Avidin-biotin System Research Articles

Targeted delivery of reduced graphene oxide nanosheets using multifunctional ultrasound nanobubbles for visualization and enhanced photothermal therapy.

Ultrasound molecular imaging as a promising strategy, which involved the use of molecularly targeted contrast agents, combined the advantages of contrast-enhanced ultrasound with the photothermal effect of reduced graphene oxide (rGO).Methods and resultsThe heparin sulfate proteoglycan glypican-3 (GPC3) is a potential molecular target for hepatocellular carcinoma (HCC). In this study, we covalently linked biotinylated GPC3 antibody to PEGylated nano-rGO to obtain GPC3-modified rGO-PEG (rGO-GPC3), and then combined rGO-GPC3 with avidinylated nanobubbles (NBs) using biotin-avidin system to prepare NBs-GPC3-rGO with photothermal effect and dispersibility, solubility in physiological environment. The average size of NBs-GPC3-rGO complex was 700.4±52.9 nm due to the polymerization of biotin-avidin system. Scanning electron microscope (SEM) showed NBs-GPC3-rGO attached to human hepatocellular carcinoma HepG2 cell. The ultrasound-targeted nanobubble destruction (UTND) technology make use of the physical energy of ultrasound exposure for the improvement of rGO delivery. Compared with other control groups, the highest nanobubble destruction efficiency of NBs-GPC3-rGO was attributed to the dissection effect of rGO on UTND. This is a positive feedback effect that leads to an increase in the concentration of rGO around the HepG2 cell. So NBs-GPC3-rGO using UTND and near-infrared (NIR) irradiation resulted in cell viability within 24 h, 48 h, 72 h lower than other treatment groups.ConclusionThis work established NBs-GPC3-rGO as an ultrasonic photothermal agent due to its suitable size, imaging capability, photothermal efficiency for visual photothermal therapy in vitro.

Angle-Scanning Surface Plasmon Resonance System with 3D Printed Components for Biorecognition Investigation

Surface plasmon resonance (SPR) is a real-time, label-free, and high-sensitive detection technology. SPR has been widely used in many applications such as biomolecular interaction analysis, environmental monitoring, and medical diagnostics. However, conventional SPR sensor systems usually require expensive equipment and complicated optics. In this paper, we have demonstrated a rapid prototyping of angle-scanning SPR for bioanalytical investigation. Rapid prototyping was attained by utilizing the FDM (fused deposition modeling) based 3D (three-dimensional) printing technology. Two rotating platforms were employed to drive the laser source and photodiode, respectively. A temperature regulation unit was incorporated to maintain the system temperature in order to reduce the temperature effect. The proposed SPR rapid prototyping yielded a refractive index resolution of 6.4×10−6 RIU (refractive index unit), and the biotin-avidin system validated the kinetics parameters measurement capability. The obtained results indicated that the FDM 3D printing has great potential for developing rapid-prototyping SPR system.

An attempt to incorporate effect of direct interaction between a ligand and explicit water molecules into MM/3D-RISM.

Endpoint methods using continuum-solvent models are widely used to estimate protein-ligand affinity. A recently developed method, MM/3D-RISM, estimates the solvation energy using statistical mechanics by 3D-RISM. This method is theoretically expected to accurately describe solvation effects and to also be less dependent on protein-ligand systems. In this study, we examined the performance of MM/3D-RISM for a set of α-thrombin inhibitors with a non-congeneric series of ligands, containing three diverse chemical scaffolds. The standard MM/3D-RISM showed a weak correlation (R2 =0.191) but correctly estimated affinity for two of the three scaffolds. However, the simplest inhibitor, benzamidine, was not ranked appropriately. From visual inspection of inhibitor-binding modes, an attempt was made to incorporate the direct interaction between a ligand and water molecules into MM/3D-RISM. A model (Model-1) dealing with directly interacting water molecules (Wat) as an independent component of a protein (R)-ligand (L) complex-formation, that is, R+L+Wat → R-L-Wat, showed a better linearity (R2 =0.422) than that of the standard MM/3D-RISM model and achieved a good ranking of all three scaffolds of α-thrombin inhibitors. Additionally, an attempt was made to model avidin-biotin system with a congeneric series of inhibitors, and results showed that both the standard MM/3D-RISM model (R2 =0.839) and Model-1 (R2 =0.695) satisfactorily estimated the affinity.

