Quartz Crystal Microbalance Biosensor Research Articles

A reusable QCM biosensor with stable antifouling nano-coating for on-site reagent-free rapid detection of E. coli O157:H7 in food products

Numerous biosensors have shown exceptional analytical performance under laboratory conditions, yet only a few are capable of on-site use with complex, non-model samples while exhibiting reliable analytical performance. Here, we present a new portable biosensor for the rapid (30 min) and accurate detection of bacterial agents in “real-world” food samples, which are originally in either solid or liquid form. The biosensor combines well-established quartz crystal microbalance (QCM) technology, with innovative terpolymer brush nano-coatings on the sensing surface to efficiently reduce non-specific fouling from food samples. Following reagent-free sample preparation, where solid food samples are homogenized, we validated the sensor's detection capabilities on native pathogenic Escherichia coli O157:H7 (E. coli O157:H7) in hamburgers, Czech dumplings, and milk. We achieved limits of detection (LOD), as low as 7.5 × 102 CFU/mL in milk, a value approaching fundamental QCM limits, using a simple direct detection assay format. The biosensor's exceptional reusability was demonstrated through 60 sequential hamburger sample injections, resulting in only a minor LOD shift toward the end of series. A 10-min sonication treatment during sample preparation significantly enhanced sensitivity for E. coli O157:H7 in hamburgers and dumplings, yielding LODs as low as 3.1 × 10³ CFU/mL and 2.6 × 10⁴ CFU/mL, respectively. For on-site analysis, we integrated the nano-coated sensing chip into a custom-built four-channel portable QCM biosensor with an optimized microfluidic system, which can be produced on a scale suitable for practical deployment.

Real-time monitoring of dephosphorylation process of phosphopeptide and rapid assay of PTP1B activity based on a 100 MHz QCM biosensing platform

The misregulation of protein phosphatases is a key factor in the development of many human diseases, notably cancers. Here, based on a 100 MHz quartz crystal microbalance (QCM) biosensing platform, the dephosphorylation process of phosphopeptide (P-peptide) caused by protein tyrosine phosphatase 1B (PTP1B) was monitored in real time for the first time and PTP1B activity was assayed rapidly and sensitively. The QCM chip, coated with a gold (Au) film, was used to immobilized thiol-labeled single-stranded 5′-phosphate-DNAs (P-DNA) through Au–S bond. The P-peptide, specific to PTP1B, was then connected to the P-DNA via chelation between Zr4+ and phosphate groups. When PTP1B was injected into the QCM flow cell where the P-peptide/Zr4+/MCH/P-DNA/Au chip was placed, the P-peptide was dephosphorylated and released from the Au chip surface, resulting in an increase in the frequency of the QCM Au chip. This allowed the real-time monitoring of the P-peptide dephosphorylation process and sensitive detection of PTP1B activity within 6 min with a linear detection range of 0.01–100 pM and a detection limit of 0.008 pM. In addition, the maximum inhibitory ratios of inhibitors were evaluated using this proposed 100 MHz QCM biosensor. The developed 100 MHz QCM biosensing platform shows immense potential for early diagnosis of diseases related to protein phosphatases and the development of drugs targeting protein phosphatases.

Electric Field-Assisted Molecularly Imprinted Polymer-Modified QCM Sensor for Enhanced Detection of Immunoglobulin.

In this study, an electric-field-assisted molecularly imprinted polymer (EFAMIP) as an enhanced form of MIP was developed to improve the MIP-modified quartz crystal microbalance (QCM) biosensors. While exerting a vertical electric field, polymerization of methacrylic acid in the presence of immunoglobulin G (IgG) as the template was initiated, and later, after the template removal process, the EFAMIPs were obtained. The polymer surface characterization was conducted by using a scanning electron microscope. The impact of electric field direction on IgG binding sites, forming either EFAMIP-Fab or EFAMIP-Fc, was assessed. Next, the static measurement results in liquid for EFAMIP-modified QCM and MIP-modified QCM were compared. While encompassing IgG, EFAMIP-modified QCMs exhibited up to a 113.5% higher frequency shift than typical MIP in time-limited detection. The final frequency shift of EFAMIP, which determines the detection limit of IgG, was improved up to 12.5% compared to typical MIP. Moreover, the EFAMIP-Fab performance was promising for the selective detection of IgG in a solution containing different types of immunoglobulins.

