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ABSTRACT: The prospect of characterizing individual nanoparticles, molecules, or DNA base pairs has generated considerable interest in resistive pulse sensing. In addition to size and concentration analysis, this technique also has the capacity to measure the charge density of objects in situations where electrophoretic forces dominate their motion. Here we present a methodology to simultaneously extract, via appropriate theoretical models, the size and ζ-potential of objects from the resistive pulse signal they generate. The methodology was demonstrated using a size-tunable elastic pore sensor to measure a complex "bimodal" suspension composed of two particle sets with different size and charge. Elastically tuning the size of the pore sensor, by stretching the elastic pore membrane, enables a larger sample size range to be analyzed, improves measurement sensitivity, and fine-tunes the forces acting on objects. This methodology represents a new approach for investigating and understanding the fundamental behavior of nanoscale dispersions.
ACS Nano 07/2012; 6(8):6990-7. · 10.77 Impact Factor
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ABSTRACT: An electrochemical immunoassay which minimizes nonspecific protein adsorption and improves detection sensitivity of proteomic cancer biomarker is described. Our technique comprises two novel features: (i) a high density terminally functionalized poly(N-isopropyl acrylamide) 'brush' layer is grown by surface initiated reversible addition fragmentation chain transfer (RAFT) polymerization method from the electrode surface in order to minimize nonspecific adsorption of serum proteins and other biomolecules, and (ii) a signal amplifying 'bionanoconjugate' comprised of graphene oxide nanosheets decorated with CdSe quantum dots and recombinant single-chain variable fragments towards MSLN, is used to 'physically' amplify the anodic stripping voltammetric signal. This method enabled a detection limit of ca. 1 pg/mL MSLN (RSD=4.6%, n=4) spiked in serum samples. Because of the simple, specific and sensitive nature of this methodology, we feel that it may find potential use in serum-based protein diagnostics.
Biosensors & bioelectronics 05/2012; 38(1):132-7. · 5.43 Impact Factor
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ABSTRACT: An empirically derived model of how the dimensions of an elastic size-tunable pore sensor change with applied membrane stretch is presented. Quantitative modeling of the pore dimensions, in conjugation with a simplified pore resistance model, enabled particle size and translocation velocity profiles to be calculated from the individual particle 'pulse' events, at any membrane stretch. Size analysis of a trimodal suspension, composed of monodisperse 220, 330 and 410 nm particles, gave rise to 3 distinguishable particle peaks with coefficient of variances below 8.2% and average size values within 2.5% of single modal dynamic light scattering measurements. Particle translocation velocity profiles, over the approximate 12 μm pore sensing zone, showed that particles entering the small pore were initially accelerated to velocities approaching 5,000 to 6,000 μm/s. They then rapidly decelerated due to the pore geometry affects on the forces driving particle translocation, being the electric field strength and fluid flow.
The Journal of Physical Chemistry C 04/2012; 116(15):8554-8561. · 4.80 Impact Factor
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ABSTRACT: Since the first reported use of a biological ion channel to detect differences in single stranded genomic base pairs in 1996, a renaissance in nanoscale resistive pulse sensors has ensued. This resurgence of a technique originally outlined and commercialized over fifty years ago has largely been driven by advances in nanoscaled fabrication, and ultimately, the prospect of a rapid and inexpensive means for genomic sequencing as well as other macromolecular characterization. In this pursuit, the potential application of these devices to characterize additional properties such as the size, shape, charge, and concentration of nanoscaled materials (10 - 900 nm) has been largely overlooked. Advances in nanotechnology and biotechnology are driving the need for simple yet sensitive individual object readout devices such as resistive pulse sensors. This review will examine the recent progress in pore-based sensing in the nanoscale range. A detailed analysis of three new types of pore sensors - in-series, parallel, and size-tunable pores - has been included. These pores offer improved measurement sensitivity over a wider particle size range. The fundamental physical chemistry of these techniques, which is still evolving, will be reviewed.
Nano Today 10/2011; 6(5):531-545. · 15.35 Impact Factor
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ABSTRACT: A multiplexed screening methodology for the rapid development of antifouling polymer surfaces is presented. An array of protein resistant polymer layers with high grafting (>100 mg m(-2)) were polymerized on optically encoded particles. Multiplexed analysis showed a 97% reduction in nonspecific protein adsorption for all polymer layers created.
