Acoustic detection of a mutation-specific Ligase Chain Reaction based on liposome amplification.

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.

Monoolein-Based Wireless Capacitive Sensor for Probing Skin Hydration.

Capacitive humidity sensors typically consist of interdigitated electrodes coated with a dielectric layer sensitive to varying relative humidity levels. Previous studies have investigated different polymeric materials that exhibit changes in conductivity in response to water vapor to design capacitive humidity sensors. However, lipid films like monoolein have not yet been integrated with humidity sensors, nor has the potential use of capacitive sensors for skin hydration measurements been fully explored. This study explores the application of monoolein-coated wireless capacitive sensors for assessing relative humidity and skin hydration, utilizing the sensitive dielectric properties of the monoolein-water system. This sensitivity hinges on the water absorption and release from the surrounding environment. Tested across various humidity levels and temperatures, these novel double functional sensors feature interdigitated electrodes covered with monoolein and show promising potential for wireless detection of skin hydration. The water uptake and rheological behavior of monoolein in response to humidity were evaluated using a quartz crystal microbalance with dissipation monitoring. The findings from these experiments suggest that the capacitance of the system is primarily influenced by the amount of water in the monoolein system, with the lyotropic or physical state of monoolein playing a secondary role. A proof-of-principle demonstration compared the sensor's performance under varying conditions to that of other commercially available skin hydration meters, affirming its effectiveness, reliability, and commercial viability.

New Insights into Aptamers: An Alternative to Antibodies in the Detection of Molecular Biomarkers.

Aptamers are short oligonucleotides with single-stranded regions or peptides that recently started to transform the field of diagnostics. Their unique ability to bind to specific target molecules with high affinity and specificity is at least comparable to many traditional biorecognition elements. Aptamers are synthetically produced, with a compact size that facilitates deeper tissue penetration and improved cellular targeting. Furthermore, they can be easily modified with various labels or functional groups, tailoring them for diverse applications. Even more uniquely, aptamers can be regenerated after use, making aptasensors a cost-effective and sustainable alternative compared to disposable biosensors. This review delves into the inherent properties of aptamers that make them advantageous in established diagnostic methods. Furthermore, we will examine some of the limitations of aptamers, such as the need to engage in bioinformatics procedures in order to understand the relationship between the structure of the aptamer and its binding abilities. The objective is to develop a targeted design for specific targets. We analyse the process of aptamer selection and design by exploring the current landscape of aptamer utilisation across various industries. Here, we illuminate the potential advantages and applications of aptamers in a range of diagnostic techniques, with a specific focus on quartz crystal microbalance (QCM) aptasensors and their integration into the well-established ELISA method. This review serves as a comprehensive resource, summarising the latest knowledge and applications of aptamers, particularly highlighting their potential to revolutionise diagnostic approaches.

Mass-Fabrication Scheme of Highly Sensitive Wireless Electrodeless MEMS QCM Biosensor with Antennas on Inner Walls of Microchannel.

Quartz-crystal-microbalance (QCM) biosensor is a typical label-free biosensor, and its sensitivity can be greatly improved by removing electrodes and wires that would be otherwise attached to the surfaces of the quartz resonator. The wireless-electrodeless QCM biosensor was then developed using a microelectro-mechanical systems (MEMS) process, although challenges remain in the sensitivity, the coupling efficiency, and the miniaturization (or mass production). In this study, we establish a MEMS process to obtain a large number of identical ultrasensitive and highly efficient sensor chips with dimensions of 6 mm square. The fundamental shear resonance frequency of the thinned AT-cut quartz resonator packaged in the microchannel exceeds 160 MHz, which is excited by antennas deposited on inner walls of the microchannel, significantly improving the electro-mechanical coupling efficiency in the wireless operation. The high sensitivity of the developed MEMS QCM biosensors is confirmed by the immunoglobulin G (IgG) detection using protein A and ZZ-tag displaying a bionanocapsule (ZZ-BNC), where we find that the ZZ-BNC can provide more effective binding sites and higher affinity to the target molecules, indicating a further enhancement in the sensitivity of the MEMS QCM biosensor. We then perform the label-free C-reactive protein (CRP) detection using the ZZ-BNC-functionalized MEMS QCM biosensor, which achieves a detection limit of 1 ng mL-1 or less even with direct detection.

