scholarly journals Nanogravimetric and Optical Characterizations of Thrombin Interaction with a Self-Assembled Thiolated Aptamer

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Jane Politi ◽  
Ilaria Rea ◽  
Fabrizia Nici ◽  
Principia Dardano ◽  
Monica Terracciano ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
High Specificity ◽  
Medical Diagnostics ◽  
Affinity Constant ◽  
Label Free ◽  
Gold Substrate ◽  
System Sensitivity ◽  
All Optical ◽  
Thiolated Aptamer

Efficient biorecognition of thrombin (TB), a serine protease with crucial role in physiological and pathological blood coagulation, is a hot topic in medical diagnostics. In this work, we investigate the ability of synthetic thrombin aptamer (TBA), immobilized on a gold substrate, to bind thrombin by two different label-free techniques: the quartz crystal microbalance (QCM) and the spectroscopic ellipsometry (SE). By QCM characterization in the range from 20 to 110 nM, we demonstrate high specificity of TBA-TB interaction and determine affinity constant (Kd) of17.7±0.3 nM, system sensitivity of0.42±0.03 Hz nM−1, and limit of detection (LOD) of240±20 pM. The interaction between TBA and TB is also investigated by SE, an all-optical method, by quantifying the thickness increase of the TBA film assembled on gold substrate. AFM characterization of TBA and TB molecules deposited on flat silicon surface is also supplied.

scholarly journals Novel Latex Microsphere Immunochromatographic Assay for Rapid Detection of Cadmium Ion in Asparagus

Foods ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 78
Author(s):  
Naifeng Xu ◽  
Qiaojuan Zhu ◽  
Jiangxiong Zhu ◽  
Jingze Jia ◽  
Xinlin Wei ◽  
...  
Keyword(s):  
Rapid Detection ◽  
Limit Of Detection ◽  
High Specificity ◽  
Test Strip ◽  
Immunochromatographic Assay ◽  
Affinity Constant ◽  
Quantitative Detection ◽  
Cadmium Ion ◽  
Copper Transporter ◽  
Knock Out

Recently, concerns about heavy metal cadmium ion (Cd2+) residue in asparagus have been frequently reported, and there is an urgent need to develop an effective, sensitive, and rapid detection method for Cd2+. In this study, we innovatively combined molecular microbiology to carry out the comparative screening of Cd2+ chelators in a green, efficient, and specific way. The knock-out putative copper-transporter gene (pca1Δ) yeast strain with high sensitivity to Cd2+ was first used to screen the Cd2+ chelator, and the optimum chelator 1-(4-Isothiocyanatobenzyl)ethylenediamine-N,N,N,N′-tetraacetic acid (ITCBE) was obtained. Additionally, a rapid latex microsphere immunochromatographic assay (LMIA) was developed, based on the obtained monoclonal antibody (mAb) with high specificity and high affinity (affinity constant Ka = 1.83 × 1010 L/mol), to detect Cd2+ in asparagus. The 50% inhibitive concentration (IC50) of test strip was measured to be 0.2 ng/mL, and the limit of detection (IC10) for qualitative (LOD, for visual observation) and quantitative detection (LOQ, for data simulation) of the test strip was 2 ng/mL and 0.054 ng/mL, respectively. In all, the developed mAb-based LMIA shows a great potential for monitoring Cd2+ in asparagus, even in vegetable samples.

Green Synthesis and Spectroscopic Studies of Ag-rGO Nanocomposites for Highly Selective Mercury (II) Sensing

2018 ◽  
Vol 9 (1) ◽  
pp. 101-108 ◽  
Author(s):  
Shubhangi J. Mane-Gavade ◽  
Sandip R. Sabale ◽  
Xiao-Ying Yu ◽  
Gurunath H. Nikam ◽  
Bhaskar V. Tamhankar
Keyword(s):  
Green Synthesis ◽  
Color Change ◽  
Limit Of Detection ◽  
Aqueous Media ◽  
Suitable Condition ◽  
Cost Effective ◽  
Spectroscopic Studies ◽  
Calibration Plot ◽  
First Time

