Laser-fabricated gold nanoparticles for lateral flow immunoassays.

Gold nanoparticles fabricated by pulsed laser ablation are an attractive alternative over those made by chemical reduction, as they offer a more reactive, chemically-bare surface. In this manuscript, we investigate the interactions of these nanoparticles with different classes of antibodies and quantify surface coverage via a fluorescence-based displacement assay. Saturation surface coverage of monoclonal antibodies was found to be almost 2× higher for particles made by pulsed laser ablation (∼28 antibodies/particle) as opposed to those made by chemical means (∼17 antibodies/particle). This higher coverage translated into ∼2× better immunoassay sensitivity, demonstrated by a sandwich lateral flow assay for human chorionic gonadotropin. This work highlights the advantages of these pulsed laser-fabricated materials and showcases their unique properties for colorimetric assay development.

An ultrasensitive universal detector based on neutralizer displacement.

Diagnostic technologies that can provide the simultaneous detection of nucleic acids for gene expression, proteins for host response and small molecules for profiling the human metabolome will have a significant advantage in providing comprehensive patient monitoring. Molecular sensors that report changes in the electrostatics of a sensor's surface on analyte binding have shown unprecedented sensitivity in the detection of charged biomolecules, but do not lend themselves to the detection of small molecules, which do not carry significant charge. Here, we introduce the neutralizer displacement assay that allows charge-based sensing to be applied to any class of molecule irrespective of the analyte charge. The neutralizer displacement assay starts with an aptamer probe bound to a neutralizer. When analyte binding occurs the neutralizer is displaced, which results in a dramatic change in the surface charge for all types of analytes. We have tested the sensitivity, speed and specificity of this system in the detection of a panel of molecules: (deoxy)ribonucleic acid, ribonucleic acid, cocaine, adenosine triphosphate and thrombin.

Nanostructured biomolecular detectors: pushing performance at the nanoscale.

Nanomaterial-based biosensing strategies offer a number of advantages over traditional molecular diagnostic and cellular analysis techniques, including signal amplification, improved sensitivity and speed, and versatile sensing schemes that can be tailored to a desired target. In this article, we highlight a variety of nanomaterial-based sensors, and discuss the advantages of different nanomaterials compositions and probes of different biomolecular classes. Recent advances in the development of optical, electrical, or electrochemical transduction mechanisms are covered, with special regard to breakthroughs in sensitivity. The works reviewed herein emphasize the improvements that nanomaterials offer in the realm of diagnostic assays and make a solid case for further advancement with automation and multiplexing.

Hybridization efficiency of molecular beacons bound to gold nanowires: effect of surface coverage and target length.

Surface-bound nucleic acid probes designed to adopt specific secondary structures are becoming increasingly important in a range of biosensing applications but remain less well characterized than traditional single-stranded probes, which are typically designed to avoid secondary structure. We report the hybridization efficiency for surface-immobilized hairpin DNA probes. Our probes are molecular beacons, carrying a 3' dye moiety and a 5' thiol for attachment to gold nanowires, which serve as both scaffolds for probe attachment and quenchers. Hybridization efficiency was dependent on probe surface coverage, reaching a maximum of ∼90% at intermediate coverages of (1-2) × 10(12) probes/cm(2) and dropping to ≤20% at higher or lower coverages. Fluorescence intensity did not track with the number of target molecules bound, and was highest for high probe coverage despite the lower bound targets per square centimeter. Backfilling with short thiolated oligoethylene glycol spacers increased hybridization efficiency at low hairpin probe coverages (∼(3-4) × 10(11) probes/cm(2)), but not at higher probe coverages (1 × 10(12)/cm(2)). We also evaluated the effect of target length by adding up to 50 nonhybridizing nucleotides to the 3' or 5' end of the complementary target sequence. Additional nucleotides on the 3' end of the complementary target sequence (i.e., the end near the nanowire surface) had a much greater impact on hybridization efficiency as compared to nucleotides added to the 5' end. This work provides guidance in designing sensors in which surface-bound probes designed to adopt secondary structures are used to detect target sequences from solution.

Encoded anisotropic particles for multiplexed bioanalysis.

Encoded anisotropic nano- and microparticles represent an exciting new class of detection and identification strategies for bioanalysis. These particles are synthesized in a number of different ways and can be encoded by shape, composition, topographical features, or optical properties. In this review, we explore synthetic methods for the formation of anisotropic encoded particles and evaluate these systems as multiplexed biosensing platforms. Suspension arrays using anisotropic particles have been used to detect a range of biological species including proteins, nucleic acids, spores, cells, and small molecules. Because in many cases a large number of codes should be obtainable, the potential exists for high levels of multiplexing (thousands or more). The bulk of work in this area to date has focused on initial proof of principle synthesis and identification; however, multiplexed bioassays have been demonstrated for a number of different anisotropic carrier particles and are beginning to be adopted in commercial assays.

