Development of a combined surface plasmon resonance/surface acoustic wave device for the characterization of biomolecules

It is known that acoustic sensor devices, if operated in liquid phase, are sensitive not just to the mass of the analyte but also to various other parameters, such as size, shape, charge and elastic constants of the analyte as well as bound and viscously entrained water. This can be used to extract valuable information about a biomolecule, particularly if the acoustic device is combined with another sensor element which is sensitive to the mass or amount of analyte only. The latter is true in good approximation for various optical sensor techniques. This work reports on the development of a combined surface plasmon resonance/surface acoustic wave sensor system which is designed for the investigation of biomolecules such as proteins or DNA. Results for the deposition of neutravidin and DNA are reported

Effect of Langmuir monolayer of bovine serum albumin protein on the morphology of calcium carbonate

Bovine serum albumin (BSA) Langmuir monolayer, as a model of biomineralization-associated proteins, was used to study its effect on regulated biomineralization of calcium carbonate. The effects of the BSA Langmuir monolayer and the concentration of the subphase solution on the nucleation and growth processes and morphology of the calcium carbonate crystal were investigated. The morphology and polymorphic phase of the resulting calcium carbonate crystals were characterized by scanning electron microscopy (SEM) and X-ray diffraction analysis (XRD). Moreover, the interaction mechanisms of the subphase solution with the BSA Langmuir monolayer were discussed. It was found that BSA Langmuir monolayer could be used as a template to successfully manipulate the polymorphic phase and crystal morphology of calcium carbonate and had obvious influence on the oriented crystallization and growth. The final morphology or aggregation mode of the calcite crystal was closely dependent on the concentration of calcium bicarbonate solution. It is expected that this research would help to better understand the mechanism of biomineralization by revealing the interactions between protein matrices and crystallization of calcium carbonate crystal.Comment: 4 pages, 4 figure

The Nucleation and Growth of Calcium Phosphate Crystals at Protein and Phosphatidylserine Liposome Surfaces

The kinetics of calcium phosphate crystal growth at the surfaces of proteins and phospholipids has been investigated using free drift and constant composition methods in supersaturated calcium phosphate solutions (relative supersaturations: with respect to hydroxyapatite, HAP, σHAP = 15.0, and with respect to octacalcium phosphate, OCP, σOCP = 1. 9). Fibrinogen and collagen molecules adsorbed at hydrophobic surfaces as well as uncross-linked collagen fibrils induce ion binding and subsequent nucleation of calcium phosphate. The formation of OCP on phosphatidylserine vesicles introduced to highly supersaturated calcium phosphate solutions probably involves the interaction of the calcium ions with the ionized carboxylic groups of the phospholipid

Acoustic Array Biochip Combined with Allele-Specific PCR for Multiple Cancer Mutation Analysis in Tissue and Liquid Biopsy

[EN] 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 104 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.This work was supported by the European Union's Horizon H2020-FETOPEN-1-2016-2017 under grant agreement no. 737212 (CATCH-U-DNA).Naoumi, N.; Michaelidou, K.; Papadakis, G.; Simaiaki, AE.; Fernández Díaz, R.; Calero-Alcarria, MDS.; Arnau Vives, A.... (2022). Acoustic Array Biochip Combined with Allele-Specific PCR for Multiple Cancer Mutation Analysis in Tissue and Liquid Biopsy. ACS Sensors. 7(2):495-503. https://doi.org/10.1021/acssensors.1c02245S4955037

High-frequency phase shift measurement greatly enhances the sensitivity of QCM immunosensors

[EN] In spite of being widely used for in liquid biosensing applications, sensitivity improvement of conventional (5-20 MHz) quartz crystal microbalance (QCM) sensors remains an unsolved challenging task. With the help of a new electronic characterization approach based on phase change measurements at a constant fixed frequency, a highly sensitive and versatile high fundamental frequency (HFF) QCM immunosensor has successfully been developed and tested for its use in pesticide (carbaryl and thiabendazole) analysis. The analytical performance of several immunosensors was compared in competitive immunoassays taking carbaryl insecticide as the model analyte. The highest sensitivity was exhibited by the 100 MHz HFF-QCM carbaryl immunosensor. When results were compared with those reported for 9 MHz QCM, analytical parameters clearly showed an improvement of one order of magnitude for sensitivity (estimated as the I50 value) and two orders of magnitude for the limit of detection (LOD): 30 µg L-1 vs 0.66 µg L-1 I50 value and 11 µg L-1 vs 0.14 µg L-1 LOD, for 9 and 100 MHz, respectively. For the fungicide thiabendazole, I50 value was roughly the same as that previously reported for SPR under the same biochemical conditions, whereas LOD improved by a factor of 2. The analytical performance achieved by high frequency QCM immunosensors surpassed those of conventional QCM and SPR, closely approaching the most sensitive ELISAs. The developed 100 MHz QCM immunosensor strongly improves sensitivity in biosensing, and therefore can be considered as a very promising new analytical tool for in liquid applications where highly sensitive detection is required.We would like to acknowledge Federico Martin and Juan Antonio Buitrago for their excellent technical assistance. This work was supported by the Spanish Ministry of Economy and Competitiveness/European Regional Development Fund (ERDF) (DETECTA IPT-2012-0154-300000 project).March Iborra, MDC.; García Narbón, JV.; Sánchez, Á.; Arnau Vives, A.; Jiménez Jiménez, Y.; García, P.; Manclus Ciscar, JJ.... (2015). High-frequency phase shift measurement greatly enhances the sensitivity of QCM immunosensors. Biosensors and Bioelectronics. (65):1-8. https://doi.org/10.1016/j.bios.2014.10.001S186

