Amount Of Biomolecules Research Articles

Recent advances in capillary electrophoresis-mass spectrometry analysis of ‍trace biomolecules

In recent years, various trace bioanalysis methods have been developed, including single-cell transcriptome analysis methods. As the sample volume and amount of biomolecules contained therein are extremely limited, development of new single-cell analysis methods require extremely high-level techniques. It is necessary to design an appropriate analysis system that integrates a highly sensitive detection system and a pretreatment protocol for minimizing sample loss, where separation method is especially important for analyzing diverse mixtures of biomolecules. Among them, capillary electrophoresis (CE) can separate biomolecules in nanoliter-scale solutions with high resolution, making it highly compatible with trace samples such as single cells. By combining with highly sensitive nano-electrospray ionization-mass spectrometry (MS), it is possible to detect nanomolar to sub-nanomolar biomolecules, which can be further improved by using online sample preconcentration methods. These highly sensitive analytical techniques have made it possible to analyze trace amounts of metabolites, proteins, lipids, etc. This review paper summarizes the research on CE-MS trace bioanalysis that has been reported to date, with a focus on single-cell analysis.

Reduced liver damage and fibrosis with combined SCD Probiotics and intermittent fasting in aged rat.

This study aimed to examine the impact of SCD Probiotics supplementation on liver biomolecule content and histological changes during a 30-day intermittent fasting (IF) program in 24-month-old male Sprague-Dawley rats. Rats underwent 18-h daily fasting and received 1 × 108 CFU of SCD Probiotics daily. Liver tissue biomolecules were analysed using FTIR Spectroscopy, LDA, and SVM techniques, while histopathological evaluations used Haematoxylin and eosin and Masson trichrome-stained tissues. Blood samples were collected for biochemical analysis. Gross alterations in the quantity of biomolecules were observed with individual or combined treatments. LDA and SVM analyses demonstrated a high accuracy in differentiating control and treated groups. The combination treatments led to the most significant reduction in cholesterol ester (1740 cm-1 ) and improved protein phosphorylation (A1239 /A2955 and A1080 /A1545 ) and carbonylation (A1740 /A1545 ). Individually, IF and SCD Probiotics were more effective in enhancing membrane dynamics (Bw2922 /Bw2955 ). In treated groups, histological evaluations showed decreased hepatocyte degeneration, lymphocyticinfiltration, steatosis and fibrosis. Serum ALP, LDH and albumin levels significantly increased in the SCD Probiotics and combined treatment groups. This study offers valuable insights into the potential mechanisms behind the beneficial effects of IF and SCD Probiotics on liver biomolecule content, contributing to the development of personalized nutrition and health strategies.

Lactic Fermentation of Broccoli (Brassica oleracea var. italica) to Enhance the Antioxidant and Antiproliferative Activities

Lactic acid bacteria (LAB) have been used for centuries to produce fermented foods. Cruciferous vegetables contain large amounts of health-promoting compounds such as glucosinolates (GLSs) and phenolics. GLSs and phenolics have been linked to antioxidant, anticancer, and immunosuppressive effects. However, it has been reported that some LAB strains are able to metabolize and enhance the activities and amounts of biomolecules through decarboxylation and/or reduction activities, with positive impacts on human diet and colorectal cancer (CRC) prevention. In the present work, the bioprocessing of broccoli by lactic fermentation was evaluated to produce a functional food using both spontaneous and induced fermentation (Levilactobacillus brevis and Lactococcus lactis as starter co-culture). Changes in the proximal composition, GLSs, and phenolic content as well as the antioxidant, antiproliferative, and immunosuppressive effect of the fermented product were evaluated in in vitro cellular models to validate their potential in CRC chemoprevention. The results demonstrated that fermented broccoli extracts increased the antioxidant activity in Caco2 cells and inhibited the proliferation of HT29 and HT116 cell lines in a concentration-dependent manner, with the best results on day 6 at a concentration of 600 µg/mL. Our findings also provide evidence that fermented broccoli could have an anti-inflammatory effect.

Myco-Facilitated Biosynthesis of Nano-Silver From Wasp Nest Fungus, Paecilomyces variotii, and Its Antimicrobial Activity Against MTCC Strains.

