Michael A. Brook

Design of gold nanoparticle-based colorimetric biosensing assays.

Gold nanoparticle (AuNP)‐based colorimetric biosensing assays have recently attracted considerable attention in diagnostic applications due to their simplicity and versatility. This Minireview summarizes recent advances in this field and attempts to provide general guidance on how to design such assays. The key to the AuNP‐based colorimetric sensing platform is the control of colloidal AuNP dispersion and aggregation stages by using biological processes (or analytes) of interest. The ability to balance interparticle attractive and repulsive forces, which determine whether AuNPs are stabilized or aggregated and, consequently, the color of the solution, is central in the design of such systems. AuNP aggregation in these assays can be induced by an “interparticle‐crosslinking” mechanism in which the enthalpic benefits of interparticle bonding formation overcome interparticle repulsive forces. Alternatively, AuNP aggregation can be guided by the controlled loss of colloidal stability in a “noncrosslinking‐aggregation” mechanism. In this case, as a consequence of changes in surface properties, the van der Waals attractive forces overcome interparticle repulsive forces. Using representative examples we illustrate the general strategies that are commonly used to control AuNP aggregation and dispersion in AuNP‐based colorimetric assays. Understanding the factors that play important roles in such systems will not only provide guidance in designing AuNP‐based colorimetric assays, but also facilitate research that exploits these principles in such areas as nanoassembly, biosciences and colloid and polymer sciences.

Rolling Circle Amplification: Applications in Nanotechnology and Biodetection with Functional Nucleic Acids

Rolling circle amplification (RCA) is an isothermal, enzymatic process mediated by certain DNA polymerases in which long single-stranded (ss) DNA molecules are synthesized on a short circular ssDNA template by using a single DNA primer. A method traditionally used for ultrasensitive DNA detection in areas of genomics and diagnostics, RCA has been used more recently to generate large-scale DNA templates for the creation of periodic nanoassemblies. Various RCA strategies have also been developed for the production of repetitive sequences of DNA aptamers and DNAzymes as detection platforms for small molecules and proteins. In this way, RCA is rapidly becoming a highly versatile DNA amplification tool with wide-ranging applications in genomics, proteomics, diagnosis, biosensing, drug discovery, and nanotechnology.

DNA Aptamer Folding on Gold Nanoparticles: From Colloid Chemistry to Biosensors

We have investigated the effect of the folding of DNA aptamers on the colloidal stability of gold nanoparticles (AuNPs) to which an aptamer is tethered. On the basis of the studies of two different aptamers (adenosine aptamer and K+ aptamer), we discovered a unique colloidal stabilization effect associated with aptamer folding: AuNPs to which folded aptamer structures are attached are more stable toward salt-induced aggregation than those tethered to unfolded aptamers. This colloidal stabilization effect is more significant when a DNA spacer was incorporated between AuNP and the aptamer or when lower aptamer surface graft densities were used. The conformation that aptamers adopt on the surface appears to be a key factor that determines the relative stability of different AuNPs. Dynamic light scattering experiments revealed that the sizes of AuNPs modified with folded aptamers were larger than those of AuNPs modified with unfolded (but largely collapsed) aptamers in salt solution. From both the electrostatic and steric stabilization points of view, the folded aptamers that are more extended from the surface have a higher stabilization effect on AuNP than the unfolded aptamers. On the basis of this unique phenomenon, colorimetric biosensors have been developed for the detection of adenosine, K+, adenosine deaminase, and its inhibitors. Moreover, distinct AuNP aggregation and redispersion stages can be readily operated by controlling aptamer folding and unfolding states with the addition of adenosine and adenosine deaminase.