Design, Fabrication and Performance Analysis of a Portable, Antenna Analyzer Based, Quartz Crystal Microbalance Measuring System with Energy Dissipation

Impedance measurements play a critical role in analyzing the electrical behavior of piezoelectric biosensors in general. Antenna analyzers are engineered to measure the specific case of input impedance for antenna systems. In this study small form factor antenna analyzer is repurposed to work as driving circuit for a Quartz Crystal Microbalance (QCM) biosensor in combination with a single board computer as an indication of how small and portable an impedance measuring system can be made, while allowing monitoring of important parameters of series and parallel resonance frequencies together with dissipation factor. A QCM crystal with a 10 MHz fundamental resonance frequency is employed to determine the limit of detection of the system in Bovine Serum Albumin (BSA) and glycerol solutions. Dissipation factor and phase angle were monitored during the experiments. Limit of detection is 20 µg/ml BSA in phosphate buffer saline (PBS) and 250 µl of glycerol in 100 ml of deionized water.

Development of gold nanospikes-modified quartz crystal microbalance biosensor for prostate specific antigen detection

Prostate-specific antigen (PSA) screening is vital for the early detection and assessment of prostate cancer treatment efficacy. Integrating nanotechnology into biosensor development allows for targeted biomarker detection. In this study, we present the fabrication of gold nanospikes (AuNS) structures on a quartz crystal microbalance (QCM) using the pulse current electrodeposition (PCD) technique. By varying the duty cycles of PCD, we achieved the desired nanostructure. The characteristics of the AuNS-modified QCM electrode were thoroughly investigated using various micro-structural characterization techniques such as x-ray diffraction (XRD), field emission scanning electron microscope (FESEM), atomic force microscope (AFM), X-ray photoelectron spectroscopy (XPS) and cyclic voltammetry (CV). Prior to biomolecule immobilization, each chemical treatment was verified through contact angle and FT-IR analyses. The performance of the developed QCM biosensor was evaluated using PSA at various concentrations. The results revealed that the frequency variation of the QCM biosensor was directly proportional to the PSA concentration. Notably, the AuNS-modified QCM biosensor exhibited the highest frequency shift at a 20 % duty cycle, demonstrating a linear detection range of 0.1–100 ng mL−1 and a limit of detection (LOD) of 24 pg mL−1. The developed QCM biosensor significantly enhances detection efficiency by increasing the number of binding sites for biomolecules through an enlarged surface area. The demonstrated sensitivity of the AuNS-modified QCM biosensor indicates its potential to address the increasing demand for early diagnosis and screening in healthcare systems. This innovative biosensor technology holds promise for improving prostate cancer detection and has broader implications for the development of highly sensitive biosensors for various biomarkers.

MEMS‐Based Ultra‐High Frequency Wireless 10 × 10 QCM Sensor Array for Biochemical Sensing Applications

The quartz crystal microbalance (QCM) biosensor is one of the devices that enables label‐free evaluation of biomolecular reactions. Commercial QCM biosensors have less than 10 channels for detection and are not suitable for the screening. The sensitivity of the QCM biosensor increases as the quartz thickness becomes thinner; however, the limit of thinning is 60–330 μm. In this study, applying micromachining technology, a QCM sensor array chip of 15 mm square that had 100 ultra‐thin quartz resonators (2.5 μm) that were operated wirelessly was developed. And we succeeded in detecting the resonance spectrum through the device evaluation. © 2024 Institute of Electrical Engineers of Japan. Published by Wiley Periodicals LLC.

Coverage Effects in Quartz Crystal Microbalance Measurements with Suspended and Adsorbed Nanoparticles.

Quartz crystal microbalance (QCM) biosensors often deal with nanoparticles suspended in the solvent at tens of nanometers above the resonator while being linked to some molecular receptor (DNA, antibody, etc.). This work presents a numerical analysis based on the immersed boundary method for the flow and QCM impedance created by an ensemble of spherical particles of radius R at varying surface coverage Θ and particle-surface gap distance Δ. The trends for the frequency Δf and dissipation ΔD shifts against Θ and Δ are shown to be determined by modifications in the structure of the perturbative flow created by the analytes. Simulations are in good agreement with a relatively large experimental database collected from the literature. Qualitative differences between the adsorbed (Δ ≈ 0) and suspended states (Δ > 0) are highlighted. In the case of adsorbed particles, deviations from the linear scaling Δf ∝ Θ are observed above Θ > 0.05 and largely depend on the specific analyte-substrate combination. Moreover, in general, ΔD(Θ) is not monotonous and usually presents a maximum around Θ ∼ 0.2. In the case of suspended analytes, the agreement with the numerical results is quantitative, indicating that the predicted scalings are universal and determined by hydrodynamics. Up to high coverage, the suspended particles present Δf ∼ Θ and ΔD ∼ Θβ, where β ≈ 0.85 is not largely dependent on R. The present findings should help forecast molecular configurations from QCM signals and have implications on QCM analyses, e.g., in the case of suspended ligands (Δf ∝ Θ), it is safe to use Δf to build Langmuir isotherms and estimate equilibrium constants. Open questions on the transition from the suspended-to-adsorbed state are discussed.