The utility of fungi as stabilizing and reducing agents in the biogenic synthesis of silver nanoparticles is striking due to the production of large quantities of biomolecules of minute toxic residuals. During the current study, sunlight- and dark-assessed silver nanoparticles were synthesized from wasp nest fungus, Paecilomyces variotii, at different pHs. Synthesized silver nanoparticles (AgNPs) at 6 pH were found to be more prominent than at 7 and 8 pHs. AgNPs were within the 20- to 90-nm range and were polygonal and elongated in shape. FTIR spectra of light-mediated AgNPs showed diverse transmittance bands than the silver nanoparticles synthesized in the dark. The synthesized AgNPs were found with diverse antimicrobial activities against pathogenic MTCC bacterial strains, i.e., Staphylococcus aureus, Vibrio parahaemolyticus, Escherichia coli, Shewanella putrefaciens, and fungus, Candida albicans. Aqueous filtrate and filtrate-mediated AgNPs combined with methanol solvent extract of yeast extract manitol broth (YEMB) had more inhibitory effects on all bacteria and Candida albicans. Furthermore, the combined effect of AgNPs and methanol solvent extract from YEMB culture filtrate was found more effective against E. coli, while AgNPs combined with methanol solvent of aqueous filtrate had inhibitory effects on E. coli and Candida albicans.

Fluorescent probes for biomolecule detection under environmental stress

The use of fluorescent probes in visible detection has been developed over the last several decades. Biomolecules are essential in the biological processes of organisms, and their distribution and concentration are largely influenced by environmental factors. Significant advances have occurred in the applications of fluorescent probes for the detection of the dynamic localization and quantity of biomolecules during various environmental stress-induced physiological and pathological processes. Herein, we summarize representative examples of small molecule-based fluorescent probes that provide bimolecular information when the organism is under environmental stress. The discussion includes strategies for the design of smart small-molecule fluorescent probes, in addition to their applications in biomolecule imaging under environmental stresses, such as hypoxia, ischemia-reperfusion, hyperthermia/hypothermia, organic/inorganic chemical exposure, oxidative/reductive stress, high glucose stimulation, and drug treatment-induced toxicity. We believe that comprehensive insight into the beneficial applications of fluorescent probes in biomolecule detection under environmental stress should enable the further development and effective application of fluorescent probes in the biochemical and biomedical fields.

Rhamnolipids as Effective Green Agents in the Destabilisation of Dolomite Suspension.

In this paper, we describe an application of mono- and dirhamnolipid homologue mixtures of a biosurfactant as a green agent for destabilisation of a dolomite suspension. Properties of the biosurfactant solution were characterised using surface tension and aggregate measurements to prove aggregation of rhamnolipids at concentrations much lower than the critical micelle concentration. Based on this information, the adsorption process of biosurfactant molecules on the surface of the carbonate mineral dolomite was investigated, and the adsorption mechanism was proposed. The stability of the dolomite suspension after rhamnolipid adsorption was investigated by turbidimetry. The critical concentration of rhamnolipid at which destabilisation of the suspension occurred most effectively was found to be 50 mg·dm−3. By analysing backscattering profiles, solid-phase migration velocities were calculated. With different amounts of biomolecules, this parameter can be modified from 6.66 to 20.29 mm·h−1. Our study indicates that the dolomite suspension is destabilised by hydrophobic coagulation, which was proved by examining the wetting angle of the mineral surface using the captive bubble technique. The relatively low amount of biosurfactant used to destabilise the system indicates the potential application of this technology for water treatment or modification of the hydrophobicity of mineral surfaces in mineral engineering.