Introduction: Herein we report the green synthesis and characterization of silverreduced graphene oxide nanocomposites (Ag-rGO) using Acacia nilotica gum for the first time. Experimental: We demonstrate the Hg2+ ions sensing ability of the Ag-rGO nanocomposites form aqueous medium. The developed colorimetric sensor method is simple, fast and selective for the detection of Hg2+ ions in aqueous media in presence of other associated ions. A significant color change was noticed with naked eye upon Hg2+ addition. The color change was not observed for cations including Sr2+, Ni2+, Cd2+, Pb2+, Mg2+, Ca2+, Fe2+, Ba2+ and Mn2+indicating that only Hg2+ shows a strong interaction with Ag-rGO nanocomposites. Under the most suitable condition, the calibration plot (A0-A) against concentration of Hg2+ was linear in the range of 0.1-1.0 ppm with a correlation coefficient (R2) value 0.9998. Results & Conclusion The concentration of Hg2+ was quantitatively determined with the Limit of Detection (LOD) of 0.85 ppm. Also, this method shows excellent selectivity towards Hg2+ over nine other cations tested. Moreover, the method offers a new cost effective, rapid and simple approach for the detection of Hg2+ in water samples.

scholarly journals Demonstration of a Label-Free and Low-Cost Optical Cavity-Based Biosensor Using Streptavidin and C-Reactive Protein

Biosensors ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 4
Author(s):  
Donggee Rho ◽  
Seunghyun Kim
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Low Cost ◽  
Optical Cavity ◽  
Sample Volume ◽  
Small Sample ◽  
Label Free ◽  
C Reactive Protein ◽  
Reactive Protein ◽  
Cavity Structure

An optical cavity-based biosensor (OCB) has been developed for point-of-care (POC) applications. This label-free biosensor employs low-cost components and simple fabrication processes to lower the overall cost while achieving high sensitivity using a differential detection method. To experimentally demonstrate its limit of detection (LOD), we conducted biosensing experiments with streptavidin and C-reactive protein (CRP). The optical cavity structure was optimized further for better sensitivity and easier fluid control. We utilized the polymer swelling property to fine-tune the optical cavity width, which significantly improved the success rate to produce measurable samples. Four different concentrations of streptavidin were tested in triplicate, and the LOD of the OCB was determined to be 1.35 nM. The OCB also successfully detected three different concentrations of human CRP using biotinylated CRP antibody. The LOD for CRP detection was 377 pM. All measurements were done using a small sample volume of 15 µL within 30 min. By reducing the sensing area, improving the functionalization and passivation processes, and increasing the sample volume, the LOD of the OCB are estimated to be reduced further to the femto-molar range. Overall, the demonstrated capability of the OCB in the present work shows great potential to be used as a promising POC biosensor.

scholarly journals Characterization of Liposomes Using Quantitative Phase Microscopy (QPM)

Pharmaceutics ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 590
Author(s):  
Jennifer Cauzzo ◽  
Nikhil Jayakumar ◽  
Balpreet Singh Ahluwalia ◽  
Azeem Ahmad ◽  
Nataša Škalko-Basnet
Keyword(s):  
Rapid Development ◽  
Fluorescent Labeling ◽  
Label Free ◽  
Batch Mode ◽  
Full Field ◽  
Quantitative Phase ◽  
Phase Microscopy ◽  
Quantitative Phase Microscopy ◽  
Tracking Ability

The rapid development of nanomedicine and drug delivery systems calls for new and effective characterization techniques that can accurately characterize both the properties and the behavior of nanosystems. Standard methods such as dynamic light scattering (DLS) and fluorescent-based assays present challenges in terms of system’s instability, machine sensitivity, and loss of tracking ability, among others. In this study, we explore some of the downsides of batch-mode analyses and fluorescent labeling, while introducing quantitative phase microscopy (QPM) as a label-free complimentary characterization technique. Liposomes were used as a model nanocarrier for their therapeutic relevance and structural versatility. A successful immobilization of liposomes in a non-dried setup allowed for static imaging conditions in an off-axis phase microscope. Image reconstruction was then performed with a phase-shifting algorithm providing high spatial resolution. Our results show the potential of QPM to localize subdiffraction-limited liposomes, estimate their size, and track their integrity over time. Moreover, QPM full-field-of-view images enable the estimation of a single-particle-based size distribution, providing an alternative to the batch mode approach. QPM thus overcomes some of the drawbacks of the conventional methods, serving as a relevant complimentary technique in the characterization of nanosystems.