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.

Sugar-Lectin Interactions for Direct and Selective Detection of Escherichia coli Bacteria Using QCM Biosensor.

Pathogenic Escherichia coli (E. coli) remains a safety concern in the preservation and quality of green leafy vegetables. Sugar-lectin interactions provide a reliable, specific, and effective sensing platform for the detection of bacteria as compared to the tedious conventional plate counting technique. Herein, we present the synthesis of 4-(N-mannosyl) benzoic acid (4-NMBA) and 4-thiophenyl-N-mannose (4-TNM) via a two-step reductive amination for the detection of E. coli using a quartz crystal microbalance (QCM) biosensor. The 4-NMBA was synthesized with mannose and para-aminobenzoic (4-PBA), while the 4-TNM was synthesized with mannose and 4-aminophenyl disulfide (4-AHP) using water and acetic acid in a 1:1 ratio. The resultant structure of mannose derivatives (4-NMBA and 4-TNM) was characterized and confirmed using analytical tools, such as Mass Spectrometer, SEM, and FTIR. The choice of ligands (mannose derivatives) is ascribed to the specific recognition of mannose to the FimH lectin of the type 1 pilus of E. coli. Furthermore, the 4-PBA and 4-AHP conjugated to mannose increase the ligand affinity to FimH lectins. The setup of the QCM biosensor was composed of modification of the crystal surface and the covalent attachment of ligands for the detection of E. coli. The piezoelectric effect (frequency shift of the quartz) was proportional to the change in mass added to the gold crystal surface. Both the 4-NMBA- and 4-TNM-coated QCM sensors had a limit of detection of 3.7 CFU/mL and 6.6 CFU/mL with a sensitivity of 2.56 × 103 ng/mL and 8.99 × 10-5 ng/mL, respectively, within the dynamic range of 103 to 106 CFU/mL. This study demonstrates the application of ligand-coated QCM biosensors as a cost-effective, simple, and label-free technology for monitoring pathogenic bacteria via molecular interactions on crystal surfaces.

Detection of β-amyloid peptide aggregates by quartz crystal microbalance based on dual-aptamer assisted signal amplification

β-amyloid peptide (Aβ) aggregates are regarded as a typical neuropathology hallmark for the diagnosis of Alzheimer's disease (AD). Aβ40 aggregates include soluble oligomers (Aβ40O) and insoluble fibrils (Aβ40F). Both of them can simultaneously bind to two different kinds of its aptamer (Apt1 and Apt2). As a mass-sensitive sensing platform, quartz crystal microbalance (QCM) converts changes in mass on the Au chip surface into frequency shift. Here, a dual-aptamer assisted Aβ40 aggregates assay was developed. Taking Aβ40O detection as an example, Apt2 was modified on the surface of Au chip by Au–S bond. Subsequently, the solution consisted of Aβ40O and gold nanoparticles-Apt1 (AuNPs-Apt1) were injected into the QCM chamber. As a result, Aβ40O was specifically recognized and captured by Apt2. AuNPs-Apt1 were also combined on the surface of the Au chip because Aβ40O can simultaneously bind to Apt1. Then, a significant frequency shift occurred because of the large weight of AuNPs. Similarly, this procedure can be used to detect Aβ40F. This QCM biosensor was able to detect Aβ40O with a range of 0.2–10 pM with a detection limit of 0.11 pM, while the linear range for Aβ40F was 0.1–10 pM with a detection limit of 0.02 pM. This QCM biosensor was simple and highly sensitive, which provided a new method for Aβ40 aggregates detection.

