Ligand Immobilization Methods for Affinity Chromatography.

There is a plethora of chromatographic supports available in different shapes, sizes, functionalities, and chemical compositions, that could be employed for the numerous applications such as purification, scavenging, and target quantification. Therefore, it becomes critical to understand the chemical nature of these materials to select optimum conditions for ligand immobilization. In this chapter, we have explained some commonly employed ligand immobilization techniques to graft the ligand on the resin surfaces. For a quick reference, we have also included some functionalities of the resins that are commercially available.

Magnetic Nanoparticles with Surface Nanopockets for Highly Selective Antibody Isolation.

Monoclonal antibodies (mAbs) are key components of revolutionary disease immunotherapies and are also essential for medical diagnostics and imaging. The impact of cost is illustrated by a price >$200,000 per year per patient for mAb-based cancer therapy. Purification represents a major issue in the final cost of these immunotherapy drugs. Protein A (PrA) resins are widely used to purify antibodies, but resin cost, separation efficiency, reuse, and stability are major issues. This paper explores a synthesis strategy for low-cost, reusable, stable PrA-like nanopockets on core-shell silica-coated magnetic nanoparticles (NPs) for IgG antibody isolation. Mouse IgG2a, a strong PrA binder, was used as a template protein, first attaching it stem-down onto the NP surface. The stem-down orientation of IgG2a on the NP surface before polymerization is critical for designing the films to bind IgGs. Following this, 1-tetraethoxysilane and four organosilane monomers with functional groups capable of mimicking binding interactions of proteins with IgG antibody stems were reacted to form a thin polymer coating on the NPs. After blocking nonspecific binding sites, removal of the mouse IgG2a provided nanopockets on the core-shell NPs that showed binding characteristics for antibodies remarkably similar to PrA. Both smooth and rough core-shell NPs were used, with the latter providing much larger binding capacities for IgGs, with an excellent selectivity slightly better than that of commercial PrA magnetic beads. This paper is the first report of IgG-binding NPs that mimic PrA selectivity. These nanopocket NPs can be used for at least 15 regeneration cycles, and cost/use was 57-fold less than a high-quality commercial PrA resin.

Kilovoltage cone-beam computed tomography imaging dose estimation and optimization: Need of daily cone-beam computed tomography.

AIM: The aim of the present study was to access the need of daily cone-beam computed tomography (CBCT) and the requirement of in-house protocols of image acquisition frequency to reduce unnecessary exposure to the patients undergoing radiotherapy treatment. MATERIALS AND METHODS: The dose delivered during CBCT procedure (On-Board Imager, Trilogy, Varian medical system, Inc., Palo Alto, California) was assessed for pelvic and head and neck region. For dose estimation, cylindrical polymethyl methacrylate phantoms of 15 cm length, 16 cm, and 32 cm diameter were used to simulate the patient's head and neck and pelvic region thickness, respectively. More than 10 cm scatterer was added on either end of this phantom. Calibrated Ionization chamber DCT10 LEMO SN 1685 iba, dosimetry, Germany (10 cm active length) was used to measure the dose Index. The doses known as cone-beam dose index (CBDI100) were estimated for all the scanning protocols (kV and mAs setting) available on the machine. In this study, image acquisition frequency to correct the setup error was optimized. In-house protocol for image acquisition frequency during treatment has been suggested to reduce the dose. It was based on the principle of as low as reasonable achievable. RESULTS: Optimized dose protocol observed was the "standard dose head" for which the CBDI100 was 2.43 mGy. Whereas for pelvic imaging, single protocol of 125 kV, 80 mA was available by which a dose of 7.61 mGy is likely to be received by the patient during scan. Maximum shift of 6 mm in lateral direction was observed to the patient of Pelvis region and 5 mm was observed in the longitudinal direction for the H and N patients. Angular shift measured in patient position was 3.8° and 3.1° for H and N and pelvic region, respectively. CONCLUSION: Three consecutive-day CBCT-imaging at the beginning of the treatment followed by once weekly CBCT and two-dimensional (2D) imaging in remaining days of treatment can be an optimized way of imaging for the patient having malignancy in the region of pelvic and abdomen. For H and N, once in a week, CBCT with standard dose head protocol, followed by 2D-imaging in remaining days can be an optimized way of imaging.