Simple and Rapid Colorimetric Biosensors Based on DNA Aptamer and Noncrosslinking Gold Nanoparticle Aggregation

Recently, gold nanoparticles (AuNPs) have emerged as novel colorimetric reporters for the detection of various substances including DNA, metal ions, and proteins. The advantages of using AuNPs include: 1) their simplicity, 2) the fact that no complicated and expensive analytical instruments are needed, and 3) the extremely high extinction coefficients ( 1000 times larger than those of organic dyes) and the strongly distance-, shape-, and size-dependent optical properties of AuNPs, which allow AuNP-based colorimetric detection to have comparable sensitivity and selectivity to conventional fluorescent detection. AuNP’s use as a colorimetric reporter relies on its unique surface plasmon resonance (SPR): the dispersed AuNP solution is red whereas the aggregated AuNP solution appears purple (or blue), a phenomenon that can be well explained by the Mie theory. Based on this principle, two general types of colorimetric assays (referred to as type I and type II in this report) have been developed. In type I assays, the color of the AuNP solution changes from red (dispersed particles) to purple (aggregates), in type II assays, the color changes from purple (aggregates) to red (dispersed particles). Mirkin and co-workers pioneered the type I assay in which AuNP was used for the detection of DNA. In their study, AuNPs that were modified with two different oligonucleotides aggregated upon the addition of the complementary DNA target, which acted as a crosslinker to result in a color change from red to purple. Liu and Lu reported a type II assay for the detection of lead ions in which the aggregated AuNPs, crosslinked by cleavable DNA enzymes, were dissociated into dispersed AuNPs in the presence of Pb . More recently, Liu and Lu have extended this concept for the detection of small organic compounds (such as ATP) by using AuNPs crosslinked by DNA aptamers. Aptamers are single-stranded (ss) DNA or RNA molecules created by in vitro selection for binding to a chosen target with high affinity and specificity. In the aptamer-based assay designed by Liu and Lu, oligonucleotide-modified AuNPs were first crosslinked by a DNA aptamer sequence to form aggregates. Upon the addition of a desirable target, the aptamer underwent a structural switch that caused the dissociation of the AuNP aggregates; this was accompanied by the purple-to-red color change. The marriage of AuNP and aptamers in these studies allows the AuNP-based assay to be generic, in principle, for any analyte for which an appropriate aptamer is available. We present here a simple and rapid colorimetric assay that exploits structure-switching DNA aptamers and the phenomenon of salt-induced, noncrosslinking AuNP aggregation. Conceptually, as shown in Figure 1A, a structure-switching DNA

Enzymatic cleavage of nucleic acids on gold nanoparticles: a generic platform for facile colorimetric biosensors.

The enzymatic cleavage of nucleic acids (DNA or DNA with a single RNA linkage) on well-dispersed gold nanoparticles (AuNPs) is exploited in the design of facile colorimetric biosensors. The assays are performed at salt concentrations such that DNA-modified AuNPs are barely stabilized by the electrostatic and steric stabilization. Enzymatic cleavage of DNA chains on the AuNP surface destabilizes the AuNPs, resulting in a rapid aggregation driven by van der Waals attraction, and a red-to-purple color change. Two different systems are chosen, DNase I (a DNA endonuclease) and 8-17 (a Pb(2+)-depedent RNA-cleaving DNAzyme), to demonstrate the utility of our assay for the detection of metal ions and sensing enzyme activities. Compared with previous studies in which AuNP aggregates are converted into dispersed AuNPs by enzymatic cleavage of DNA crosslinkers, the present assay is technically simpler. Moreover, the accessibility of DNA to biomolecular recognition elements (e.g. enzymes) on well-dispersed AuNPs in our assay appears to be higher than that embedded inside aggregates. This biosensing system should be readily adaptable to other enzymes or substrates for detection of analytes such as small molecules, proteases and their inhibitors.