Development of a quartz crystal microbalance-based immunosensor for the early detection of mesothelin in cancer

A robust real-time flow injection assay using Quartz Crystal Microbalance (QCM) biosensor has been developed for the detection of mesothelin, an antigen expressed on various malignant tumors including mesothelioma and ovarian cancers. A QCM sensor chip functionalized with self-assembled monolayer of cysteamine used to fabricate a sensitive immunosensor. Mesothelin specific antibody was immobilized on cysteamine modified gold surface of quartz crystal using N-ethyl-N’-(3-dimethylaminopropyl) carbodiimide and N-hydroxy succinimide coupling. Further, ethanolamine was used as a blocking reagent to prevent the non-specific adsorption on antibody functionalized gold surface. Various concentrations of mesothelin was tested and the resonant frequency variation of the crystal was observed by a quartz crystal microbalance until a stable response is obtained. A good correlation between the frequency changes and concentration of mesothelin tested was observed and the QCM biosensor detects mesothelin in the linear range of 100pg/mLto 50 ng/mL. Thus, QCM assay could be a promising technique for early diagnosis of mesothelioma, ovarian and pancreatic adenocarcinoma.

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 streptavidin-100 nm AuNPs through biotin-avidin interaction. The frequency shifts of 103 CFU/mL showed -28.04 Hz, and consequently improved the limit of detection.

A quartz crystal microbalance biosensor based on polyethylenimine-modified gold electrode to detect hepatitis B biomarker

Biomarkers-based QCM-biosensors are suitable tools for the label-free detection of infectious diseases. In the current study, a QCM-biosensor was developed for the detection of HBsAg. Briefly, anti-HBsAg antibodies were covalently bound to the primary amines after PEI and thiolated-PEI surface modifications of gold-electrode. After RSM optimization, the statistical analysis revealed no significant difference between the immobilization yields of modified layers. Therefore, the PEI-modified QCM-biosensor was selected for further analysis. The PEI-surface was evaluated by FESEM, AFM, ATR-FTIR, and CA measurement. The surface hydrophilicity and its roughness were increased after PEI-coating. Also, FTIR confirmed the PEI-layering on the gold-surface. RSM optimization increased the antibody immobilization yield up to 80%. The QCM-biosensor showed noteworthy results with a wide dynamic range of 1–1 × 103 ng/mL, LOD of 3.14 ng/mL, LOQ of 9.52 ng/mL, and detection capability in human-sera, which were comparable with the ELISA. The mean accuracy of the QCM-biosensor was obtained at 91% when measured by the spike recovery test using human-sera. The biosensor was completely regenerated using 50 mM NaOH and 1% SDS. The benefits provided by the developed biosensor such as broad dynamic range, sensitivity, selectivity, stability, regenerate ability, and low cost suggest its potential application for the non-invasive and timely monitoring of HBV-biomarker.

Protein adsorption on amino-acid-conjugated self-assembled molecule-modified SiO2 surfaces

Protein adsorption has a crucial effect on biocompatibility during the interaction of biomaterial surfaces and the biological environment. It is significant to understand and control the interactions among biomaterials and proteins for several biomedical applications. Surface engineering plays a significant role in determining biocompatibility by tuning the effects directly on proteins. In this study, amino acid (histidine and leucine) conjugated self-assembled molecules were synthesized and used to modify silicon dioxide (SiO2) surfaces to investigate protein adsorption behavior. Silicon dioxide surfaces were modified with (3-aminopropyl)triethoxysilane-conjugated histidine and leucine amino acids. Modified silicon dioxide surfaces were characterized by water contact angle measurements and X-ray photoelectron spectroscopy analysis. Protein adsorption (human serum albumin, fibrinogen and immunoglobulin G) on silicon dioxide-coated crystals was investigated in situ by using a quartz crystal microbalance (QCM) biosensor. From the results, model proteins showed different selectivities to the amino-acid-conjugated silicon dioxide-coated crystals depending on the type of the amino acid and concentration. Consequently, this controlled chemistry on the surface of biomaterials has a great potential to manipulate protein adsorption and enhance the biocompatibility of biomaterials for various biomedical applications.