Laser microdissection: sample preparation and applications

Laser microdissection (LM) is a rapid, easy, one-step, and efficient method of conserving and isolating single cell or cell clusters from fixed tissue sections under direct microscopic visualization. LM is currently the only method that can be used to isolate homogeneous cells from heterogeneous tissue. This method was first developed for tumor analysis. Multiple generations of LM instruments are available on the market. For instance, the Veritas™ microdissection combines the LM system based on the IR with UV laser cutting. The desired cells can be harvested specific and wanted cells can be harvested directly by cutting target cells abroad from unwanted cells. The extracted cells are then utilized in various disciplines of experimental and clinical biology, such as genomic analysis and proteomics. LM is a an efficient and powerful tool that facilitates the analysis of small amounts of molecules isolated from a complex tissue, thus offering new opportunities to understand physiological and fundamental processes. Here, we describe a method of preparing paraffin sections of maize roots for laser microdissection to three parts (stele, cortex and outer layers) by LM using a microwave embedding method. This approach allows RNA to be extracted from each type of tissue separately. The RNA integrity of our samples following LM ranged between 7 and 7.2, indicating that this RNA can be reliably used for further analysis. The work reported in this paper highlights how advances in protocols and methods have made LM a powerful and sometimes essential tool for genomic and proteomic analyses of tiny amounts of biomolecules extracted from a few cells isolated from a complex tissue in their physiological context, thus offering a new pathway to understand fundamental, physiological and/or patho-physiological processes.

Transition of microbial community structures after development of membrane fouling in membrane bioreactors (MBRs)

Although membrane fouling is a major issue when operating membrane bioreactors (MBRs), information regarding MBR performance and the sludge microbiome after the development of fouling remains limited. For the present study, two MBRs were operated for approximately 1 month under conditions of membrane fouling to investigate the effects of highly stressed environments on the sludge microbiome. After the development of fouling, a Collimonas-related operational taxonomic unit (OTU) was highly dominant in both reactors (relative abundances were ⁓ 63%) and this predomination caused a precipitous decline in the diversity indices of the sludge microbiomes. Because the excessive predomination by limited numbers of OTUs can lead to reductions in the adaptability to environmental changes, monitoring microbial diversity may be a valuable indicator for maintaining the robustness of a sludge microbiome. While, the decrease in the abundance of the Collimonas-related OTU resulted in the predomination of distinct microorganisms in each of the reactors despite being operated under the same conditions; this finding indicates existence of strong pressure to perturb the microbiomes. Detailed analyses suggested that the availability of terminal electron acceptors and competitive interactions between microbes via the secretion of extracellular proteins appeared to differentiate the structures of the respective microbial communities. During the extracellular proteins were secreted in the sludge, considerable portion of microbes were dead and large amounts of biomolecules seemed to be released; resultantly facilitated the predomination of fermentative anaerobes in one reactor as they use organic substances but not inorganic terminal electron acceptors to generate ATP under anaerobic conditions.

Combination of area controllable sensing surface and bipolar electrode-electrochemiluminescence approach for the detection of tetracycline

A novel area controllable biosensing interface is designed on glassy carbon bead (GCB) and used for measurement of tetracycline (TET) in closed bipolar electrode-electrochemiluminescence (ECL) device. One face of GCB is modified with Au particles and the covered area is varied from 0 to 45.3% by tuning the external voltage during bipolar electrodeposition process in a home-made open bipolar electrochemical cell. It enables the conjugation of various amounts of biomolecules on Au/GCB. Then DNA walker and methylene blue (MB) labeled DNA (MB-DNA) are conjugated on Au surface for the following combination of aptamer. In the presence of target, aptamer partially hybridized with DNA walker will be released from Au surface, leading to the formation of dsDNA between DNA walker and MB-DNA. As a result, MB-DNA with recognition site for nicking endonuclease (Nb.BbvCI) in dsDNA is cleft into two segments by Nb.BbvCI. Meanwhile, the liberated DNA walker is triggered to continue the degradation of many MB-DNA. Due to the excellent electrochemical performance of MB, it is reduced at the cathode of BPE to amplify the ECL signal at the anode of BPE in a closed BPE-ECL platform. When the covered area of GCB by Au particles is enlarged from 10.1 to 45.3%, the change of ECL intensity could be increased 2.7-fold. The linear range for the detection of tetracycline is from 1 × 10−12 to 1 × 10−5 M with the detection limit of 6.0 × 10−13 M. Hence, controllable sensing area on a single glassy carbon bead, the amplification effect of DNA walker and MB make this approach possess many advantages over traditional biosensor.