Sugar-modified silanes: precursors for silica monoliths

Sugarsilanes, alkoxysilanes derived from sugars and sugar alcohols including glycerol, sorbitol, maltose and dextran, were hydrolyzed to prepare monolithic, mesoporous silicas. Unlike conventional alkoxysilanes such as tetramethylorthosilicate (TMOS) and tetraethylorthosilicate (TEOS), the sol–gel hydrolysis and cure rates of sugarsilanes were very sensitive to ionic strength, but not to pH: comparable rates of gelation were observed for any specific compound at constant ionic strength over a pH range of about 5.5–11. Reduced levels of shrinkage when compared to TEOS (65% for diglycerylsilane (DGS)-derived silica; 50% for monosorbitylsilane (MSS)-derived silica) were also observed provided that the residual sugars were not washed or pyrolyzed from the silica monolith. Pore sizes in the dried silica monoliths (∼2–3 nm diameter) were marginally increased by the addition of non-functional polyethylene oxide (PEO) (mesopore sizes: no PEO, 3.1 nm; 4 wt% PEO MW 2000, 10000, 3.3 and 3.5 nm, respectively): the protein Human Serum Albumin did not act as a porogen. PEO terminated with Si(OEt)3 groups (TES-PEO), however, was very efficient at increasing mesopore size (TES-PEO MW 200 and 10000, led to pores of average diameter 3.7 and 6.1 nm, respectively). The addition of a multivalent metal such as Mg2+ to the sol increased the pore sizes of glycerol silane-derived silica, but led to decreased sizes in silica prepared from TEOS. These changes in cure chemistry and final properties are attributed to a distortion of the silica cure equilibrium by the multidentate sugar ligands.

Hydrophobization of wood surfaces: covalent grafting of silicone polymers

Abstract The hydrophilicity of Maritime pine wood surfaces was modified by silicone, an extremely hydrophobic material. A generic method for the introduction of a variety of silicones at the surface of pre-treated wood was developed. The initial treatment of wood with maleic anhydride and allyl glycidyl ether resulted in oligoesterified wood bearing terminal alkenes. The hydro- osilylation of these groups, performed with hydride-terminated silicones, led to very hydrophobic surfaces, even after extensive soxhlet extraction with good solvents for silicones. Thze presence of silicon, only at the surface of hydrosilylated wood, was confirmed by ESCA. The silicones appear to be attached to the wood by covalent bonds.

Surface properties of PEO–silicone composites: reducing protein adsorption

Silicone-based polymers with reduced protein adsorption were successfully prepared by incorporating mono- or bifunctional poly(ethylene oxide) (PEO) derivatives, respectively, into PDMS during rubber formation using classic room temperature vulcanization chemistry. Characterization of the films by water contact-angle measurements and XPS showed that the PEO was present on the film surface, with greater amounts of PEO at the interface modified with monofunctional PEO. Scanning electron microscopy showed the PEO domains segregated into regular zigzag patterns on the PEO-modified surfaces. Significant reductions in the adsorption of fibrinogen, albumin and lysozyme were observed on both PEO-modified surfaces, although the monofunctional PEO surfaces performed much better in this regard. The reductions in protein adsorption were comparable for all three proteins on both surfaces, suggesting that molecular mass of the protein is not a significant factor in determining the magnitude of protein deposition. Western blot studies showed that the adsorption of proteins from plasma to the monofunctional PEO-modified surfaces was also significantly reduced and surprisingly selective, with very few bands noted relative to the control surfaces and those modified with bifunctional PEO.

Immobilization of bacteriophages on modified silica particles

Bacteriophages are selective anti-bacterial agents, which are receiving increasing acceptance by regulatory agencies for use both in the food industry and in clinical settings for biocontrol. While immobilized phage could be particularly useful to create antimicrobial surfaces, current immobilization strategies require chemical bioconjugation to surfaces or more difficult processes involving modification of their head proteins to express specific binding moieties, for example, biotin or cellulose binding domains; procedures that are both time and money intensive. We report that morphologically different bacteriophages, active against a variety of food-borne bacteria: Escherichia coli; Salmonella enterica; Listeria monocytogenes; and Shigella boydii, will effectively physisorb to silica particles, prepared by silica surface modification with poly(ethylene glycol), carboxylic acid groups, or amines. The phages remain infective to their host bacteria while adsorbed on the surface of the silica particles. The number of infective phage bound to the silica is enhanced by the presence of ionic surfaces, with greater surface charge - to a maximum - correlating with greater concentration of adsorbed phage. Above the maximum charge concentration, the number of active phage drops.

Simple and rapid colorimetric enzyme sensing assays using non-crosslinking gold nanoparticle aggregation

Non-crosslinking gold nanoparticle (AuNP) aggregation induced by the loss (or screen) of surface charges is applied for enzymatic activity sensing and potentially inhibitor screening.