Chemical Communications 09/2011; 47(34):9687-9. · 6.17 Impact Factor
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ABSTRACT: The protein resistance of dextran and dextran-poly(ethylene glycol) (PEG) copolymer films was examined on an organosilica particle-based assay support. Comb-branched dextran-PEG copolymer films were synthesized in a two step process using the organosilica particle as a solid synthetic support. Particles modified with increasing amounts (0.1-1.2 mg m(-2)) of three molecular weights (10,000, 66,900, 400,000 g mol(-1)) of dextran were found to form relatively poor protein-resistant films compared to dextran-PEG copolymers and previously studied PEG films. The efficacy of the antifouling polymer films was found to be dependent on the grafted amount and its composition, with PEG layers being the most efficient, followed by dextran-PEG copolymers, and dextran alone being the least efficient. Immunoglobulin gamma (IgG) adsorption decreased from ∼5 to 0.5 mg m(-2) with increasing amounts of grafted dextran, but bovine serum albumin (BSA) adsorption increased above monolayer coverage (∼2 mg m(-2)) indicating ternary adsorption of the smaller protein within the dextran layer.
Biofouling 05/2011; 27(5):497-503. · 4.43 Impact Factor
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Small 10/2010; 6(23):2653-8. · 8.35 Impact Factor
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ABSTRACT: A 10-fold improvement in the signal-to-noise (S/N) ratio of an optically encoded silica particle-based immunoassay was achieved through incorporating a protein resistant poly(ethylene glycol) (PEG) surface layer and optimizing antibody immobilization conditions. PEG was activated using 2,2,2-trifluoroethanesulfonyl chloride (tresyl) and required a minimum reaction time of 1.5 h. The activated PEG had a reactive half-life of approximately 5 h when stored in acidified dimethyl sulfoxide (DMSO). By increasing the protein incubation time and concentration, a maximum antibody loading on the particle surface of 1.6 x 10(-2) molecules per nm(2) was achieved. The assay S/N ratio was assessed using a multiplexed multicomponent optically encoded species-specific immunoassay. Encoded particles were covalently grafted or nonspecifically coated with either bovine or mouse IgG for the simultaneous detection of complementary anti-IgG "target" or uncomplementary anti-IgG "noise". The versatility and potential as a serum-based assay platform was demonstrated by immobilizing either a polyclonal antibody or an engineered single-chain variable fragment (scFv) capture probe on particles for the detection of the ovarian cancer biomarker, mesothelin (MSLN). The MLSN antigen was spiked into PBS buffer or 50% human serum. Both capture probe orientations, and media conditions showed similar low level detection limits of 5 ng/mL; however, a 40% decrease in maximum signal intensity was observed for assays run in 50% serum.
Langmuir 12/2009; 25(23):13510-5. · 4.19 Impact Factor
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ABSTRACT: The synthesis, characterization, and use of dendron-like poly(ethylene glycol)-lysine (PEG-Lys) copolymers as an intermediate layer for biomolecular diagnostic signal enhancement is presented. Solid phase Fmoc-peptide synthesis was used to synthesize polymers with one, two, and three PEG-Lys comonomer units in both a linear and first and second-generation dendronic structure directly onto organosilica microspheres. The microsphere surface loadings (number of free amine sites) were modified and quantified through an innovative use of the protecting groups of coupled amino acids. Surfaces with 0.1-100% of the original loading corresponding to 0.3-270 nmol/m2 of free amines were achieved. The influence of polymer structure and surface loading (grafting density) on the signal-to-noise of the microsphere-based molecular diagnostic was assessed measuring the difference in the signal of a model protease digestion assay and reduction in the nonspecific adsorption of bovine serum albumin. Increasing the polymer grafting density and the addition of dendronic branching were both found to increase the assay signal and reduce the nonspecific protein adsorption.
Biomacromolecules 02/2009; 10(2):360-5. · 5.48 Impact Factor
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ABSTRACT: The concept of optically encoding particles for solid phase organic synthesis has existed in the literature for several years. However, there remains a significant challenge to producing particles that are capable of withstanding harsh solvents and reagents whilst maintaining the integrity and range of the optical encoding. In this study, a new generation of fluorescently encoded support particles was used for both solid phase peptide synthesis and on-particle analysis of proteolysis in a multiplexed, flow cytometric assay. The success of the assay was demonstrated through the use of a model protease, trypsin. Our results show that the use of solid supports with high peptide yield, high swellability in water and high penetration of the enzyme into the interior of the particle is not absolutely necessary for proteolysis assays.