Immunoglobulin-Immobilized Quartz Crystal Microbalance for Staphylococcus Aureus Real-Time Detection

Quartz crystal microbalance (QCM) sensor is still a high-precision surface sensing technique. Here, we described an immunoglobulin G (IgG) biofunctionalized QCM sensor for <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">Staphylococcus aureus</i> (S. aureus) sensing application. In the case of the QCM sensor, a gold-titanium (Au/Ti) electrode layer was fabricated by magnetron sputtering and sequentially immobilized with thoil self-assembled monolayer (SAM) and IgG gripper. The selectivity test showed that the rabbit IgG-based QCM sensor exhibited a good sensitivity performance. IgG immobilization optimal experiment was conducted by adjusting parameters including immobilization time, temperature, buffer pH and IgG concentration. The lower limit of detection and sensitivity of the IgG-based QCM biosensor toward <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">S. aureus</i> were suggested to be <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$1.0\times {10}^{{4}}$ </tex-math></inline-formula> CFU/mL and 562.5 Hz/ng, respectively. Furthermore, the real-time monitoring and frequency response of IgG immobilization process and its sensitive adsorption behavior to <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">S. aureus</i> were performed in droplet environment. By comparison, the average sensitivity of IgG-immobilized QCM sensor in liquid phase is 2-3.5 times higher than that in air. Overall, it is indicative that IgG-based QCM sensors do have great potential for biological monitoring applications.

Development and Assessment of Regeneration Methods for Peptide-Based QCM Biosensors in VOCs Analysis Applications.

Cleaning a quartz crystal microbalance (QCM) plays a crucial role in the regeneration of its biosensors for reuse. Imprecise removal of a receptor layer from a transducer’s surface can lead to unsteady operation during measurements. This article compares three approaches to regeneration of the piezoelectric transducers using the electrochemical, oxygen plasma and Piranha solution methods. Optimization of the cleaning method allowed for evaluation of the influence of cleaning on the surface of regenerated biosensors. The effectiveness of cleaning the QCM transducers with a receptor layer in the form of a peptide with the KLLFDSLTDLKKKMSEC-NH2 sequence was described. Preliminary cleaning was tested for new electrodes to check the potential impact of the cleaning on deposition and the transducer’s operation parameters. The effectiveness of the cleaning was assessed via the measurement of a resonant frequency of the QCM transducers. Based on changes in the resonant frequency and the Sauerbrey equation, it was possible to evaluate the changes in mass adsorption on the transducer’s surface. Moreover, the morphology of the QCM transducer’s surface subjected to the selected cleaning techniques was presented with AFM imaging. The presented results confirm that each method is suitable for peptide-based biosensors cleaning. However, the most invasive seems to be the Piranha method, with the greatest decrease in performance after regeneration cycles (25% after three cycles). The presented techniques were evaluated for their efficiency with respect to a selected volatile compound, which in the future should allow reuse of the biosensors in particular applications, contributing to cost reduction and extension of the sensors’ lifetime.

Recent Advances in Quartz Crystal Microbalance Biosensors Based on the Molecular Imprinting Technique for Disease-Related Biomarkers

The molecular imprinting technique is a quickly developing field of interest regarding the synthesis of artificial recognition elements that enable the specific determination of target molecule/analyte from a matrix. Recently, these smart materials can be successfully applied to biomolecule detection in biomimetic biosensors. These biosensors contain a biorecognition element (a bioreceptor) and a transducer, like their biosensor analogs. Here, the basic difference is that molecular imprinting-based biosensors use a synthetic recognition element. Molecular imprinting polymers used as the artificial recognition elements in biosensor platforms are complementary in shape, size, specific binding sites, and functionality to their template analytes. Recent progress in biomolecular recognition has supplied extra diagnostic and treatment methods for various diseases. Cost-effective, more robust, and high-throughput assays are needed for monitoring biomarkers in clinical settings. Quartz crystal microbalance (QCM) biosensors are promising tools for the real-time and quick detection of biomolecules in the past two decades A quick, simple-to-use, and cheap biomarkers detection technology based on biosensors has been developed. This critical review presents current applications in molecular imprinting-based quartz crystal microbalance biosensors for the quantification of biomarkers for disease monitoring and diagnostic results.

Kinetics and interaction studies of anti-tetraspanin antibodies and ICAM-1 with extracellular vesicle subpopulations using continuous flow quartz crystal microbalance biosensor