Preparation of dual-targeted pH-sensitive DOX prodrug-microbubble complex and drug release experiment in vitro

Objective To prepare dual-targeted pH-sensitive DOX prodrug-microbubble complex and explore the characterization of complex with ultrasound as well as drug release in vitro. Methods Dual-targeted ligands, cRGD and folate were conjugated with heparin using carbodiimide method, and then the dual-targeted pH-sensitive DOX prodrug was synthesized by coupling DOX via a pH-sensitive hydrazone bond. The prodrug was combined with microbubbles to prepare complex by biotin-avidin system. The characterization of complex with/without ultrasound was investigated for size, morphology and drug loaded capacity.In vitro drug release manner of complex with/without at different pH was analyzed. Results DOX content of the prodrug determined by UV Spectrophotometry was about 18.9%. Dynamic laser light scattering analysis(DLS), corresponding to transmission electron microscope(TEM) findings, revealed its inhomogeneous size distribution [mean size (159.7±24.5)nm and (1 089.0±174.9)nm]. However, the complex was dispersed into uniform fragment after ultrasound irradiation[mean size (155.9±29.8)nm, polymer dispersity index(PDI) 0.22, Zeta potential -(20.6±3.4)mV]. The cumulative release rate of DOX from both complex and complex with ultrasound at pH 5.0 were much faster than that at pH 7.4, displaying a pH-triggered release manner. Conclusions Dual-targeted pH-sensitive DOX prodrug-microbubble complex displays excellent drug release activity in acid environment. Uniform fragment and smaller particle size of complex could be achieved via ultrasound irradiation, promoting DOX accumulation within tumor tissue and facilitating in vivo antitumor ability. Key words: Sonication; Doxorubicin; Microbubbles; Targeted therapy

Characterization of the functions and proteomes associated with membrane rafts in chicken sperm.

Cellular membranes are heterogeneous, and this has a great impact on cellular function. Despite the central role of membrane functions in multiple cellular processes in sperm, their molecular mechanisms are poorly understood. Membrane rafts are specific membrane domains enriched in cholesterol, ganglioside GM1, and functional proteins, and they are involved in the regulation of a variety of cellular functions. Studies of the functional characterization of membrane rafts in mammalian sperm have demonstrated roles in sperm-egg binding and the acrosomal reaction. Recently, our biochemical and cell biological studies showed that membrane rafts are present and might play functional roles in chicken sperm. In this study, we isolated membrane rafts from chicken sperm as a detergent-resistant membranes (DRM) floating on a density gradient in the presence of 1% Triton X-100, and characterized the function and proteomes associated with these domains. Biochemical comparison of the DRM between fresh and cryopreserved sperm demonstrated that cryopreservation induces cholesterol loss specifically from membrane rafts, indicating the functional connection with reduced post-thaw fertility in chicken sperm. Furthermore, using an avidin-biotin system, we found that sperm DRM is highly enriched in a 60 KDa single protein able to bind to the inner perivitelline layer. To identify possible roles of membrane rafts, quantitative proteomics, combined with a stable isotope dimethyl labeling approach, identified 82 proteins exclusively or relatively more associated with membrane rafts. Our results demonstrate the functional distinctions between membrane domains and provide compelling evidence that membrane rafts are involved in various cellular pathways inherent to chicken sperm.

Structural Characterization of the Avidin Interactions with Fluorescent Pyrene-Conjugates: 1-Biotinylpyrene and 1-Desthiobiotinylpyrene.

Avidin is a tetrameric protein that belongs to the calycin superfamily. It has been studied mainly because of its extraordinary affinity to biotin, which led to a wide range of applications based on the avidin-biotin system. In the present study, we report the first crystal structures of avidin in a complex with two novel fluorescent pyrene derivatives: 1-biotinylpyrene (B9P) and 1-desthiobiotinylpyrene (D9P). The crystal structures were solved by molecular replacement using the coordinates of avidin molecule as a starting model and the final models of avidin/B9P and avidin/D9P were refined to resolutions of 2.0 Å and 2.1 Å, respectively. Our data reveal changes in loop conformation as well as in overall fold and quaternary arrangement of the avidin upon the binding of these fluorescent probes. Moreover, the crystal structures allowed analysis of the details of the interactions between the protein and the pyrene derivatives. Structural description of the complexes will contribute to the design of conjugates for expanding the capabilities of avidin–biotin technology.