A novel molecular imprinted QCM sensor based on MoS2NPs-MWCNT nanocomposite for zearalenone determination.

Zearalenone (ZEN) is a mycotoxin that has a carcinogenic effect and is often found at a high rate in frequently consumed foods. In this study, a characteristic molecular imprinted quartz crystal microbalance (QCM) sensor based on molybdenum disulfide nanoparticle (MoS2NPs)-multiwalled carbon nanotube (MWCNT) nanocomposite (MoS2NPs-MWCNTs) is presented for selective determination of ZEA in rice samples. Firstly, molybdenum disulfide nanoparticle (MoS2NP)-multiwalled carbon nanotube nanocomposites were characterized by using microscopic, spectroscopic, and electrochemical techniques. Then, ZEA-imprinted QCM chip was prepared in the presence of methacryloylamidoglutamicacid (MAGA) as monomer, N,N'-azobisisobutyronitrile (AIBN) as initiator, and ZEA as target molecule by using UV polymerization. The sensor revealed a linearity toward ZEA in the range 1.0-10.0 ng L-1 with a detection limit (LOD) of 0.30 ng L-1. The high repeatability, reusability, selectivity, and stability of the developed sensor enable reliable ZEA detection in rice samples.

Adsorption of Different Ionic Types of Polyacrylamide on Montmorillonite Surface: Insight from QCM-D and Molecular Dynamic Simulation.

This study investigates the interaction between montmorillonite and polyacrylamide (PAM) with different ionic types using quartz crystal microbalance with dissipation monitoring (QCM-D) and molecular dynamics (MD) simulations. The goal was to understand the effect of ionicity and ionic type on polymer deposition on montmorillonite surfaces. The results of the QCM-D analysis showed that a decrease in pH led to an increase in the adsorption of montmorillonite on the alumina surface. The ranking of adsorption mass on alumina and pre-adsorbed montmorillonite alumina surfaces was found to be cationic polyacrylamide (CPAM) > polyacrylamide (NPAM) > anionic polyacrylamide (APAM). The study also found that CPAM had the strongest bridging effect on montmorillonite nanoparticles, followed by NPAM, while APAM had a negligible bridging effect. The MD simulations showed that ionicity had a significant influence on the adsorption of polyacrylamides. The cationic functional group N(CH3)3+ had the strongest attraction interaction with the montmorillonite surface, followed by the hydrogen bonding interaction of the amide functional group CONH2, and the anionic functional group COO- had a repulsive interaction. The results suggest that at high ionicity levels, CPAM can be adsorbed on the montmorillonite surface, while at low ionicity levels, APAM may still be adsorbed with a strong coordination trend.

Dual-Mode and Label-Free Detection of Exosomes from Plasma Using an Electrochemical Quartz Crystal Microbalance with Dissipation Monitoring.

The biomolecular contents of extracellular vesicles, such as exosomes, have been shown to be crucial in intercellular communication and disease propagation. As a result, there has been a recent surge in the exploration of novel biosensing platforms that can sensitively and specifically detect exosomal content such as proteins and nucleic acids, with a view toward application in diagnostic assays. Here, we demonstrate dual-mode and label-free detection of plasma exosomes using an electrochemical quartz crystal microbalance with dissipation monitoring (EQCM-D). The platform adopts a direct immunosensing approach to effectively capture exosomes via their surface protein expression of CD63. By combining QCM-D with a tandem in situ electrochemical impedance spectroscopy measurement, we are able to demonstrate relationships between mass, viscoelasticity and impedance inducing properties of each functional layer and analyte. In addition to lowering the limit of detection (by a factor of 2-4) to 6.71 × 107 exosome-sized particles (ESP) per mL in 25% v/v serum, the synergy between dissipation and impedance response introduces improved sensing specificity by offering further distinction between soft and rigid analytes, thereby promoting EQCM-D as an important technique for exosome analysis.