Curvature effects in DNA:Au nanoparticle conjugates.

DNA-coated Au nanoparticles have myriad applications as versatile building blocks in nanomaterials assembly, powerful amplification tags for bioanalysis, and promising new approaches to medical therapeutics. Characterization, control, and a thorough understanding of the DNA surface interface are essential in the development of these conjugates. A new paper in this issue explores the impact of nanosphere diameter on DNA adsorption and demonstrates that particle curvature plays an important role in controlling the DNA surface density. The study proposes a model that can be used to predict DNA packing on nonspherical particles and validates it using Au nanorods. This work paves the way for improved understanding of the DNA:Au interface in these versatile bioconjugates.

Molecular beacon-metal nanowire interface: effect of probe sequence and surface coverage on sensor performance.

We report the effect of surface coverage and sequence on the performance of 5' thiolated, 3' fluorophore-labeled DNA hairpin probes bound to Au/Ag striped ("barcoded") metal nanowires. Coverage was controlled by varying probe concentration, buffer ionic strength, and by addition of short hydroxy-terminated alkanethiol diluent molecules during probe assembly onto the nanowire surface. Surface dilution of the surface-bound probes with a omega-hydroxyl alkanethiol, a commonly accepted practice in the surface-bound DNA literature, did not appreciably improve sensor performance as compared to similar probe coverages without hydroxyalkanethiol diluents; this finding underscores the differences between the molecular beacon probes used here and more traditional nonfluorescent, random coil probes. We found that intermediate probe coverage of approximately 10 (12) molecules/cm (2) gave the best discrimination between presence and absence of a target sequence. Because we are interested in multiplexed assays, we also compared several beacon probe sequences having different stabilities for secondary structure formation in solution; we found that both probe surface coverage and sensor performance varied for different probe sequences. When five different molecular beacon probes, each bound to barcoded nanowires, were used in a multiplexed, wash-free assay for target oligonucleotides corresponding to viral nucleic acid sequences, these differences in probe performance did not prevent accurate target identification. We anticipate that the findings described here will also be relevant to other applications involving molecular beacons or other structured nucleic acid probes immobilized on metal surfaces.

Metallic barcodes for multiplexed bioassays.

Both detection and postdiagnosis monitoring are critical for cancer isolation and treatment. Particle-based sensing strategies could help to address these medical needs. This review describes barcoded metallic nanowires as particle scaffolds for multiplexed detection of antigens or nucleic acids. Barcode patterns are compositionally encoded as stripes of gold and silver metal along the nanowire length during fabrication by templated electrodeposition. Particle identification is accomplished using reflectance optical microscopy and can be coupled with fluorescence readout of antigen- or nucleic acid-binding events. Several approaches to multiplexed biodetection based on barcoded nanowires will be described and the potential for these particles in cancer detection will be discussed.

Coupling molecular beacons to barcoded metal nanowires for multiplexed, sealed chamber DNA bioassays.

We have combined molecular beacon (MB) probes with barcoded metal nanowires to enable no-wash, sealed chamber, multiplexed detection of nucleic acids. Probe design and experimental parameters important in nanowire-based MB assays are discussed. Loop regions of 24 bases and 5 base pair stem regions in the beacon probes gave optimal performance. Our results suggest that thermodynamic predictions for secondary structure stability of solution-phase MB can guide probe design for nanowire-based assays. Dengue virus-specific probes with predicted solution-phase DeltaG of folding in 500 mM buffered NaCl of approximately -4 kcal/mol performed better than those with DeltaG > -2 or < -6 kcal/mol. Buffered 300-500 mM NaCl was selected after comparison of several buffers previously reported for similar types of assays, and 200-500 mM NaCl was found to be the optimal ionic strength for the hybridization temperatures (25 and 50 degrees C) and probe designs used here. Target binding to the surface as a function of solution concentration fit a Sips isotherm with Kd = 1.7 +/- 0.3 nM. The detection limit was approximately 100 pM, limited by incomplete quenching. Single base mismatches could be discriminated from fully complementary targets. Oligonucleotide target sequences specific for human immunodeficiency, hepatitis C, and severe acute respiratory viruses were assayed simultaneously in a no-wash, sealed chamber, multiplexed experiment in which each of three probe sequences was attached to a different pattern of encoded nanowires. Finally, we demonstrated that probe-coated nanowires retain their selectivity and sensitivity in a triplexed assay after storage for over 3 months.