Maskless localized patterning of biomolecules on carbon nanotube microarray functionalized by ultrafine atmospheric pressure plasma jet using biotin-avidin system

Ultrafine plasma jet is a promising technology with great potential for nano- or micro-scale surface modification. In this letter, we demonstrated the use of ultrafine atmospheric pressure plasma jet (APPJ) for patterning bio-immobilization on vertically aligned carbon nanotube (CNT) microarray platform without a physical mask. The biotin-avidin system was utilized to demonstrate localized biomolecule patterning on the biosensor devices. Using ±7.5 kV square-wave pulses, the optimum condition of plasma jet with He/NH3 gas mixture and 2.5 s treatment period has been obtained to functionalize CNTs. The functionalized CNTs were covalently linked to biotin, bovine serum albumin (BSA), and avidin-(fluorescein isothiocyanate) FITC, sequentially. BSA was necessary as a blocking agent to protect the untreated CNTs from avidin adsorption. The localized patterning results have been evaluated from avidin-FITC fluorescence signals analyzed using a fluorescence microscope. The patterning of biomolecules on the CNT microarray platform using ultrafine APPJ provides a means for potential application of microarray biosensors based on CNTs.

Rapid recognition and functional analysis of membrane proteins on human cancer cells using atomic force microscopy

Understanding the physicochemical properties of cell surface signalling molecules is important for us to uncover the underlying mechanisms that guide the cellular behaviors. Atomic force microscopy (AFM) has become a powerful tool for detecting the molecular interactions on individual cells with nanometer resolution. In this paper, AFM peak force tapping (PFT) imaging mode was applied to rapidly locate and visually map the CD20 molecules on human lymphoma cells using biochemically sensitive tips. First, avidin–biotin system was used to test the effectiveness of using PFT imaging mode to probe the specific molecular interactions. The adhesion images obtained on avidin-coated mica using biotin-tethered tips obviously showed the recognition spots which corresponded to the avidins in the simultaneously obtained topography images. The experiments confirmed the specificity and reproducibility of the recognition results. Then, the established procedure was applied to visualize the nanoscale organization of CD20s on the surface of human lymphoma Raji cells using rituximab (a monoclonal anti-CD20 antibody)-tethered tips. The experiments showed that the recognition spots in the adhesion images corresponded to the specific CD20–rituximab interactions. The cluster sizes of CD20s on lymphoma Raji cells were quantitatively analyzed from the recognition images. Finally, under the guidance of fluorescence recognition, the established procedure was applied to cancer cells from a clinical lymphoma patient. The results showed that there were significant differences between the adhesion images obtained on cancer cells and on normal cells (red blood cell). The CD20 distributions on ten cancer cells from the patient were quantified according to the adhesion images. The experimental results demonstrate the capability of applying PFT imaging to rapidly investigate the nanoscale biophysical properties of native membrane proteins on the cell surface, which is of potential significance in developing novel biomarkers for cancer diagnosis and drug development.

Optimal preparation of the complex of paclitaxel loaded heparin nanoparticles and microbubbles and screening for the optimized ultrasound condition for different breast cancer cells

Objective To investigate the optimal preparation of the complex of paclitaxel-loaded heparin nanoparticles and microbubbles and screen the optimized ultrasound (US) condition for highly aggressive breast cancer cells MDA-MB-231 and less aggressive breast cancer cells MCF-7. Methods Paclitaxel-loaded nanoparticles were prepared using heparin as nanocarrier, and biotinlyated microbubbles were prepared by mechanical shaking. Paclitaxel-loaded nanoparticles was conjugated with microbubbles to prepare the complex using avidin-biotin system. The optimized method was screened. Breast cancer cell MDA-MB-231 and MCF-7 were treated with the prepared complex and different ultrasonic parameters including irradiation intensity and exposure duration were evaluated by cellular uptake experiments. Results The drug loading in the complex was 106.7 μg/108 microbubbles when the paclitaxel-nanoparticle concentration was 2 mg/ml. For MDA-MB-231 cells, the optimal irradiation intensity was 1.0 W/cm2 and the optimal exposure duration was 40 s, and they were 1.5W/cm2 and 40s for MCF-7 cells, respectively. Conclusions The complex with high drug loading weight, composed of paclitaxel-loaded heparin nanoparticles and microbubbles, was successfully prepared, and it was found the optimal ultrasound condition was different in two breast cancer cells. Key words: Ultrasonography; Paclitaxel-nanoparticle; Microbubbles; Tumor cells