Scalable bacterial production of moldable and recyclable biomineralized cellulose with tunable mechanical properties

Sustainable structural materials with excellent impact-resistance properties are urgently needed but challenging to produce, especially in a scalable fashion and with control over 3D shape. Here, we show that bacterial cellulose (BC) and bacterially precipitated calcium carbonate self-assemble into a layered structure reminiscent of tough biomineralized materials in nature (nacre, bone, dentin). The fabrication method consists of biomineralizing BC to form an organic/inorganic mixed slurry, in which calcium carbonate crystal size is controlled with bacterial poly(γ-glutamic acid) and magnesium ions. This slurry self-assembles into a layered material that combines high toughness and high impact and fire resistance. The rapid fabrication is readily scalable, without involving toxic chemicals. Notably, the biomineralized BC can be repeatedly recycled and molded into any desired 3D shape and size using a simple kitchen blender and sieve. This fully biodegradable composite is well suited for use as a component in daily life, including furniture, helmets, and protective garments.The authors thank Ward Groutars and Elvin Karana for useful discussions. K.Y. is supported financially by the China Scholarship Council (CSC no.201706630001). S.B. is funded by the Air Force Office of Scientific Research, Asian Office of Aerospace Research and Development (grant no. FA2386-18-1-4059)

A Facile Strategy for In Situ Core-Template-Functionalizing Siliceous Hollow Nanospheres for Guest Species Entrapment

The shell wall-functionalized siliceous hollow nanospheres (SHNs) with functional molecules represent an important class of nanocarriers for a rich range of potential applications. Herein, a self-templated approach has been developed for the synthesis of in situ functionalized SHNs, in which the biocompatible long-chain polycarboxylates (i.e., polyacrylate, polyaspartate, gelatin) provide the framework for silica precursor deposition by simply controlling chain conformation with divalent metal ions (i.e., Ca2+, Sr2+), without the intervention of any external templates. Metal ions play crucial roles in the formation of organic vesicle templates by modulating the long chains of polymers and preventing them from separation by washing process. We also show that, by in situ functionalizing the shell wall of SHNs, it is capable of entrapping nearly an eightfold quantity of vitamin Bc in comparison to the bare bulk silica nanospheres. These results confirm the feasibility of guest species entrapment in the functionalized shell wall, and SHNs are effective carriers of guest (bio-)molecules potentially for a variety of biomedical applications. By rationally choosing the functional (self-templating) molecules, this concept may represent a general strategy for the production of functionalized silica hollow structures

Specific Binding and Mineralization of Calcified Surfaces by Small Peptides

Several small (<25aa) peptides have been designed based on the sequence of the dentin phosphoprotein, one of the major noncollagenous proteins thought to be involved in the mineralization of the dentin extracellular matrix during tooth development. These peptides, consisting of multiple repeats of the tripeptide aspartate-serine-serine (DSS), bind with high affinity to calcium phosphate compounds and, when immobilized, can recruit calcium phosphate to peptide-derivatized polystyrene beads or to demineralized human dentin surfaces. The affinity of binding to hydroxyapatite surfaces increases with the number of (DSS)n repeats, and though similar repeated sequences—(NTT)n, (DTT)n, (ETT)n, (NSS)n, (ESS)n, (DAA)n, (ASS)n, and (NAA)n—also showed HA binding activity, it was generally not at the same level as the natural sequence. Binding of the (DSS)n peptides to sectioned human teeth was shown to be tissue-specific, with high levels of binding to the mantle dentin, lower levels of binding to the circumpulpal dentin, and little or no binding to healthy enamel. Phosphorylation of the serines of these peptides was found to affect the avidity, but not the affinity, of binding. The potential utility of these peptides in the detection of carious lesions, the delivery of therapeutic compounds to mineralized tissues, and the modulation of remineralization is discussed