Novel epoxy-silica nanoparticles to develop non-enzymatic colorimetric probe for analytical immuno/bioassays.

We have developed a novel method to develop epoxy silica nanoparticles (EfSiNP) in a single pot. High surface coverage of epoxy functional groups between 150 and 57000 molecules per particles (∼1013-1016 molecules/mL of 200 nm EfSiNPs) was achieved for different preparation conditions. We then created a red colored probe by conjugating Fuchsin dye to the epoxy functionalities of EfSINPs. Anti-mouse IgG was co-immobilized with Fuchsin and their ratios were optimized for achieving optimum ratios by testing those in functional assays. Dye to antibody ratios were in good negative correlation with a coefficient of -1.00 measured at a confidence level of over 99%. We employed the developed non-enzymatic colorimetric immunonanoprobe for detecting mouse IgG in a direct immunoassay format. We achieved a sensitivity of 427 pg/mL with the assay.

Epitope-Resolved Detection of Peanut-Specific IgE Antibodies by Surface Plasmon Resonance Imaging.

Peanut allergy can be life-threatening and is mediated by allergen-specific immunoglobulin E (IgE) antibodies. Investigation of IgE antibody binding to allergenic epitopes can identify specific interactions underlying the allergic response. Here, we report a surface plasmon resonance imaging (SPRi) immunoassay for differentiating IgE antibodies by epitope-resolved detection. IgE antibodies were first captured by magnetic beads bearing IgE ϵ-chain-specific antibodies and then introduced into an SPRi array immobilized with epitopes from the major peanut allergen glycoprotein Arachis hypogaea h2 (Ara h2). Differential epitope responses were achieved by establishing a binding environment that minimized cross-reactivity while maximizing analytical sensitivity. IgE antibody binding to each Ara h2 epitope was distinguished and quantified from patient serum samples (10 µL each) in a 45 min assay. Excellent correlation of Ara h2-specific IgE values was found between ImmunoCAP assays and the new SPRi method.

Albumin removal from human serum using surface nanopockets on silica-coated magnetic nanoparticles.

Selective removal of albumin from human serum is an essential step prior to proteomic analyses, especially when using mass spectrometry. Here we report stable synthetic nanopockets on magnetic nanoparticle surfaces that bind to human serum albumin (HSA) with high affinity and specificity. The nanopockets are created by templating HSA on 200 nm silica-coated paramagnetic nanoparticles using polymer layers made using 4 organo-silane monomers. These monomers have amino acid-like side chains providing hydrophobic, hydrophilic and H-bonding interactions that closely mimic features of binding sites on antibodies. The binding capacity of the material was 21 mg HSA g-1, and consistently removed ∼88% albumin from human serum in multiple repeated use.

Site-selective orientated immobilization of antibodies and conjugates for immunodiagnostics development.

Immobilized antibody systems are the key to develop efficient diagnostics and separations tools. In the last decade, developments in the field of biomolecular engineering and crosslinker chemistry have greatly influenced the development of this field. With all these new approaches at our disposal, several new immobilization methods have been created to address the main challenges associated with immobilized antibodies. Few of these challenges that we have discussed in this review are mainly associated to the site-specific immobilization, appropriate orientation, and activity retention. We have discussed the effect of antibody immobilization approaches on the parameters on the performance of an immunoassay.

Fast nucleation for silica nanoparticle synthesis using a sol-gel method.