scholarly journals Microfluidic Tools for Enhanced Characterization of Therapeutic Stem Cells and Prediction of Their Potential Antimicrobial Secretome

Antibiotics ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 750
Author(s):  
Pasquale Marrazzo ◽  
Valeria Pizzuti ◽  
Silvia Zia ◽  
Azzurra Sargenti ◽  
Daniele Gazzola ◽  
...  
Keyword(s):  
Stem Cells ◽  
Defense Mechanisms ◽  
Live Cells ◽  
Label Free ◽  
Bacterial Diseases ◽  
Prospective Application ◽  
Therapy Strategies ◽  
Stromal Stem Cells ◽  
Different Sources

Antibiotic resistance is creating enormous attention on the development of new antibiotic-free therapy strategies for bacterial diseases. Mesenchymal stromal stem cells (MSCs) are the most promising candidates in current clinical trials and included in several cell-therapy protocols. Together with the well-known immunomodulatory and regenerative potential of the MSC secretome, these cells have shown direct and indirect anti-bacterial effects. However, the low reproducibility and standardization of MSCs from different sources are the current limitations prior to the purification of cell-free secreted antimicrobial peptides and exosomes. In order to improve MSC characterization, novel label-free functional tests, evaluating the biophysical properties of the cells, will be advantageous for their cell profiling, population sorting, and quality control. We discuss the potential of emerging microfluidic technologies providing new insights into density, shape, and size of live cells, starting from heterogeneous or 3D cultured samples. The prospective application of these technologies to studying MSC populations may contribute to developing new biopharmaceutical strategies with a view to naturally overcoming bacterial defense mechanisms.

scholarly journals Characterization of Recombinant Adeno-Associated Viruses (rAAVs) for Gene Therapy Using Orthogonal Techniques

Pharmaceutics ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 586
Author(s):  
Liam Cole ◽  
Diogo Fernandes ◽  
Maryam T. Hussain ◽  
Michael Kaszuba ◽  
John Stenson ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Light Scattering ◽  
Dynamic Light Scattering ◽  
Viral Vector ◽  
Structural Characteristics ◽  
Genetic Material ◽  
Label Free ◽  
Gene Therapies ◽  
Multiple Challenges

Viruses are increasingly used as vectors for delivery of genetic material for gene therapy and vaccine applications. Recombinant adeno-associated viruses (rAAVs) are a class of viral vector that is being investigated intensively in the development of gene therapies. To develop efficient rAAV therapies produced through controlled and economical manufacturing processes, multiple challenges need to be addressed starting from viral capsid design through identification of optimal process and formulation conditions to comprehensive quality control. Addressing these challenges requires fit-for-purpose analytics for extensive characterization of rAAV samples including measurements of capsid or particle titer, percentage of full rAAV particles, particle size, aggregate formation, thermal stability, genome release, and capsid charge, all of which may impact critical quality attributes of the final product. Importantly, there is a need for rapid analytical solutions not relying on the use of dedicated reagents and costly reference standards. In this study, we evaluate the capabilities of dynamic light scattering, multiangle dynamic light scattering, and SEC–MALS for analyses of rAAV5 samples in a broad range of viral concentrations (titers) at different levels of genome loading, sample heterogeneity, and sample conditions. The study shows that DLS and MADLS® can be used to determine the size of full and empty rAAV5 (27 ± 0.3 and 33 ± 0.4 nm, respectively). A linear range for rAAV5 size and titer determination with MADLS was established to be 4.4 × 1011–8.7 × 1013 cp/mL for the nominally full rAAV5 samples and 3.4 × 1011–7 × 1013 cp/mL for the nominally empty rAAV5 samples with 3–8% and 10–37% CV for the full and empty rAAV5 samples, respectively. The structural stability and viral load release were also inferred from a combination of DLS, SEC–MALS, and DSC. The structural characteristics of the rAAV5 start to change from 40 °C onward, with increasing aggregation observed. With this study, we explored and demonstrated the applicability and value of orthogonal and complementary label-free technologies for enhanced serotype-independent characterization of key properties and stability profiles of rAAV5 samples.