고감도 나노-바이오센서를 위한 나노로드 전극 표면 개질에 관한 연구

많은 생체 친화적인 센서들 중에서 avidin-biotin system은 높은 상호 특이적인 친화성으로 인하여 많은 생물학적인 응용 연구에 이용되어 왔다. 효과적인 avidin-biotin 바이오센서 개발을 위해 avidin-biotin 간의 상호 반응성을 증대시키기 위해서는 높은 표면적을 가지는 전극이 필요하다. 본 연구에서는 이러한 목적을 위해 gold nanorods electrode를 사용하였다. 전기화학적인 특성은 cyclic voltammetry (CV)와 electrochemical impedance spectroscopy (EIS)를 가지고 redox couple <TEX>$[Fe(CN)_6</TEX><TEX>]</TEX><TEX>^{3-/4-}$</TEX>를 사용하여 다양한 biotin의 농도에 따라 분석되었다. 결론적으로 nanorod의 전극은 1 ng/mL보다 낮은 biotin의 농도도 감지할 수 있음을 보였다. Among many kinds of bioaffinity sensors, the avidin-biotin system has been widely used in a variety of biological applications due to the specific and high affinity interaction of the system. In this work, gold nanorods with high surface area were explored as electrodes in order to amplify the signal response from the avidin-biotin interaction which can be further utilized for avidin-biotin biosensors. Electrochemical performance of electrodes modified with nanorods and functionalized with avidin in response to interactions with biotin at various concentrations using <TEX>$[Fe(CN)_6</TEX><TEX>]</TEX><TEX>^{3-/4-}$</TEX> couple as the redox probe were investigated using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). A very low biotin concentration of less than 1 ng/mL could be detected using the electrodes modified with nanorods.

Nanopore as a Sensor Based on Avidin-Biotin System

This work aims to design a highly selective biosensor, able to detect selectively a molecule with a label or with an antigen/antibody system, and without the use of a microscope or a laser, and thus building a low cost detection system.To make this we have explored different strategies used to fashion artificial nanopore in order to mimic biological ligand-gated channel. This strategy is based on PEG like-avidin tethering in track etched nanopore modified by atomic layer deposition (ALD). Track etched combined with ALD was chosen to control their geometry and the nanopore diameter (cylindrical) and to functionalize them with polymers and biomolecules.Then we have investigated the sensing aspect more in details. (i) the size of the spacer, with a long or short PEG chain, (ii) compare avidin and streptavidin, (iii) biotinylated proteins: IgG and BSA, (iv) and the addition of an anti-body (anti BSA).This new strategy to tailor artificial ligand-gated nanopore permits to consider a simple and low cost biosensor for antibody detection.

BMP6 and BMP4 expression in patients with cancer-related anemia and its relationship with hepcidin and s-HJV.

In the present study, we investigated BMP6 and BMP4 expression in patients with cancer-related anemia (CRA) as well as its relationship with hepcidin and s-HJV. The avidin-biotin system enzyme-linked immunosorbent assay was used to test serum levels of BMP6, BMP4, s-HJV, and hepcidin in 53 cancer patients with anemia and 52 control cancer patients without anemia. Serum levels of BMP6 and hepcidin in the anemia group were 434.53 ± 212.11 ng/mL and 5.68 ± 3.89 μg/L, respectively. In the non-anemia cancer group, serum BMP6 and hepcidin levels were 334.37 ± 171.32 ng/mL and 4.60 ± 2.28 μg/L, which were significantly lower than the levels for the CRA group (P < 0.05). In addition, the serum level of s-HJV was 0.69 ± 0.28 ng/mL in the CRA group, which was significantly lower compared to that for the non-anemia group (1.07 ± 1.00 ng/mL, P < 0.01). There were no significant differences in BMP4 expression between the two groups. BMP6 was negatively correlated with s-HJV and Hb (r = -0.2536 and -0.2949, P < 0.01), but was not correlated with hepcidin. Similarly, BMP4 expression was not correlated with Hb, s-HJV, or hepcidin. Our study shows that patients with CRA had high expression of BMP6 and hepcidin and low expression of s-HJV. BMP6 was found to be negatively correlated with s-HJV; both regulate hepcidin expression and play important roles in the development of anemia.