We have developed a method that for the first time allowed us to synthesize silica particles in 20 minutes using a sol-gel preparation. Therefore, it is critically important to understand the synthesis mechanism and kinetic behavior in order to achieve a higher degree of fine tuning ability during the synthesis. In this study, we have employed our ability to modulate the physical nature of the reaction medium from sol-gel to emulsion, which has allowed us to halt the reaction at a particular time; this has allowed us to precisely understand the mechanism and chemistry of the silica polymerization. The synthesis medium is kept quite simple with tetraethyl orthosilicate (TEOS) as a precursor in an equi-volumetric ethanol-water system and with sodium hydroxide as a catalyst. Synthesis is performed under ambient conditions at 20 °C for 20 minutes followed by phasing out of any unreacted TEOS and polysilicic acid chains via their emulsification with supersaturated water. We have also demonstrated that the developed particles with various sizes can be used as seeds for further particle growth and other applications. Luminol, a chemiluminescent molecule, has been entrapped successfully between the layers of silica and was demonstrated for the chemiluminescence of these particles.

Sodium hydroxide catalyzed monodispersed high surface area silica nanoparticles.

Understanding of the synthesis kinetics and our ability to modulate medium conditions allowed us to generate nanoparticles via an ultra-fast process. The synthesis medium is kept quite simple with tetraethyl orthosilicate (TEOS) as precursor and 50% ethanol and sodium hydroxide catalyst. Synthesis is performed under gentle conditions at 20 °C for 20 min Long synthesis time and catalyst-associated drawbacks are most crucial in silica nanoparticle synthesis. We have addressed both these bottlenecks by replacing the conventional Stober catalyst, ammonium hydroxide, with sodium hydroxide. We have reduced the overall synthesis time from 20 to 1/3 h, ~60-fold decrease, and obtained highly monodispersed nanoparticles with 5-fold higher surface area than Stober particles. We have demonstrated that the developed NPs with ~3-fold higher silane can be used as efficient probes for biosensor applications.

Electrochemistry-based approaches to low cost, high sensitivity, automated, multiplexed protein immunoassays for cancer diagnostics.

Early detection and reliable diagnostics are keys to effectively design cancer therapies with better prognoses. The simultaneous detection of panels of biomarker proteins holds great promise as a general tool for reliable cancer diagnostics. A major challenge in designing such a panel is to decide upon a coherent group of biomarkers which have higher specificity for a given type of cancer. The second big challenge is to develop test devices to measure these biomarkers quantitatively with high sensitivity and specificity, such that there are no interferences from the complex serum or tissue matrices. Lastly, integrating all these tests into a technology that does not require exclusive training to operate, and can be used at point-of-care (POC) is another potential bottleneck in futuristic cancer diagnostics. In this article, we review electrochemistry-based tools and technologies developed and/or used in our laboratories to construct low-cost microfluidic protein arrays for the highly sensitive detection of a panel of cancer-specific biomarkers with high specificity which at the same time has the potential to be translated into POC applications.

Sequential glycan profiling at single cell level with the microfluidic lab-in-a-trench platform: a new era in experimental cell biology.

It is now widely recognised that the earliest changes that occur on a cell when it is stressed or becoming diseased are alterations in its surface glycosylation. Current state-of-the-art technologies in glycoanalysis include mass spectrometry, protein microarray formats, techniques in cytometry and more recently, glyco-quantitative polymerase chain reaction (Glyco-qPCR). Techniques for the glycoprofiling of the surfaces of single cells are either limited to the analysis of large cell populations or are unable to handle multiple and/or sequential probing. Here, we report a novel approach of single live cell glycoprofiling enabled by the microfluidic "Lab-in-a-Trench" (LiaT) platform for performing capture and retention of cells, along with shear-free reagent loading and washing. The significant technical improvement on state-of-the-art is the demonstration of consecutive, spatio-temporally profiling of glycans on a single cell by sequential elution of the previous lectin probe using their corresponding free sugar. We have qualitatively analysed glycan density on the surface of individual cells. This has allowed us to qualitatively co-localise the observed glycans. This approach enables exhaustive glycoprofiling and glycan mapping on the surface of individual live cells with multiple lectins. The possibility of sequentially profiling glycans on cells will be a powerful new tool to add to current glycoanalytical techniques. The LiaT platform will enable cell biologists to perform many high sensitivity assays and also will also make a significant impact on biomarker research.