Studies on Thiol Etching of Gold by Using QCM-D Sensor as the Sacrificial Probe.

There is still a lack of deep understanding on the reaction kinetics and mechanism of thiol etching of gold. Herein, by using the sensor of quartz crystal microbalance (QCM) as the sacrificial probe, the etching reaction of gold has been studied by employing cysteamine (CS) as a typical thiol etchant. The etching reaction is verified as diffusion-controlled and shows a half-order reaction kinetics. It is demonstrated that intact thiol and amino on CS are both crucial for its etching ability to gold. Applied potentials can affect the electron transfer and hence can be used to regulate the gold etching. Our results also reveal that only two carbon atoms of the spacer between thiol and amino on CS are very critical to the excellent etching ability. This work exhibits a new route to explore the thiol etching reaction of gold and elucidates the reaction kinetics and mechanism.

Protein Desorption Kinetics Depends on the Timescale of Observation.

The presence of so-called reversible and irreversible protein adsorption on solid surfaces is well documented in the literature and represents the basis for the development of nanoparticles and implant materials to control interactions in physiological environments. Here, using a series of complementary single-molecule tracking approaches appropriate for different timescales, we show that protein desorption kinetics is much more complex than the traditional reversible-irreversible binary picture. Instead, we find that the surface residence time distribution of adsorbed proteins transitions from power law to exponential behavior when measured over a large range of timescales (10-2-106 s). A comparison with macroscopic results obtained using a quartz crystal microbalance suggested that macroscopic measurements have generally failed to observe such nonequilibrium phenomena because they are obscured by ensemble-averaging effects. These findings provide new insights into the complex phenomena associated with protein adsorption and desorption.

A QCM immunosensor employing signal amplification strategies by enlarging the size of nanoparticles using gold or silver staining.

We present quartz crystal microbalance (QCM) immunosensors for the detection of alpha-fetoprotein (AFP) in a human serum immunoassay with high sensitivity. In this study, we employed three types of signal amplification strategies using size enlargement and/or increase in mass of gold and titanium dioxide nanoparticles. Since the basic principle of the QCM sensor is to measure the change in resonance frequency according to the mass change caused by the molecular interactions on the sensor surface, we were able to quantitatively analyze AFP by sandwich immunoassay using gold or titanium dioxide nanoparticles conjugated with anti-AFP detection antibodies and the subsequent three signal amplification techniques in a similar manner. The signal amplification technologies provide the size expansion of gold nanoparticles by gold or silver staining reaction and mass enhancement by photocatalytic silver staining of titanium dioxide nanoparticles. The limit of detections (LODs) of the AFP immunoassay in human serum by the gold and silver staining-mediated signal amplifications for gold nanoparticles were 56 and 87 pg mL-1, respectively, but by the photocatalytic silver staining signal amplification for titanium dioxide nanoparticles was 118 pg mL-1. This means that the signal amplification method through size enhancement by gold staining of gold nanoparticles further improved the detection ability of the QCM immunosensor.

Precision Temperature Control System with Low EMI for Applications in Analyzing Thermal Properties of Highly Sensitive Piezoelectric Sensors.