scholarly journals Surface Plasmon Resonance Assay for Label-Free and Selective Detection of HIV-1 p24 Protein

Biosensors ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 180
Author(s):  
Lucia Sarcina ◽  
Giuseppe Felice Mangiatordi ◽  
Fabrizio Torricelli ◽  
Paolo Bollella ◽  
Zahra Gounani ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Covalent Binding ◽  
Hiv Infections ◽  
Selectivity Ratio ◽  
Selective Detection ◽  
Label Free ◽  
Self Assembled Monolayer ◽  
Label Free Detection ◽  
P24 Protein ◽  
Hiv 1

The early detection of the human immunodeficiency virus (HIV) is of paramount importance to achieve efficient therapeutic treatment and limit the disease spreading. In this perspective, the assessment of biosensing assay for the HIV-1 p24 capsid protein plays a pivotal role in the timely and selective detection of HIV infections. In this study, multi-parameter-SPR has been used to develop a reliable and label-free detection method for HIV-1 p24 protein. Remarkably, both physical and chemical immobilization of mouse monoclonal antibodies against HIV-1 p24 on the SPR gold detecting surface have been characterized for the first time. The two immobilization techniques returned a capturing antibody surface coverage as high as (7.5 ± 0.3) × 1011 molecule/cm2 and (2.4 ± 0.6) × 1011 molecule/cm2, respectively. However, the covalent binding of the capturing antibodies through a mixed self-assembled monolayer (SAM) of alkanethiols led to a doubling of the p24 binding signal. Moreover, from the modeling of the dose-response curve, an equilibrium dissociation constant KD of 5.30 × 10−9 M was computed for the assay performed on the SAM modified surface compared to a much larger KD of 7.46 × 10−5 M extracted for the physisorbed antibodies. The chemically modified system was also characterized in terms of sensitivity and selectivity, reaching a limit of detection of (4.1 ± 0.5) nM and an unprecedented selectivity ratio of 0.02.

scholarly journals Immortalization and Characterization of Rat Lingual Keratinocytes in a High-Calcium and Feeder-Free Culture System Using ROCK Inhibitor Y-27632

2021 ◽  
Vol 22 (13) ◽  
pp. 6782
Author(s):  
Zixing Chen ◽  
Wenmeng He ◽  
Thomas Chun Ning Leung ◽  
Hau Yin Chung
Keyword(s):  
Calcium Imaging ◽  
Cultured Cells ◽  
Rho Kinase ◽  
Culture Method ◽  
Taste Bud ◽  
Label Free ◽  
Sprague Dawley ◽  
Rock Inhibitor ◽  
High Calcium

Cultured keratinocytes are desirable models for biological and medical studies. However, primary keratinocytes are difficult to maintain, and there has been little research on lingual keratinocyte culture. Here, we investigated the effect of Y-27632, a Rho kinase (ROCK) inhibitor, on the immortalization and characterization of cultured rat lingual keratinocyte (RLKs). Three Y-27632–supplemented media were screened for the cultivation of RLKs isolated from Sprague–Dawley rats. Phalloidin staining and TUNEL assay were applied to visualize cytoskeleton dynamics and cell apoptosis following Y-27632 removal. Label-free proteomics, RT-PCR, calcium imaging, and cytogenetic studies were conducted to characterize the cultured cells. Results showed that RLKs could be conditionally immortalized in a high-calcium medium in the absence of feeder cells, although they did not exhibit normal karyotypes. The removal of Y-27632 from the culture medium led to reversible cytoskeletal reorganization and nuclear enlargement without triggering apoptosis, and a total of 239 differentially expressed proteins were identified by proteomic analysis. Notably, RLKs derived from the non-taste epithelium expressed some molecular markers characteristic of taste bud cells, yet calcium imaging revealed that they rarely responded to tastants. Collectively, we established a high-calcium and feeder-free culture method for the long-term maintenance of RLKs. Our results shed some new light on the immortalization and differentiation of lingual keratinocytes.

