bacteriophage adaptational evolution.