A low electromagnetic interference (EMI), precision temperature control system for sensitive piezoelectric sensors stabilization and their thermal characteristics research was proposed. Quartz crystal microbalance (QCM) was chosen as the device to be tested. Recently, QCMs found use in many fields of study such as biology, chemistry, and aerospace. They often operate in harsh environments and are exposed to many external factors including temperature fluctuations, to which QCMs are highly susceptible. Such disturbances can cause undesirable resonant frequency shifts resulting in measurement errors that are difficult to eliminate. The proposed solution enables measurements of QCMs thermal characteristics, effectiveness evaluation of temperature compensation methods, and testing of the frequency stability. As a part of the developed solution, two independent temperature regulators were used: first to maintain the QCM crystal at desired temperature, and second to keep the QCM oscillator circuit at fixed temperature. The single regulator consists of a thermoelectric module (TEC) used for both heating and cooling. Two considered TEC driving methods were compared in terms of EMI and their impact on the QCM signal quality. The proposed system was examined for its temperature stabilization capability showing high stability of 11 mKp-p for one hour and the setpoint accuracy of ±15 mK in the full temperature range.

Characterization of Thermal Gradient Effects on a Quartz Crystal Microbalance.

Quartz crystal microbalances are widely used sensors with applications for the detection of very-low-mass deposition in many different fields, from contamination monitoring in the high vacuum of deep space missions to the monitoring of biological activity or pollution using specifically designed active substrates. These sensors are very stable over time; nevertheless, their sensitivity to the temperature is well known, and different implementations have been devised to correct it, e.g., through compensation with a dual crystal. This paper deals with the effects of temperature on QCM but separates the case of uniform crystal temperature from the case of in-plane temperature gradients considering a QCM based on quartz crystals with deposited film resistors used as both RTDs and heaters. This configuration allows both an accurate temperature measurement and efficient thermal control, allowing the achievement of crystals temperatures in the order of 400 °C higher than the environment with a low power dissipation of the order of 1 W. The film resistors deposited around the electrodes allow directly measuring the average crystal temperature and directly delivering power to the crystal for thermal control. The localized delivery of the heat nevertheless also determines uncommon temperature fields on the crystal, and thus, an analysis of both the effects of temperature on the new microbalance was performed. The temperature gradient has strong effects on the frequency; therefore, along with the temperature, the thermal gradients have tobe compensated. The calibration of the QCM thermometers and the assessment of the achievable measurement accuracy were performed, as well as the determination of the frequency-temperature relationship. The comparison between frequency changes in the case of uniform temperature and those observed while using crystal heaters proved that temperature gradients have a strong effect on the crystal frequency. To identify the temperature field on the crystal surface of a QCM crystal, the gold coating of the deposited films was removed to achieve an emissivity acceptable for thermal imaging with an IR camera. Moreover, image processing for emissivity correction was developed. In order to correlate the temperature gradient with the frequency variation, a test campaign was performed to measure the frequency changes derived from different power levels delivered to the crystal heaters. From this test campaign and thermal analysis, the effect of the thermal gradient was assessed.

Advances in the Mass Sensitivity Distribution of Quartz Crystal Microbalances: A Review.

A quartz crystal microbalance (QCM) is a typical acoustic transducer that undergoes a frequency shift due to changes in the mass of its surface. Its high sensitivity, robustness, small size design, and digital output have led to its widespread development for application in the fields of chemistry, physics, biology, medicine, and surface science. Mass sensitivity is one of the vital parameters and forms the basis for quantitative analysis using QCMs. This review firstly introduces the importance, definition, calculation, and measuring method of the mass sensitivity and then focuses on reviewing the influence of electrode parameters (including electrode shape, electrode diameter, electrode thickness, electrode material, etc.) on the mass sensitivity distribution of QCMs. Finally, the effect of the operating frequency on the mass sensitivity of QCMs is also analyzed.

Detection of Salmonella Typhimurium with Gold Nanoparticles Using Quartz Crystal Microbalance Biosensor.

