Search

The aim of this work was to measure and compare the binding constants of antibody immunoglobulin G (IgG) to bacterial cell wall proteins, streptococcal protein G and Staphylococcus aureus protein A, using an acoustic wave sensor. Devices, which used shear-horizontal acoustic waves propagating in a waveguide configuration at 108 and 155 MHz, were employed in the detection of apparent IgG binding constants at the solid-liquid interface in the range of 6.7-667 nM IgG. Real-time data during IgG-protein G and IgG-protein A binding yielded apparent association constants of 3.29 x [10.sup.4] and 8.02 x [10.sup.3] [M.sup.-1] [s.sup.-1] leading to equilibrium constants of 1.13 x [10.sup.8] and 2.90 x [10.sup.7] [M.sup.-1], respectively. The measured apparent rate constants are consistent with literature reports of higher affinity of protein G for IgG. Furthermore, protein binding through the Fc region of IgG is suggested to occur below 333 nM, while different mechanisms are suggested to occur above 333 nM. For the first time, nonequilibrium studies of IgG-protein G and A binding at a solid-liquid interface has yielded valuable quantitative kinetic information about binding mechanisms. The promise of this detection method is shown by providing quick determination of binding constants with low sample volumes.

Compares the binding constants of antibody immunoglobulin with bacterial cell wall proteins G and A using acoustic wave sensor. Detection of apparent immunoglobulin G (IgG) binding constants with low sample volumes; Nonequilibrium studies of IgG; Consistency among the measured apparent rate constants.

The goal of this study is to test a novel device and methodology based on the "Pebble" platform and real-time quantitative colorimetric loop-mediated isothermal amplification (qcLAMP) during SARS-CoV-2 detection using crude samples and extracted RNA. The new method employs an inexpensive lightweight device aimed toward rapid point-of-care testing. An extensive evaluation was performed consisting of 1,693 clinical samples across five independent clinical testing centers. Positive colorimetric results were observed within 20 minutes of testing. At a 20-minute time-to-positive cut-off, the specificity is 98.5% with a diagnostic accuracy of 91.9%, compared to qPCR assays. Our findings indicate that the SARS-CoV-2 qcLAMP diagnostic assay in conjunction with the Pebble device is ideal for point-of-care/near-patient testing.IMPORTANCEHere, we describe our analyses and validation of a novel real-time quantitative colorimetric loop-mediated isothermal amplification (qcLAMP) device, available under the name "Pebble" and associated SARS-CoV-2 diagnostic qcLAMP assay for clinical diagnostic use. The analyses were performed in five independent testing sites across Europe using clinical samples from the associated clinical sites and support the use of "pebble" and associated kit in the diagnostic environment.

Background: Verticilium dahliae is the most important wilt pathogen of olive trees with a broad host range causing devastating diseases currently without any effective chemical control. Traditional detection methodologies are based on symptoms-observation or lab-detection using time consuming culturing or molecular techniques. Therefore, there is an increasing need for portable tools that can detect rapidly V. dahliae in the field., Results: In this work, we report the development of a novel method for the rapid, reliable and on-site detection of V. dahliae using a newly designed isothermal LAMP assay and crude extracts of olive wood. For the detection of the fungus, LAMP primers were designed targeting the internal transcribed spacer (ITS) region of the rRNA gene. The above assay was combined with a purpose-built prototype portable device which allowed real time quantitative colorimetric detection of V. dahliae in 35 min. The limit of detection of our assay was found to be 0.8 fg/μl reaction and the specificity 100% as indicated by zero cross-reactivity to common pathogens found in olive trees. Moreover, detection of V. dahliae in purified DNA gave a sensitivity of 100% (Ct < 30) and 80% (Ct > 30) while the detection of the fungus in unpurified crude wood extracts showed a sensitivity of 80% when multisampling was implemented. The superiority of the LAMP methodology regarding robustness and sensitivity was demonstrated when only LAMP was able to detect V. dahliae in crude samples from naturally infected trees with very low infection levels, while nested PCR and SYBR qPCR failed to detect the pathogen in an unpurified form., Conclusions: This study describes the development of a new real time LAMP assay, targeting the ITS region of the rRNA gene of V. dahliae in olive trees combined with a 3D-printed portable device for field testing using a tablet. The assay is characterized by high sensitivity and specificity as well as ability to operate using directly crude samples such as woody tissue or petioles. The reported methodology is setting the basis for the development of an on-site detection methodology for V. dahliae in olive trees, but also for other plant pathogens., (© 2024. The Author(s).)

Single nucleotide variants (SNVs) play a crucial role in understanding genetic diseases, cancer development, and personalized medicine. However, existing ligase-based amplification and detection techniques, such as Rolling Circle Amplification and Ligase Detection Reaction, suffer from low efficiency and difficulties in product detection. To address these limitations, we propose a novel approach that combines Ligase Chain Reaction (LCR) with acoustic detection using highly dissipative liposomes. In our study, we are using LCR combined with biotin- and cholesterol-tagged primers to produce amplicons also modified at each end with a biotin and cholesterol molecule. We then apply the LCR mix without any purification directly on a neutravidin modified QCM device Au-surface, where the produced amplicons can bind specifically through the biotin end. To improve sensitivity, we finally introduce liposomes as signal enhancers. For demonstration, we used the detection of the BRAF V600E point mutation versus the wild-type allele, achieving an impressive detection limit of 220 aM of the mutant target in the presence of the same amount of the wild type. Finally, we combined the assay with a microfluidic fluidized bed DNA extraction technology, offering the potential for semi-automated detection of SNVs in patients' crude samples. Overall, our LCR/acoustic method outperforms other LCR-based approaches and surface ligation biosensing techniques in terms of detection efficiency and time. It effectively overcomes challenges related to DNA detection, making it applicable in diverse fields, including genetic disease and pathogen detection.