DNA Detection Using SiC Nanowire Based Transistor

The fast and direct detection of small quantities of biomolecules improves early medical diagnosis of certain serious diseases as cancers and can be used to detect in situ the presence of pathogenic viruses or GMOs for food industry, protection environmental and bio-defense. Numerous research projects are conducted on nanoelectronic devices that can perform such detection with high sensitivity using nanostructures. Currently, these devices are made from Silicon nanowires [1]. For these applications, Silicon Carbide (SiC) material can advantageously replace Silicon as this semiconductor is now known to be biocompatible and to show a high chemical inertness [2]. Here, we present the electrical detection of DNA using a SiC Nanowire Field Effect Transistor (NWFET). The NWFETs are fabricated and then functionalized with DNA molecules. Between each step of the functionalization process, I-V characteristic measurements are performed. Comparative and simultaneous measurements are carried out on two SiC NWFETs: one is the sensor and the second one is used as a reference. Some interesting properties of the sensor are studied for the first time which opens the way to future developments of SiC nanowire based sensors.

Developing Fully-Integrated Biosensing Systems on the Laboratory Benchtop

Fully-integrated biosensing systems that detect the presence, absence, and quantity of biomolecules (nucleic acids and proteins) in clinical samples have a wide range of applications in clinical diagnosis of cancers, infectious diseases, and hereditary diseases. Furthermore, handheld and inexpensive biosensors that can be operated with a high degree of automation by novice users make it possible for these systems to be applied to point-of-care (POC) settings where the current state-of-the-art diagnostic tests, designed for centralized laboratories, are not applicable. In order to develop handheld, automated, and inexpensive biosensing systems applicable to the point-of-care, it is essential to combine multiple devices for sample preparation and biomolecular detection on the same platform. This requires multiple materials and structures having a wide range of properties and length-scales to be integrated on the same platform. Furthermore, translation of such multi-material systems from the laboratory to the market indicates the need for inexpensive, rapid, and scalable fabrication methods for creating tunable materials and structure. We have developed an integrated fabrication process for creating all-electrical biosensing platforms using methods available on the laboratory benchtop. In order to create electrodes that are structurally tunable from the 10 nm to the 10 mm length-scale and meet the conductivity demands of sample preparation and biosensing devices, we have combined methods including craft cutting, ink-jet printing, self-assembly, electroless deposition, and electrodeposition to deposit and pattern application-specific materials on polymer substrates. In addition, we have used shape memory polymers to further tune the properties of the patterned electrodes such as surface texture, film thickness, and conductivity. Materials characterization results obtained using techniques including scanning electron microscopy, atomic force microscopy, and four-point-probe method indicate that the fabrication method results in electrodes of the appropriate configuration (micro-coils, interdigitated electrodes, electrode arrays), surface texture (tunable from tens of nanometer to tens of microns), and conductivity (>5% of the bulk) for use in an all-electrical biosensing platform. Using the abovementioned fabrication methods, we have developed three classes of devices for bacterial lysis, magnetic manipulation of cells and biomolecules, and detection of nucleic acids. Bacterial lysis devices that include an interdigitated array of microelectrodes decorated with electrodeposited three-dimensional nano-pillars offer high efficiency lysis (> 95%) at low applied potentials (4 V). The magnetic manipulation devices containing on-polymer magnetic coils coupled with a ferromagnetic capture layer generate forces required for manipulating super-paramagnetic microbeads (1-10 pN) at low applied currents (< 50 mA). Furthermore, we have created a fully-integrated electro-fluidic device for electrochemical detection of nucleic acids. Given that all of these devices are created using the rapid, inexpensive and benchtop-based integrated fabrication process presented here, we expect this method to be applicable to the rapid prototyping and fabrication of several fully-integrated biosensing systems for POC diagnostic applications.

Concept and Design of SOI Nanogap Based on Capacitive Sensor for &lt;i&gt;Escherichia coli&lt;/i&gt; O157:H7 Detection

Early warning detection method for food-borne pathogenEscherichiacoli(E.coli) especially a harmful serotype O157:H7 has become an important public health issue in food industrial due to its frequent outbreak around the world. Serotype O157:H7E.coliis an enterhemorrhagic virulence group ofE.colithat can be found in contaminated food such as raw meat, unpasteurized milk, raw tomatoes, cucumber, and leafy salad or in animal and media which spread the pathogen. Consumption of a small amount of O157:H7E.coliwithin 10 to 24 hours can caused severe illness such as diarrhea, nausea, vomiting, fever, headache and muscular pain. Therefore, there is a need for higher sensitivity and selectivity and rapid method to detect the deadly pathogen in early stage and to prevent the spread and infection to human. Nanogap based biosensor has become an emerging advance method for detecting a nanometer size biomolecules that cannot be seen to the human eyes. Nanogap is a combination of two electrodes with nanometer gap. The novelty of nanogap is its ability to detect a small amount of biomolecules that trap between the gaps by observing their electrical properties changes. In this paper, we present a concept and design of SOI nanogap based capacitance sensor for O157:H7E.colidetection using AutoCAD software.