scholarly journals Electrokinetic Formation of “Microbridges” for Protein Biomarkers as Sensors

2007 ◽  
Vol 12 (5) ◽  
pp. 311-317 ◽  
Author(s):  
Vindhya Kunduru ◽  
Shalini Prasad
Keyword(s):  
Electric Fields ◽  
Microelectrode Array ◽  
Protein Detection ◽  
High Specificity ◽  
Detection Methods ◽  
Label Free ◽  
Protein Biomarkers ◽  
Detection Scheme ◽  
Polystyrene Beads ◽  
Conducting Path

We demonstrate a technique to detect protein biomarkers contained in vulnerable coronary plaque using a platform-based microelectrode array (MEA). The detection scheme is based on the property of high specificity binding between antibody and antigen similar to most immunoassay techniques. Rapid clinical diagnosis can be achieved by detecting the amount of protein in blood by analyzing the protein's electrical signature. Polystyrene beads which act as transportation agents for the immobile proteins (antigen) are electrically aligned by application of homogenous electric fields. The principle of electrophoresis is used to produce calculated electrokinetic movement among the anti-C-reactive protein (CRP), or in other words antibody funtionalized polystyrene beads. The electrophoretic movement of antibody-functionalized polystyrene beads results in the formation of “Microbridges” between the two electrodes of interest which aid in the amplification of the antigen—antibody binding event. Sensitive electrical equipment is used for capturing the amplified signal from the “Microbridge” which essentially behaves as a conducting path between the two electrodes. The technique circumvents the disadvantages of conventional protein detection methods by being rapid, noninvasive, label-free, repeatable, and inexpensive. The same principle of detection can be applied for any receptor—ligand-based system because the technique is based only on the volume of the analyte of interest. Detection of the inflammatory coronary disease biomarker CRP is achieved at concentration levels spanning over the lower microgram/milliliter to higher order nanogram/milliliter ranges.

scholarly journals Label-Free Electrochemical Biosensor Based on Au@MoS₂–PANI for Escherichia coli Detection

Chemosensors ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 49
Author(s):  
Pushap Raj ◽  
Man Hwan Oh ◽  
Kyudong Han ◽  
Tae Yoon Lee
Keyword(s):  
Escherichia Coli ◽  
Bacterial Infections ◽  
Limit Of Detection ◽  
Bacterial Pathogens ◽  
Cost Effective ◽  
Covalent Immobilization ◽  
Label Free ◽  
Self Assembled Monolayer ◽  
Detection Range ◽  
Linear Detection

Bacterial infections have become a significant challenge in terms of public health, the food industry, and the environment. Therefore, it is necessary to address these challenges by developing a rapid, cost-effective, and easy-to-use biosensor for early diagnosis of bacterial pathogens. Herein, we developed a simple, label-free, and highly sensitive immunosensor based on electrochemical detection using the Au@MoS₂–PANI nanocomposite. The conductivity of the glassy carbon electrode is greatly enhanced using the Au@MoS₂–PANI nanocomposite and a self-assembled monolayer of mercaptopropionic acid on the gold nanoparticle surface was employed for the covalent immobilization of antibodies to minimize the nonspecific adsorption of bacterial pathogens on the electrode surface. The biosensor established a high selectivity and sensitivity with a low limit of detection of 10 CFU/mL, and detected Escherichia coli within 30 min. Moreover, the developed biosensor demonstrated a good linear detection range, practical utility in urine samples, and electrode regenerative studies.

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