Protein Microarrays with Novel Microfluidic Methods: Current Advances.

Microfluidic-based micromosaic technology has allowed the pattering of recognition elements in restricted micrometer scale areas with high precision. This controlled patterning enabled the development of highly multiplexed arrays multiple analyte detection. This arraying technology was first introduced in the beginning of 2001 and holds tremendous potential to revolutionize microarray development and analyte detection. Later, several microfluidic methods were developed for microarray application. In this review we discuss these novel methods and approaches which leverage the property of microfluidic technologies to significantly improve various physical aspects of microarray technology, such as enhanced imprinting homogeneity, stability of the immobilized biomolecules, decreasing assay times, and reduction of the costs and of the bulky instrumentation.

A chemical quenching- and physical blocking-based method to minimize process-mediated aggregation of antibody-crosslinked nanoparticles for imaging application.

Critical limitation of nanoparticles (NP) is their aggregation after functionalisation and antibody cross-linking. We analysed the cause of this aggregation with respect to functionalities (carboxyls and amines) on the NP surface. We have devised a low cost novel method to reduce such aggregations during protein cross-linking and validated it by probing the platelet surface with platelet surface-specific anti-CD41 antibody conjugated NPs.

Dendrimer driven self-assembly of SPR active silver-gold nanohybrids.

A fourth generation PAMAM dendrimer has been successfully employed for the development of a single step synthesis strategy for self-assembled Ag-Au nanohybrid structures. The surface plasmon resonance properties and the degree of self-assembly of the nanohybrid are strongly correlated with the stoichiometry of the metals which gives rise to enhanced plasmonic properties. The enhanced plasmonic response of the nanohybrids is modeled and is validated experimentally in a model HRP (horseradish peroxidise) bioassay carried out on an SPR-based biochip platform.

Synthesis and characterization of model silica-gold core-shell nanohybrid systems to demonstrate plasmonic enhancement of fluorescence.

In this work, gold-silica plasmonic nanohybrids have been synthesized as model systems which enable tuning of dye fluorescence enhancement/quenching interactions. For each system, a dye-doped silica core is surrounded by a 15 nm spacer region, which in turn is surrounded by gold nanoparticles (GNPs). The GNPs are either covalently conjugated via mercapto silanization to the spacer or encapsulated in a separate external silica shell. The intermediate spacer region can be either dye doped or left undoped to enable quenching and plasmonic enhancement effects respectively. The study indicates that there is a larger enhancement effect when GNPs are encapsulated in the outer shell compared to the system of external conjugation. This is due to the environmental shielding provided by shell encapsulation compared to the exposure of the GNPs to the solvent environment for the externally conjugated system. The fluorescence signal enhancement of the nanohybrid systems was evaluated using a standard HRP-anti-HRP fluorescence based assay platform.

Nanosphere lithography-based platform for developing rapid and high sensitivity microarray systems.

A novel gold nanoarray (NA)-based platform was developed for microarray applications. This novel approach is based upon the principle of nanosphere lithography and can be used for one-step antibody immobilization. The developed platform was checked by functionalizing with cysteine followed by capturing biotinylated antibody and detecting it with dye-conjugated steptravidin. An immunoassay was performed with spiked samples containing human fetuin A antigen. The minimum limits of detection (LOD) of human fetuin A for NA-based and conventional microarray platforms were 50 pg/mL and 50 ng/mL, respectively. The developed approach was highly reproducible and unlike conventional microarray approaches the use of a spotting system was omitted because immobilization was controlled and directed on the predefined arrays. This approach could be an ideal alternative for developing microarrays. And, the ease of the strategy also allows the high throughput production of the microarrays.

Synthesis and characterization of a Noble metal Enhanced Optical Nanohybrid (NEON): a high brightness detection platform based on a dye-doped silica nanoparticle.