Biosensors Based on Plasmonics

Because of the sensitivity to refractive index changes, plasmonics nanostructures have been investigated broadly for the bio-molecules detection through the excitations of the propagating surface plasmon resonance (PSPR) or localized surface plasmon resonance (LSPR). PSPR sensors can detect sub-monolayer quantities of bio-molecules at the gold film surface and provide real-time data through continuous optical measurements. LSPR sensors could be more sensitive to local refractive index changes and the factors of nanoparticle material, shape and size are all interrelated and contribute to the refractive index sensitivities.

A Nanoscale Nuclear Magnetic Resonance System for Signature-Based Detection of Biomolecules

The need to incorporate advanced engineering tools in biology, biochemistry and medicine is in great demand. With the rapid emergence of nanotechnologies during the past decade, it is becoming hard to deny the great impact the new technologies can make on our everyday life and especially the fields of biology and medicine. In this review article, we discuss one such technology, nanoliter-volume nuclear magnetic resonance spectroscopy (NMR), which could be used for chemical signature-based detection of ultra-small quantities of biomolecules. NMR spectroscopy has been used by biochemists for over two decades. However, conventional NMR spectrometers are relatively costly, bulky and often require special laboratory environment for their proper operation. Such NMR systems require ultra-high magnetic field, i.e., of the order of several Tesla, in a relatively large volume and therefore require special shielding and installation. The major challenge for successful scaling of the conventional NMR systems down to the nanoscale range is the difficulty in maintaining adequate (for reasonable signal detection) signal-to-noise ratio (SNR). This paper discusses a new technology aimed to develop NMR spectrometers that not only could be scaled down to the nanoscale range but also could operate with adequate SNR at field strengths as low as 1/4 Tesla. The largest physical component of this system is a ring-shaped permanent magnet that has an outer diameter of 40 mm. The system was designed via computer simulations to consider the magnetostatic interactions between relatively components of the system (magnets, coils, sensors, shields, and so on) and also spin dependent nanoscale magnetic devices which could be used to further boost the net signal to noise ratio of the system. Particularly, a special tunneling magneto-resistive sensor array was designed to achieve the goal of further increasing SNR. The computational tools include direct digital synthesis to generate RF signals with an ultra-high frequency precision, digital signal processing to filter and condition the retrieved NMR signal and a special backpropagation neural network based computer chip to find the optimum match for the NMR spectrum of a sample under investigation. This portable NMR system is intended to analyze samples with volumes as small as a few hundreds of nanoliters at room temperature. In the future, the discussed technology could be further developed to detect a wide range of chemicals at a single-molecule level.

INTEGRATION OF NANOPARTICLES WITH PROTEIN MICROARRAYS

A variety of DNA, protein or cell microarray devices and systems have been developed and commercialized. In addition to the biomolecule related analysis, they are also being used for pharmacogenomic research, infectious and genetic disease and cancer diagnostics, and proteomic and cellular analysis.1 Currently, microarray is fabricated on a planar surface; this limits the amount of biomolecules that can be bounded on the surface. In this work, a planar protein microarray chip with nonplanar spot surface was fabricated to enhance the chip performance. A nonplanar spot surface was created by first coating the silica nanoparticles with albumin and depositing them into the patterned microwells. The curve surfaces of the nanoparticles increase the surface area for immobilization of proteins, which helps to enhance the detection sensitivity of the chip. Using this technique, proteins are immobilized onto the nanoparticles before they are deposited onto the chip, and therefore the method of protein immobilization can be customized at each spot. Furthermore, a nonplanar surface promotes the retention of native protein structure better than planar surface.2 The technique developed can be used to produce different types of microarrays, such as DNA, protein and antibody microarrays.