Demonstration of the Salmonella Typhimurium detection system was shown utilizing a quartz crystal microbalance (QCM) biosensor and signal enhancement by gold nanoparticles. In this study, a benchtop system of a QCM biosensor was utilized for the detection of Salmonella Typhimurium. It was designed with a peristaltic pump system to achieve immobilization of antibodies, detection of Salmonella, and the addition of gold nanoparticles to the sensor. As a series of biochemical solutions were introduced to the surface, the proposed system was able to track the changes in the resonant frequency which were proportional to the variations of mass on the sensor. For antibody immobilization, polyclonal antibodies were immobilized via self-assembled monolayers to detect Salmonella O-antigen. Subsequently, Salmonella Typhimurium was detected by antibodies and the average frequency before and after detecting Salmonella was compared. The highest frequency shifts were −26.91 Hz for 109 CFU/mL while the smallest frequency shift was −3.65 Hz corresponding to 103 CFU/mL. For the specificity tests, non-Salmonella samples such as E. coli, Listeria, and Staphylococcus resulted in low cross-reactivity. For signal amplification, biotinylated antibodies reacted to Salmonella followed by streptavidin­100 nm AuNPs through biotin-avidin interaction. The frequency shifts of 103 CFU/mL showed −28.04 Hz, and consequently improved the limit of detection.

Design and Analysis of Quartz Crystal Microbalance with a New Ring-Shaped Interdigital Electrode.

In this paper, a new type of ring-shaped interdigital electrode is proposed to improve the accuracy and repeatability of quartz crystal microbalance. The influence of different types of single finger, dot finger, dot double-finger electrodes on mass sensitivity distribution as well as the optimal proportion of finger and gap width are obtained through multi-physical coupling simulation. The results show that the design criteria of interdigital electrodes will not change with the increase in the number of fingers. The gap width should obey the decrease order from central to edge and be about twice the width of finger. The width of the outermost finger and the radius of the middle dot electrode should be maintained at about 0.4 and 0.2 times of the total electrode radius. An experiment was carried out to verify that the quartz wafer with a dot double-finger electrode has high quality factors and less modal coupling, which can satisfy the engineering application well. As a conclusion, this study provides a design idea for the electrode to maintain a uniform distribution of quartz crystal microbalance mass sensitivity.

Oxytocin-Selective Nanogel Antibody Mimics.

Oxytocin imprinted polymer nanoparticles were synthesized by glass bead supported solid phase synthesis, with NMR and molecular dynamics studies used to investigate monomer-template interactions. The nanoparticles were characterized by dynamic light scattering, scanning- and transmission electron microscopy and X-ray photoelectron spectroscopy. Investigation of nanoparticle-template recognition using quartz crystal microbalance-based studies revealed sub-nanomolar affinity, kd ≈ 0.3 ± 0.02 nM (standard error of the mean), comparable to that of commercial polyclonal antibodies, kd ≈ 0.02-0.2 nM.

Integration of Adenylate Kinase 1 with Its Peptide Conformational Imprint.

In the present study, molecularly imprinted polymers (MIPs) were used as a tool to grasp a targeted α-helix or ß-sheet of protein. During the fabrication of the hinge-mediated MIPs, elegant cavities took shape in a special solvent on quartz crystal microbalance (QCM) chips. The cavities, which were complementary to the protein secondary structure, acted as a peptide conformational imprint (PCI) for adenylate kinase 1 (AK1). We established a promising strategy to examine the binding affinities of human AK1 in conformational dynamics using the peptide-imprinting method. Moreover, when bound to AK1, PCIs are able to gain stability and tend to maintain higher catalytic activities than free AK1. Such designed fixations not only act on hinges as accelerators; some are also inhibitors. One example of PCI inhibition of AK1 catalytic activity takes place when PCI integrates with an AK19-23 ß-sheet. In addition, conformation ties, a general MIP method derived from random-coil AK1133-144 in buffer/acetonitrile, are also inhibitors. The inhibition may be due to the need for this peptide to execute conformational transition during catalysis.

Study on the Preparation of Estrone Molecularly Imprinted Polymers and Their Application in a Quartz Crystal Microbalance Sensor via a Computer-Assisted Design.