Loop-mediated isothermal amplification (LAMP) is a low-technology molecular assay that is highly adaptable to point-of-care (POC) applications. However, achieving sensitive naked-eye detection of the amplified target in a crude sample is challenging. Herein, we report a simple yet highly efficient and sensitive methodology for the colorimetric visualization of a single target copy in saliva using chitosan-capped gold nanoparticles (Chit-AuNPs) synthesized via a green chemistry approach. The presence or absence of free Chit in the Chit-AuNPs solution was shown to affect LAMP colorimetric detection oppositely: the observed stabilization in the negative samples and aggregation in the positive samples in the presence of free Chit were reversed in the case of neat Chit-AuNPs. The mechanism of the two assays was investigated and attributed to electrostatic and depletion effects exerted between the Chit-AuNPs, free Chit, and the solution components. The developed contamination-free, one-tube assay successfully amplified and detected down to 1-5 cfu of Salmonella and 10 copies of SARS-CoV-2 per reaction (25 μL) used, respectively, as model DNA and RNA targets in the presence of 20% saliva, making the method suitable for POC applications. Compared to the commonly used pH-sensitive dyes, Chit-AuNPs are shown to have an enhanced sensitivity toward naked-eye colorimetric observation owing to the direct detection of DNA amplicons. Thus, this is a simple, highly sensitive, fast, and versatile naked-eye detection methodology that could be coupled to any LAMP or RT-LAMP assay, avoiding the need of using complicated sample pretreatments and/or AuNPs long and laborious functionalization processes.

Protein function can be modulated by phase transitions in their material properties, which can range from liquid- to solid-like; yet, the mechanisms that drive these transitions and whether they are important for physiology are still unknown. In the model plant Arabidopsis, we show that developmental robustness is reinforced by phase transitions of the plasma membrane-bound lipid-binding protein SEC14-like. Using imaging, genetics, and in vitro reconstitution experiments, we show that SEC14-like undergoes liquid-like phase separation in the root stem cells. Outside the stem cell niche, SEC14-like associates with the caspase-like protease separase and conserved microtubule motors at unique polar plasma membrane interfaces. In these interfaces, SEC14-like undergoes processing by separase, which promotes its liquid-to-solid transition. This transition is important for root development, as lines expressing an uncleavable SEC14-like variant or mutants of separase and associated microtubule motors show similar developmental phenotypes. Furthermore, the processed and solidified but not the liquid form of SEC14-like interacts with and regulates the polarity of the auxin efflux carrier PINFORMED2. This work demonstrates that robust development can involve liquid-to-solid transitions mediated by proteolysis at unique plasma membrane interfaces., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Liquid biopsy, in particular circulating tumor DNA (ctDNA) analysis, has paved the way for a new noninvasive approach to cancer diagnosis, treatment selection and follow-up. As a crucial step in the analysis, the extraction of the genetic material from a complex matrix needs to meet specific requirements such as high specificity and low loss of target. Here, we developed a new generation of microfluidic fluidized beds (FBs) that enable the efficient extraction and preconcentration of specific ctDNA sequences from human serum with flow rates up to 15 µL/min. We first demonstrated that implementation of a vibration system inducing flow rate fluctuations combined with a mixture of different bead sizes significantly enhanced bead homogeneity, thereby increasing capture efficiency. Taking advantage of this new generation of high-throughput magnetic FBs, we then developed a new method to selectively capture a double-stranded (dsDNA) BRAF mutated DNA sequence in complex matrices such as patient serum. Finally, as proof of concept, ligation chain reaction (LCR) assays were performed to specifically amplify a mutated BRAF sequence, allowing the detection of concentrations as low as 6 × 10 4 copies/µL of the mutated DNA sequence in serum., Competing Interests: Conflict of interestThe authors declare no competing interests., (© Aerospace Information Research Institute, Chinese Academy of Sciences 2023.)

Regular screening of point mutations is of importance to cancer management and treatment selection. Although techniques like next-generation sequencing and digital polymerase chain reaction (PCR) are available, these are lacking in speed, simplicity, and cost-effectiveness. The development of alternative methods that can detect the extremely low concentrations of the target mutation in a fast and cost-effective way presents an analytical and technological challenge. Here, an approach is presented where for the first time an allele-specific PCR (AS-PCR) is combined with a newly developed high fundamental frequency quartz crystal microbalance array as biosensor for the amplification and detection, respectively, of cancer point mutations. Increased sensitivity, compared to fluorescence detection of the AS-PCR amplicons, is achieved through energy dissipation measurement of acoustically "lossy" liposomes binding to surface-anchored dsDNA targets. The method, applied to the screening of BRAF V600E and KRAS G12D mutations in spiked-in samples, was shown to be able to detect 1 mutant copy of genomic DNA in an excess of 10 4 wild-type molecules, that is, with a mutant allele frequency (MAF) of 0.01%. Moreover, validation of tissue and plasma samples obtained from melanoma, colorectal, and lung cancer patients showed excellent agreement with Sanger sequencing and ddPCR; remarkably, the efficiency of this AS-PCR/acoustic methodology to detect mutations in real samples was demonstrated to be below 1% MAF. The combined high sensitivity and technology-readiness level of the methodology, together with the ability for multiple sample analysis (24 array biochip), cost-effectiveness, and compatibility with routine workflow, make this approach a promising tool for implementation in clinical oncology labs for tissue and liquid biopsy.