Molecular BioSystems 08/2008; 4(7):774-8. · 3.53 Impact Factor
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ABSTRACT: In a proof of concept study, we created a small focused fluorescent hexapeptide library onto 14 multiplexed barcoded sets of silica particles to probe the substrate recognition specificity of West Nile and Dengue virus proteases. A flow cytometric analysis demonstrated that the optical signature of each bead population remained distinguishable throughout the solid-phase peptide synthesis and proteolytic assay. As expected, both proteases displayed a narrow specificity for lysine and arginine residues in the P(1) and P(2) substrate positions. This open-ended platform enables the fast and simultaneous identification of peptide substrates and is applicable to other proteases.
Analytical Biochemistry 06/2008; 376(1):151-3. · 3.00 Impact Factor
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ABSTRACT: This study presents the use of flow cytometry as a high-throughput quantifiable technique to study multicomponent adsorption interactions between proteins and surfaces. Flow cytometry offers the advantage of high-throughput analysis of multiple parameters on a very small sampling scale. This enables flow cytometry to distinguish between individual adsorbent particles and adsorbate components within a suspension. As a proof of concept study, the adsorption of three proteins--bovine serum albumin (BSA), bovine immunoglobulin gamma (IgG) and fibrinogen--onto five surface-modified organosilica microsphere surfaces was used as a model multicomponent system for analysis. By uniquely labeling each protein and solid support type with spectrally distinguishable fluorescent dyes, the adsorption process could be "multiplexed" allowing for simultaneous screening of multiple adsorbate (protein) and adsorbent (particle surface) interactions. Protein adsorption experiments quantified by flow cytometry were found to be comparable to single-component adsorption studies by solution depletion. Quantitative distribution of the simultaneous competitive adsorption of BSA and IgG indicated that, at concentrations below surface saturation, both proteins adsorbed onto the surface. However, at concentrations greater than surface saturation, BSA preferentially adsorbed. Multiplexed particle suspensions of optically encoded particles were modified to produce a positively and negatively charged surface, a grafted 3400 MW poly(ethylene glycol) layer, or a physisorbed BSA or IgG layer. It was observed that adsorption was rapid and irreversible on all of the surfaces, and preadsorbed protein layers were the most effective in preventing further protein adsorption.
Langmuir 03/2008; 24(4):1204-11. · 4.19 Impact Factor
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ABSTRACT: The use and advantage of flow cytometry as a particle-by-particle, low sampling volume, high-throughput screening technique for quantitatively examining the non-specific adsorption of proteins onto surfaces is presented. The adsorption of three proteins: bovine serum albumin (BSA), immunoglobulin gamma (IgG) and protein G, incubated at room temperature for 2 h onto organosilica particles modified with poly(ethylene glycol) (PEG) of increasing MW (2000, 3400, 6000, 10,000 and 20,000 g mol(-1)) and grafted amounts (0.14-1.4 mg m(-2)) was investigated as a model system. Each protein exhibited Langmuir-like, high affinity monolayer limited adsorption on unmodified particles with the proteins reaching surface saturation at 1.8, 4.0 and 2.5 mg m(-2) for BSA, IgG and protein G, respectively. Protein adsorption on PEG-modified surfaces was found to decrease with increasing amounts of grafted polymer. PEG grafting amounts >0.6 mg m(-2) effectively prevented the adsorption of the larger two proteins (BSA and IgG) while a PEG grafting amount >1.3 mg m(-2) was required to prevent the adsorption of the smaller protein G.
Biofouling 01/2008; 24(4):267-73. · 4.43 Impact Factor
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ABSTRACT: Organosilane hybrid materials are of interest in the development of diagnostic devices and drug-delivery applications. Here we report a spectroscopic study involving the chemical and structural modification of thiol-functionalised organosilica particles with aminosilane to produce a bifunctional silica hybrid. The aminosilane was revealed to be distributed throughout the microsphere as opposed to being surface-localised as is commonly reported for modifications of pure silica. Spectroscopic methods including NMR, XPS, Ninhydrin and gravimetric measurements were employed to investigate the surface and internal elemental composition of the particles independently. A multiplexed model bioassay is presented to demonstrate the advantage of organosilane bifunctionality, enabling separate covalent attachment strategies for both homogeneous incorporation of fluorescent dyes and surface-specific biomolecule attachment. This study represents an advance in the understanding of organosilane chemistry resulting in versatile materials with a range of functionalities for covalent attachment.