Improving the Immunogenicity of the Mycobacterium bovis BCG Vaccine by Non-Genetic Bacterial Surface Decoration Using the Avidin-Biotin System.

Current strategies to improve the current BCG vaccine attempt to over-express genes encoding specific M. tuberculosis (Mtb) antigens and/or regulators of antigen presentation function, which indeed have the potential to reshape BCG in many ways. However, these approaches often face serious difficulties, in particular the efficiency and stability of gene expression via nucleic acid complementation and safety concerns associated with the introduction of exogenous DNA. As an alternative, we developed a novel non-genetic approach for rapid and efficient display of exogenous proteins on bacterial cell surface. The technology involves expression of proteins of interest in fusion with a mutant version of monomeric avidin that has the feature of reversible binding to biotin. Fusion proteins are then used to decorate the surface of biotinylated BCG. Surface coating of BCG with recombinant proteins was highly reproducible and stable. It also resisted to the freeze-drying shock routinely used in manufacturing conventional BCG. Modifications of BCG surface did not affect its growth in culture media neither its survival within the host cell. Macrophages phagocytized coated BCG bacteria, which efficiently delivered their surface cargo of avidin fusion proteins to MHC class I and class II antigen presentation compartments. Thereafter, chimeric proteins corresponding to a surrogate antigen derived from ovalbumin and the Mtb specific ESAT6 antigen were generated and tested for immunogenicity in vaccinated mice. We found that BCG displaying ovalbumin antigen induces an immune response with a magnitude similar to that induced by BCG genetically expressing the same surrogate antigen. We also found that BCG decorated with Mtb specific antigen ESAT6 successfully induces the expansion of specific T cell responses. This novel technology, therefore, represents a practical and effective alternative to DNA-based gene expression for upgrading the current BCG vaccine.

Highly Disordered Array of Silicon Nanowires: an Effective and Scalable Approach for Performing and Flexible Electrochemical Biosensors.

The direct integration of disordered arranged and randomly oriented silicon nanowires (SiNWs) into ultraflexible and transferable electronic circuits for electrochemical biosensing applications is proposed. The working electrode (WE) of a three-electrode impedance device, fabricated on a polyimide (PI) film, is modified with SiNWs covered by a thin Au layer and functionalized to bind the sensing element. The biosensing behavior is investigated through the ligand-receptor binding of biotin-avidin system. Impedance measurements show a very efficient detection of the avidin over a broad range of concentrations from hundreds of micromolar down to the picomolar values. The impedance response is modeled through a simple equivalent circuit, which takes into account the unique WE morphology and its modification with successive layers of biomolecules. This approach of exploiting highly disordered SiNW ensemble in biosensing proves to be very promising for the following three main reasons: first, the system morphology allows high sensing performance; second, these nanostructures can be built via scalable and transferable fabrication methodology allowing an easy integration on non-conventional substrates; third, reliable modeling of the sensing response can be developed by considering the morphological and surface characteristics over an ensemble of disordered NWs rather than over individual NWs.

Biotin-avidin mediates the binding of adipose-derived stem cells to a porous β-tricalcium phosphate scaffold: Mandibular regeneration.