Continuous flow quartz crystal microbalance (QCM) was utilized to study binding kinetics between EV subpopulations (exomere- and exosome-sized EVs) and four affinity ligands: monoclonal antibodies against tetraspanins (anti-CD9, anti-CD63, and anti-CD81) and recombinant intercellular adhesion molecule-1 (ICAM-1) or CD54 protein). High purity CD9+, CD63+, and CD81+ EV subpopulations of <50 nm exomeres and 50–80 nm exosomes were isolated and fractionated using our recently developed on-line coupled immunoaffinity chromatography – asymmetric flow field-flow fractionation system. Adaptive Interaction Distribution Algorithm (AIDA), specifically designed for the analysis of complex biological interactions, was used with a four-step procedure for reliable estimation of the degree of heterogeneity in rate constant distributions. Interactions between exomere-sized EVs and anti-tetraspanin antibodies demonstrated two interaction sites with comparable binding kinetics and estimated dissociation constants Kd ranging from nM to fM. Exomeres exhibited slightly higher affinity compared to exosomes. The highest affinity with anti-tetraspanin antibodies was achieved with CD63+ EVs. The interaction of EV subpopulations with ICAM-1 involved in cell internalization of EVs was also investigated. EV – ICAM-1 interaction was also of high affinity (nM to pM range) with overall lower affinity compared to the interactions of anti-tetraspanin antibodies and EVs. Our findings proved that QCM is a valuable label-free tool for kinetic studies with limited sample concentration, and that advanced algorithms, such as AIDA, are crucial for proper determination of kinetic heterogeneity. To the best of our knowledge, this is the first kinetic study on the interaction between plasma-derived EV subpopulations and anti-tetraspanin antibodies and ICAM-1.

Machine learning approach to optimization of parameters for impedance measurements of Quartz Crystal Microbalance to improve limit of detection

Impedance spectroscopy allows real time monitoring of fundamental resonance frequency and harmonics of AT cut Quartz Crystal Microbalance (QCM) biosensors. Acquisition of accurate and high resolution spectroscopic data requires optimization of parameters, which is rarely performed as it requires an excessive number of experiments. In this work, machine learning clustering and classification algorithms are applied in an attempt to minimize the number of experiments by designating a small representative subset of chosen values of parameters, while preserving the variation of those values. Our results indicated it is possible to reduce the number of experiments required by more than 10 fold. The constructed model has both an unsupervised clustering part (k-means algorithm) and a supervised classification part (Support Vector Machine algorithm), working independently. From those two different approaches 83% compatibility is obtained. Such optimization can improve limit of detection (LOD) of glycerol concentrations by a factor of 12 using a QCM with a fundamental resonance frequency of 10 MHz. This approach can be applied not only to biosensors but also to chemical sensors involving resonance frequency monitoring by means of impedance spectroscopy, and has the potential to be expanded to systems with different transducer types, monitoring resonance by other means.

Biosensors for the Detection of Bacillus anthracis.

Bacillus anthracis, present in two forms of vegetative cells and spores, is a pathogen that infects humans through contact with infected animals or contaminated animal products and is also maliciously used in terrorist acts. Therefore, a rapid and sensitive test for B. anthracis is necessary but challenging. The challenge comes from the following aspects: an accurate distinction of B. anthracis from other Bacillus species due to their high genomic similarity and the horizontal gene transfer between Bacillus members; direct detection of the B. anthracis spores without damaging them for component extraction to avoid the risk of spore atomization; and the rapid detections of B. anthracis in complex samples, such as soil and suspicious powders, without sample pretreatments and expensive large-scale equipment. Although culturing B. anthracis from samples is the conventional method for the detection of B. anthracis, it is time-consuming and the detection results would not be easy to interpret because many Bacillus species share similar phenotypic features such as a lack of motility and hemolysis, resistance to gamma phages, and so on. Intensive and extensive effort has been expended to develop reliable detection technologies, among which biosensors exhibit comprehensive advantages in terms of sensitivity, specificity, and portability. Here, we briefly review the research progress, providing highlights of the latest achievements and our own practice and experience. The contents can be summarized in three aspects: the discovery of detection targets, including genes, toxins, and other components; the creation of molecular recognition elements, such as monoclonal antibodies, single-chain antibody fragments, specific peptides, and aptamers; and the design and construction of biosensing systems by the integration of appropriate molecular recognition elements and transducer devices. These sensor devices have their own characteristics and different principles. For example, the surface plasmon resonance biosensor and quartz crystal microbalance biosensor are very sensitive, while the multiplex PCR-on-a-chip can detect multitargets. Biosensors for direct spore detection are highly recommended because they are not only fast but also avoid contamination from aerosol-containing spores. The introduction of nanotechnology has significantly improved the performance of biosensors. Superparamagnetic nanoparticles and phage-displayed gold nanoparticle ligand peptides have made the results of spore detection visible to the naked eye. Because of space constraints, many advanced biosensors for B. anthracis are not described in detail but are cited as references. Although biosensors provide a variety of options for various application scenarios, the challenges have not been fully addressed, which leaves room for the development of more advanced and practical B. anthracis detection means.