Nanotechnology-based techniques are being widely evaluated in medical testing and could provide a new generation of diagnostic assays due to their high degrees of sensitivity, high specificity, multiplexing capabilities, and ability to operate without enzymes. In this article, we have modified a nanoparticle-based biobarcode amplification (BCA) assay for early and sensitive detection of HIV-1 capsid (p24) antigen by using antip24 antibody-coated microplates to capture viral antigen (p24) and streptavidin-coated nanoparticle-based biobarcode DNAs for signal amplification, followed by detection using a chip-based scanometric method. The modified BCA assay exhibited a linear dose-dependent pattern within the detection range of 0.1 to 500 pg/ml and was approximately 150-fold more sensitive than conventional enzyme-linked immunosorbent assay (ELISA). No false positive results were observed in 30 HIV-1-negative samples, while all 45 HIV-1 RNA positive samples were found HIV-1 p24 antigen positive by the BCA assay. In addition, the BCA assay detected HIV-1 infection 3 days earlier than ELISA in seroconversion samples. Preliminary evaluation based on testing a small number of samples indicates that the HIV-1 p24 antigen BCA may provide a new tool for sensitive and early detection of HIV-1 p24 antigen in settings where HIV-1 RNA testing is currently not routinely performed.

Staphylococcus aureus (SA) is one of the most important human pathogens, causing both nosocomial and community-acquired infections (1)(2). The occurrence of methicillin-resistant SA (MRSA) has increased steadily worldwide and now accounts for a substantial portion of all staphylococcal infections in US hospitals (3). To develop preventive measures, a rapid screening method, along with accurate and timely identification of MRSA, is essential. The existing techniques for doing so are either time-intensive (culturing of bacteria on selective media), relatively insensitive (use of latex agglutination), or expensive and easily susceptible to operator error (such as PCR). We describe a method designed for clinical laboratories using oligonucleotides conjugated to gold nanoparticles. We avoid radioactivity, fluorescence, or target amplification (such as PCR), and use a simple and rapid hybridization-based approach in a microarray format, with ClearRead™ technology to detect specific genomic sequences (4). The ClearRead procedure involves a two-step process: the first involves the hybridization of target to oligonucleotides conjugated to gold nanoparticles as well as oligonucleotides attached to a solid matrix; the second step involves the catalytic deposition of silver on the gold nanoparticle, providing a sixfold amplification of signal (Fig. 1A⇓ ) (4)(5). The differentiation at isothermal hybridization temperatures is a result of the sharp melting transitions characteristic of nanoparticle probes (5). Previous methods based on this property (6)(7) have required the use of PCR and have not directly assayed for genomic DNA. Our assay, on the other hand, requires minimal amounts of genomic DNA (∼500 ng, or ∼108 DNA molecules) and has been used to reliably identify MRSA from liquid cultures, based on the detection of the mecA and tuf genes. Resistance to methicillin is mediated by the presence of penicillin-binding protein 2a, encoded by the mecA gene (8)(9). We are …

DNA microarrays are powerful tools to detect changes in transcript abundance in multiple samples in parallel. However, detection of differential transcript levels requires a reproducible sample (target) preparation method in addition to a high-performance microarray. Therefore, we optimized a target-preparation method that converts the poly(A)(+) RNA fraction of total RNA into complementary DNA, then generates biotin-labeled complementary RNA from the cDNA. We measured the efficiency of incorporation of biotin-containing nucleotides by an enzymatic digestion, followed by resolution via analytical high-performance liquid chromatography (HPLC). When the target was hybridized to a sensitive and reproducible microarray platform, low coefficients of variation in both hybridization intensities and differential expression ratios across target preparations were observed. Nearly identical hybridization intensities and expression ratios are observed regardless of whether poly(A)(+)-enriched RNA or total RNA is used as the starting material. We show the ability to discern biological and production variability through the use of different lots of commercial samples as visualized by hierarchical clustering. Automation of the target-preparation procedure shows equivalence to the manual procedure, reproducible yields of target, and low variability as measured by hybridization to microarrays. Most importantly, RNA mixing experiments show a linear and quantitative amplification in probe hybridization signals for >6000 genes across the entire signal range.

DNA microarrays enable users to obtain information on differences in transcript abundance on a massively parallel scale. Recently, however, data analyses have revealed potential pitfalls related to image acquisition, variability and misclassifications in replicate measurements, cross-hybridization and sensitivity limitations. We have generated a series of analytical tools to address the manufacturing, detection and data analysis components of a microarray experiment. Together, we have used these tools to optimize performance in an expression profiling study. We demonstrate three significant advantages of the Motorola CodeLink platform: sensitivity of one copy per cell, coefficients of variation of 10% in the hybridization signals across slides and across target preparations, and specificity in distinguishing highly homologous sequences. Slides where oligonucleotide probes are spotted in 6-fold redundancy were used to demonstrate the effect of replication on data quality. Lastly, the differential expression ratios obtained with the CodeLink expression platform were validated against those obtained with quantitative reverse transcription-PCR assays for 54 genes.

Conventional ankyrins are cortical cytoskeletal proteins that form an ankyrin-spectrin meshwork underlying the plasma membrane. We report here the unusual structure of a novel ankyrin (AO13 ankyrin, 775,369 Da, 6994 aa, pI = 4.45) that is required for proper axonal guidance in Caenorhabditis elegans. AO13 ankyrin contains the ANK repeat and spectrin-binding domains found in other ankyrins, but differs from all others in that the acidic carboxyl region contains six blocks of serine/threonine/glutamic acid/proline rich (STEP) repeats separated by seven hydrophobic domains. The STEP repeat blocks are composed primarily of sequences related to ETTTTTTVTREHFEPED(E/D)X(n)VVESEEYSASGSPVPSE (E/K)DVE(H/R)VI, and the hydrophobic domains contain sequences related to PESGEESDGEGFGSKVLGFAKK[AGMVAGGVVAAPVALAAVGA]KAAYDALKKDDDEE, which includes a potential transmembrane domain (in brackets). Recombinant protein fragments of AO13 ankyrin were used to prepare polyclonal antisera against the spectrin-binding domain (AO271 Ab), the conventional ankyrin regulatory domain (AO280 Ab), the AO13 ankyrin STEP domain (AO346 Ab), the AO13 ankyrin STEP + hydrophobic domain (AO289 Ab), and against two carboxyl terminal domain fragments (AO263 Ab and AO327 Ab). Western blot analysis with these Ab probes demonstrated multiple protein isoforms. By immunofluorescence microscopy, the antispectrin-binding and regulatory domain (AO271 and AO280) antibodies recognized many cell types, including neurons, and stained the junctions between cells. The AO13 ankyrin-specific (AO289 and AO346) antibodies showed a neurally restricted pattern, staining nerve processes and the periphery of neural cell bodies. These results are consistent with a role for AO13 ankyrin in neural development.