The present study aimed to investigate the properties of a promising bone scaffold for bone repair, which consisted of a novel composite of adipose-derived stem cells (ADSCs) attached to a porous β-tricalcium phosphate (β-TCP) scaffold with platelet-rich plasma (PRP). The β-TCP powder was synthesized and its composition was determined using X-ray diffraction and Fourier transform infrared spectroscopy. The surface morphology and microstructure of the fabricated porous β-TCP scaffold samples were analyzed using light and scanning electron microscopy, and their porosity and compressive strength were also evaluated. In addition, the viability of rabbit ADSCs incubated with various concentrations of the β-TCP extraction fluid was analyzed. The rate of attachment and the morphology of biotinylated ADSCs (Bio-ADSCs) on avidin-coated β-TCP (Avi-β-TCP), and untreated ADSCs on β-TCP, were compared. Furthermore, in vivo bone-forming abilities were determined following the implantation of group 1 (Bio-ADSCs/Avi-β-TCP) and group 2 (Bio-ADSCs/Avi-β-TCP/PRP) constructs using computed tomography, and histological osteocalcin (OCN) and alkaline phosphatase (ALP) expression analyses in a rabbit model of mandibulofacial defects. The β-TCP scaffold exhibited a high porosity (71.26±0.28%), suitable pore size, and good mechanical strength (7.93±0.06 MPa). Following incubation with β-TCP for 72 h, 100% of viable ADSCs remained. The avidin-biotin binding system significantly increased the initial attachment rate of Bio-ADSCs to Avi-β-TCP in the first hour (P<0.01). Following the addition of PRP, group 2 exhibited a bony-union and mandibular body shape, newly formed bone and increased expression levels of OCN and ALP in the mandibulofacial defect area, as compared with group 1 (P<0.05). The results of the present study suggested that the novel Bio-ADSCs/Avi-β-TCP/PRP composite may have potential application in bone repair and bone tissue engineering.

Bioconjugation efficiency of plasma-functionalized carbon-encapsulated iron nanoparticles with biotin–avidin system

The bioconjugation efficiency of carbon-encapsulated iron nanoparticles (Fe@C NPs) with a typical diameter of 20–40 nm functionalized by plasma was studied, aiming at their applications to highly-sensitive virus detection system. The Fe@C NPs were functionalized with amino group, and the population of amino groups on the nanoparticles was varied by changing the plasma operating conditions. Radio frequency wave-excited inductively-coupled Ar and ammonia gas plasmas were used to introduce amino groups onto the surface of Fe@C NPs. The efficiency in bioconjugation was examined with biotin–avidin system. Initially the amino modified Fe@C NPs were immobilized with biotins, and then the interaction of it with the avidin molecules was analyzed using absorption and fluorescence spectroscopy techniques. From the results of these studies, it was found that the plasma functionalized Fe@C NP indicated a highly-efficient bioconjugation property using biotin–avidin system. Furthermore, the present results suggest a feasibility of highly-sensitive virus capturing system using the plasma-functionalized Fe@C NPs.

Natural infection of phlebotomines (Diptera: Psychodidae) by Leishmania (Leishmania) amazonensis in an area of ecotourism in Central-Western Brazil.

BackgroundBonito municipality, known as an area of ecoturism, in Mato Grosso do Sul state, Brazil, is also a focus of visceral and cutaneous leishmaniases, with cases registered in both human and canine populations. This study sought to investigate natural infection by flagellate forms of Leishmania in phlebotomines of the urban area of Bonito.FindingsSand flies were collected fortnightly from October 2005 to July 2006 with modified automatic light traps installed in peridomiciles and animal shelters in the center and on the outskirts of the city. The females were dissected and their guts observed under an optical microscope. A total of 1977 specimens were captured, Lutzomyia longipalpis (88.4 %) and Bichromomyia flaviscutelata (3.0 %) being the most frequent species. Bi. flaviscutellata was found infected by flagellates that were identified as Leishmania (Leishmania) amazonensis by indirect immunofluorescence reaction, employing monoclonal antibodies and the biotin-avidin system. This is the first report of natural infection by L. amazonensis in Bi. flaviscutellata in a Brazilian urban area.ConclusionsAs Bi. flaviscutellata is only slightly attracted by humans, the transmission of L. amazonensis in the study area may have a zoonotic character; however, the sympatric occurrence of this parasite and Lu. longipalpis should be taken into consideration by the local health authorities since this sand fly has already been found with L. amazonensis DNA in a focus of canine visceral leishmaniasis in Bonito municipality.

PEG Functionalization of Whispering Gallery Mode Optical Microresonator Biosensors to Minimize Non-Specific Adsorption during Targeted, Label-Free Sensing.