Integration of an Amorphous Silicon Thin Film Transistor with a Microchannel Electrophoresis for Protein Identification

Amorphous silicon thin-film transistors have been prepared and used to drive a microchannel electrophoresis device for protein identification. The complete system was fabricated on a glass substrate using only three photomasks. Proteins of three different sizes (14.2, 29, and 45 kDa) were successfully separated from a microliter solution and in situ identified in a short period of 1000 s. The thin film transistor also suppressed the current perturbation of the microchannel electrophoresis device in the early operation stage. This low-cost chip is potentially applicable to the identification of micro or nano amount of biomolecules from a complicated solution.

Alpha-particle energy loss measurement of microgram depositions of biomolecules.

A commercially available alpha-particle spectrometer and 210Po alpha-particle source were used to determine the mass of microgram quantities of biomolecules. Samples were deposited in microliter volumes on thin silicon nitride windows and dried. The energy loss of the alpha-particles after traversing the sample was converted to a mass using tabulated alpha-particle stopping powers. The measurement was absolute, independent of biomolecule species, and no standards were needed for quantitation. The method has a dynamic range of 0.1-100 microg for deposits of diameter 1-2 mm. The precision varies from approximately 20% at 100 ng to a few percent at 5-100 microg. The silicon nitride windows allow multimodal analysis of the same quantified sample, including PIXE probing of elemental abundances, molecular identification by mass spectrometry, and isotopic quantitation of interactions. The method was used with accelerator mass spectrometry to quantify specific activities of microgram quantities of 14C-labeled proteins.

Impact of protein adsorption on the geometry of microfluidics devices

“Lab-on-a-chip” microfluidics devices manipulate biological fluids, which contain significant quantities of biomolecules, in particular proteins and DNA, and even living cells. As the dimensions of these devices continue to decrease and approach the sub-micron range, and as the trend towards “disposable” devices continues, the impact of the inevitable adsorption of biomolecules becomes more important. In this paper we estimated the protein-adsorption-related sensitivity of the geometry of a rectangular micron-sized channel. The estimation the thickness of the adsorbed protein layer versus processing parameters, i.e., protein concentration in the fluid; ionic strength of fluid; and surface tension of the walls, is based on a proposed semi-empirical model for protein adsorption. The model, derived from the data contained in a biomolecule adsorption database, uses the concept of a “generic protein”, i.e., a protein with molecular properties averaged over the range of data present in the database. The estimation of protein-adsorption-related impact on the geometry of a rectangular micron-sized channel, i.e., narrowing of the micro-channel, increases dramatically below a threshold value of approximately 1.5–2 μm.

Blood residues on archaeological objects — a conservation perspective

AbstractThe impact of human handling on studies of blood residues is demonstrated by experimental work undertaken on modern stone tools. The results are examined within the context of excavation techniques and conservation treatments that may influence laboratory analyses, with special emphasis on the consequences of washing and handling stone artifacts. The results of experimental work are compared with those obtained from a large assemblage of stone tools that was excavated along the shore of the Anacostia River in Washington DC. Because the amounts of biomolecules found on excavated materials are small, more sensitive biochemical techniques are being applied to archaeological artifacts. The implications of organic residue analysis for archaeologists, conservators and collections managers are considered, with emphasis on appropriate methods of excavation, treatment procedures and materials for storage. This paper focuses on the issues of immediate concern to conservators, and the impact that washing and...

Intracellular proteins as Tumor markers

With the development of new and sensitive techniques for detection of very minute quantities of biomolecules now it is possible to identify the changes in the levels of gene regulatory enzymes and other substances. Silver staining in SDS-PAGE (14). Radio receptor [RRA] and Radio immuno assays [RIA] (75) enzyme linked iummuno assays [ELISA] (76). Western; Northern and Southern blotting techniques (77,78) among others can detect upto picogram quantities. Cellular proteins such as cytoskeletal proteins, membrane bound proteins (37), chromatin associated proteins and gene regulatory enzymes which are not soluble in ordinary aqueous buffers and are non-secretory in nature can now be detected in needle biopsies by the above mentioned techniques. Steroid hormone receptor assays are routinely done in many clinical biochemistry laboratories in order to decide whether the tumor from breast, cervix or uterus are hormone dependent or not.