A highly bright and photostable, fluorescent nanohybrid particle is presented which consists of gold nanoparticles (GNPs) embedded in dye-doped silica in a core-shell configuration. The dye used is the near-infrared emitting 4,5-benzo-5'-(iodoacetaminomethyl)-1',3,3,3',3'-pentamethyl-1-(4-sulfobutyl) indodicarbo cyanine. The nanohybrid architecture comprises a GNP core which is separated from a layer of dye molecules by a 15 nm buffer layer and has an outer protective, undoped silica shell. Using this architecture, a brightness factor of 550 has been achieved compared to the free dye. This hybrid system, referred to as Noble metal Enhanced Optical Nanohybrid (NEON) in this paper, is the first nanohybrid construct to our knowledge which demonstrates such tunable fluorescence property. NEON has enhanced photostability compared to the free dye and compared to a control particle without GNPs. Furthermore, the NEON particle, when used as a fluorescent label in a model bioassay, shows improved performance over assays using a conventional single dye molecule label.

Nano-structured arrays for multiplex analyses and Lab-on-a-Chip applications.

Nanospheres lithographic (NSL) method has been used to fabricate nano-structured arrays (NAs) of hexagonally close-packed gold (Au) using polystyrene beads [PS, diameter ∼300 nm] as mask. The developed NA was incorporated with a customized and cheap microfluidics system to demonstrate its applicability as an alternative easy and efficient platform for multiplex analysis and Lab-on-a-Chip applications. The chip functionality was demonstrated with horseradish peroxidase (HRP) and anti-HRP antibody as model for recognition system. The enzyme-linked immunosorbent assay (ELISA) performed on fabricated protein biochip had a detection limit 100 pg/mL for HRP. The antibody chip was also checked for the shelf-life and it was found that these chips could be stored for 50 days when stored at 4°C without any significant loss of activity. Therefore, NAs based protein biochip with the correct microfluidics could find huge potential application in diagnostics and biosensing technology.

Multi-layered plasma-polymerized chips for SPR-based detection.

The surface functionalization of a noble metal is crucial in a surface plasmon resonance-based biomolecular detection system because the interfacial coating must retain the activity of immobilized biomolecules while enhancing the optimal loading. We present here a one-step, room-temperature, high-speed, gas-phase plasma polymerization process for functionalizing gold substrates using siloxane as an adhesion layer and acrylic acid as a functional layer. Siloxane- and thiol-based coatings were compared for their performance as adhesion and the interfacial layer for subsequent functionalization. An in situ sequential deposition of siloxane and acrylic acid resulted in a 7-fold increase in carboxylic functionality surfacial content compared to films deposited with thiol-containing precursors. Grading of the layer composition achieved as a consequence of ion-induced mixing on the surface coating under the application of the plasma is confirmed through secondary ion mass spectroscopic studies. DNA hybridization assays were demonstrated on gold/glass substrates using surface plasmon enhanced ellipsometry and the applicability of this coating for protein immunoassays were demonstrated with plasma functionalized gold/plastic substrates in Biacore 3000 SPR instrument.

Label-free optical characterization methods for detecting amine silanization-driven gold nanoparticle self-assembly.

Fluorescence lifetime correlation spectroscopy (FLCS) is presented as a single-step label-free detection method for probing the amine silanization-driven spontaneous 3D self-assembly of freestanding gold nanoparticles (GNPs) in solution. Unlike the conventional methods of studying self-assembly, for example, UV-vis spectroscopy and electron microscopy, FLCS utilizes the intrinsic gold fluorescence. The significance of this approach is to amalgamate the measurement of optical and hydrodynamic size properties simultaneously to achieve a more coherent description of the self-assembly pathway. GNP self-assembly has two-stage kinetics. Electrostatic interaction drives the initial amine silanization, and this is followed by siloxane bond formation between hydrolyzed ethoxy groups of GNP-attached APTES, resulting in the formation of micrometer-sized superstructures. The self-assembly has resulted in a 5-fold increase in the fluorescence lifetime (FL), and the FLCS study has shown an 8- to 10-fold increase in the diffusion coefficient using the pure diffusion model. This result is consistent with the transmission electron microscopy (TEM) observation, which shows a few hundred fold increase in the diameter due to assembly formation by the GNPs.