Whispering Gallery Mode (WGM) optical microresonator biosensors are a powerful tool for targeted detection of analytes at extremely low concentrations. However, in complex environments, non-specific adsorption can significantly reduce their signal to noise ratio, limiting their accuracy. To overcome this, poly(ethylene glycol) (PEG) can be employed in conjunction with appropriate recognition elements to create a nonfouling surface capable of detecting targeted analytes. This paper investigates a general route for the addition of nonfouling elements to WGM optical biosensors to reduce non-specific adsorption, while also retaining high sensitivity. We use the avidin-biotin analyte-recognition element system, in conjunction with PEG nonfouling elements, as a proof-of-concept, and explore the extent of non-specific adsorption of lysozyme and fibrinogen at multiple concentrations, as well as the ability to detect avidin in a concentration-dependent fashion. Ellipsometry, contact angle measurement, fluorescence microscopy, and optical resonator characterization methods were used to study non-specific adsorption, the quality of the functionalized surface, and the biosensor’s performance. Using a recognition element ratio to nonfouling element ratio of 1:1, we showed that non-specific adsorption could be significantly reduced over the controls, and that high sensitivity could be maintained. Due to the frequent use of biotin-avidin-biotin sandwich complexes in functionalizing sensor surfaces with biotin-labeled recognition elements, this chemistry could provide a common basis for creating a non-fouling surface capable of targeted detection. This should improve the ability of WGM optical biosensors to operate in complex environments, extending their application towards real-world detection.

Bone Tissue Engineering by Using Calcium Phosphate Glass Scaffolds and the Avidin–Biotin Binding System

Highly porous and interconnected scaffolds were fabricated using calcium phosphate glass (CPG) for bone tissue engineering. An avidin-biotin binding system was used to improve osteoblast-like cell adhesion to the scaffold. The scaffolds had open macro- and micro-scale pores, and continuous struts without cracks or defects. Scaffolds prepared using a mixture (amorphous and crystalline CPG) were stronger than amorphous group and crystalline group. Cell adhesion assays showed that more cells adhered, with increasing cell seeding efficiency to the avidin-adsorbed scaffolds, and that cell attachment to the highly porous scaffolds significantly differed between avidin-adsorbed scaffolds and other scaffolds. Proliferation was also significantly higher for avidin-adsorbed scaffolds. Osteoblastic differentiation of MG-63 cells was observed at 3days, and MG-63 cells in direct contact with avidin-adsorbed scaffolds were positive for type I collagen, osteopontin, and alkaline phosphatase gene expression. Osteocalcin expression was observed in the avidin-adsorbed scaffolds at 7days, indicating that cell differentiation in avidin-adsorbed scaffolds occurred faster than the other scaffolds. Thus, these CPG scaffolds have excellent biological properties suitable for use in bone tissue engineering.

Buoyancy-activated cell sorting using targeted biotinylated albumin microbubbles.

Cell analysis often requires the isolation of certain cell types. Various isolation methods have been applied to cell sorting, including florescence-activated cell sorting and magnetic-activated cell sorting. However, these conventional approaches involve exerting mechanical forces on the cells, thus risking cell damage. In this study we applied a novel isolation method called buoyancy-activated cell sorting, which involves using biotinylated albumin microbubbles (biotin-MBs) conjugated with antibodies (i.e., targeted biotin-MBs). Albumin MBs are widely used as contrast agents in ultrasound imaging due to their good biocompatibility and stability. For conjugating antibodies, biotin is conjugated onto the albumin MB shell via covalent bonds and the biotinylated antibodies are conjugated using an avidin-biotin system. The albumin microbubbles had a mean diameter of 2μm with a polydispersity index of 0.16. For cell separation, the MDA-MB-231 cells are incubated with the targeted biotin-MBs conjugated with anti-CD44 for 10 min, centrifuged at 10g for 1 min, and then allowed 1 hour at 4°C for separation. The results indicate that targeted biotin-MBs conjugated with anti-CD44 antibodies can be used to separate MDA-MB-231 breast cancer cells; more than 90% of the cells were collected in the MB layer when the ratio of the MBs to cells was higher than 70:1. Furthermore, we found that the separating efficiency was higher for targeted biotin-MBs than for targeted avidin-incorporated albumin MBs (avidin-MBs), which is the most common way to make targeted albumin MBs. We also demonstrated that the recovery rate of targeted biotin-MBs was up to 88% and the sorting purity was higher than 84% for a a heterogenous cell population containing MDA-MB-231 cells (CD44+) and MDA-MB-453 cells (CD44–), which are classified as basal-like breast cancer cells and luminal breast cancer cells, respectively. Knowing that the CD44+ is a commonly used cancer-stem-cell biomarker, our targeted biotin-MBs could be a potent tool to sort cancer stem cells from dissected tumor tissue for use in preclinical experiments and clinical trials.