Computer simulations are widely used for the selection of conditions for the synthesis of molecularly imprinted polymers and can rapidly reduce the experimental cycle time and save labor and materials. In this paper, estrone molecularly imprinted polymers (E1-MIPs) are designed at the M062X/6-311+G(d,p) level with itaconic acid (IA) as the functional monomer. The imprinted molar ratio between E1 and IA was optimized, cross-linkers and solvents were screened, and the nature of interactions between E1 and IA was explored. The simulated results showed that pentaerythritol triacrylate was the best cross-linker. Meanwhile, when the imprinted molar ratio between E1 and IA was 1:4, the E1-IA complex had the largest amount of hydrogen bonds, the lowest binding energy, and the strongest stability. Using the simulation results as guidance, the E1-MIPs were prepared to modify the electrons of a quartz crystal microbalance (QCM) sensor. The experimental studies showed that the E1-MIPs-QCM sensor had the highest adsorption capacity to E1 in comparison with their analogues, and the lowest detection value of the sensor was 16.00 µg/L. The computer simulations and experimental studies could provide guidance for synthesize novel E1-MIPs materials. It also could provide important references and directions for the application of E1-MIPs.

The Role of Cytoskeleton Revealed by Quartz Crystal Microbalance and Digital Holographic Microscopy.

The connection between cytoskeleton alterations and diseases is well known and has stimulated research on cell mechanics, aiming to develop reliable biomarkers. In this study, we present results on rheological, adhesion, and morphological properties of primary rat cardiac fibroblasts, the cytoskeleton of which was altered by treatment with cytochalasin D (Cyt-D) and nocodazole (Noc), respectively. We used two complementary techniques: quartz crystal microbalance (QCM) and digital holographic microscopy (DHM). Qualitative data on cell viscoelasticity and adhesion changes at the cell-substrate near-interface layer were obtained with QCM, while DHM allowed the measurement of morphological changes due to the cytoskeletal alterations. A rapid effect of Cyt-D was observed, leading to a reduction in cell viscosity, loss of adhesion, and cell rounding, often followed by detachment from the surface. Noc treatment, instead, induced slower but continuous variations in the rheological behavior for four hours of treatment. The higher vibrational energy dissipation reflected the cell's ability to maintain a stable attachment to the substrate, while a cytoskeletal rearrangement occurs. In fact, along with the complete disaggregation of microtubules at prolonged drug exposure, a compensatory effect of actin polymerization emerged, with increased stress fiber formation.

Aptasensor for paraquat detection by gold nanoparticle colorimetric method.

This study aimed to develop an aptasensor for paraquat detection by gold nanoparticles. The specific aptamer for paraquat was selected by using the SELEX process via capillary electrophoresis. Sixty-three aptamer candidates were amplified by asymmetric PCR and screened for paraquat binding using gold nanoparticles (AuNPs). Aggregation of AuNPs was specifically induced by desorption of paraquat binding aptamers from the surface of AuNPs as a result of aptamer-target interaction leading to the color change from red to purple. Aptamer 77F with the following sequence: 5'-AGGCTTACACCTGAAAAGCGGCTTAATTTACACTACTGTAT-3' was selected as a highly specific aptamer for paraquat. The detection limit of paraquat was 0.267 µg/mL by colorimetry and 1.573 µg/mL by the quartz crystal microbalance (QCM) technique. This aptamer showed specificity for paraquat by colorimetry. Dimethyl phophite, diethyl phophite and O,O diethyl thiophosphate potassium salt did not react by colorimetry but, exhibited a weak nonspecific reaction by QCM. This is first time that an aptasensor was used for detection of paraquat based on QCM. However, the aptasensor based on the colorimetric method simply uses a generation of a signal that can be detected by the naked eye. This technique is rapid, low cost easy to use and suitable for on-site detection of herbicide residue.