Hollow silica microspheres for buoyancy-assisted separation of infectious pathogens from stool.

Separation of cells and microorganisms from complex biological mixtures is a critical first step in many analytical applications ranging from clinical diagnostics to environmental monitoring for food and waterborne contaminants. Yet, existing techniques for cell separation are plagued by high reagent and/or instrumentation costs that limit their use in many remote or resource-poor settings, such as field clinics or developing countries. We developed an innovative approach to isolate infectious pathogens from biological fluids using buoyant hollow silica microspheres that function as "molecular buoys" for affinity-based target capture and separation by floatation. In this process, antibody functionalized glass microspheres are mixed with a complex biological sample, such as stool. When mixing is stopped, the target-bound, low-density microspheres float to the air/liquid surface, which simultaneously isolates and concentrates the target analytes from the sample matrix. The microspheres are highly tunable in terms of size, density, and surface functionality for targeting diverse analytes with separation times of ≤2min in viscous solutions. We have applied the molecular buoy technique for isolation of a protozoan parasite that causes diarrheal illness, Cryptosporidium, directly from stool with separation efficiencies over 90% and low non-specific binding. This low-cost method for phenotypic cell/pathogen separation from complex mixtures is expected to have widespread use in clinical diagnostics as well as basic research.

Optical systems for point-of-care diagnostic instrumentation: analysis of imaging performance and cost.

One of the key elements in point-of-care (POC) diagnostic test instrumentation is the optical system required for signal detection and/or imaging. Many tests which use fluorescence, absorbance, or colorimetric optical signals are under development for management of infectious diseases in resource limited settings, where the overall size and cost of the device is of critical importance. At present, high-performance lenses are expensive to fabricate and difficult to obtain commercially, presenting barriers for developers of in vitro POC tests or microscopic image-based diagnostics. We recently described a compact "hybrid" objective lens incorporating both glass and plastic optical elements, with a numerical aperture of 1.0 and field-of-view of 250 µm. This design concept may potentially enable mass-production of high-performance, low-cost optical systems which can be easily incorporated in the readout path of existing and emerging POC diagnostic assays. In this paper, we evaluate the biological imaging performance of these lens systems in three broad POC diagnostic application areas; (1) bright field microscopy of histopathology slides, (2) cytologic examination of blood smears, and (3) immunofluorescence imaging. We also break down the fabrication costs and draw comparisons with other miniature optical systems. The hybrid lenses provided images with quality comparable to conventional microscopy, enabling examination of neoplastic pathology and infectious parasites including malaria and cryptosporidium. We describe how these components can be produced at below $10 per unit in full-scale production quantities, making these systems well suited for use within POC diagnostic instrumentation.

A new bio-nanochip sensor aids oral cancer detection.

A diagnostic cytology-on-a-chip technique rapidly detects pre-malignant and malignant cells with high sensitivity and specificity.

Current development of saliva/oral fluid-based diagnostics.

Saliva can be easily obtained in medical and non-medical settings, and contains numerous bio-molecules, including those typically found in serum for disease detection and monitoring. In the past two decades, the achievements of high-throughput approaches afforded by biotechnology and nanotechnology allow for disease-specific salivary biomarker discovery and establishment of rapid, multiplex, and miniaturized analytical assays. These developments have dramatically advanced saliva-based diagnostics. In this review, we discuss the current consensus on development of saliva/oral fluid-based diagnostics and provide a summary of recent research advancements of the Texas-Kentucky Saliva Diagnostics Consortium. In the foreseeable future, current research on saliva based diagnostic methods could revolutionize health care.

Nano-bio-chip sensor platform for examination of oral exfoliative cytology.

Oral cancer is a deadly and disfiguring disease that could greatly benefit from new diagnostic approaches enabling early detection. In this pilot study, we describe a nano-bio-chip (NBC) sensor technique for analysis of oral cancer biomarkers in exfoliative cytology specimens, targeting both biochemical and morphologic changes associated with early oral tumorigenesis. Here, oral lesions from 41 dental patients, along with normal epithelium from 11 healthy volunteers, were sampled using a noninvasive brush biopsy technique. Specimens were enriched, immunolabeled, and imaged in the NBC sensor according to previously established assays for the epidermal growth factor receptor (EGFR) biomarker and cytomorphometry. A total of 51 measurement parameters were extracted using custom image analysis macros, including EGFR labeling intensity, cell and nuclear size, and the nuclear-to-cytoplasmic ratio. Four key parameters were significantly elevated in both dysplastic and malignant lesions relative to healthy oral epithelium, including the nuclear area and diameter (P < 0.0001), the nuclear-to-cytoplasmic ratio (P < 0.0001), and EGFR biomarker expression (P < 0.03). Further examination using logistic regression and receiver operating characteristic curve analyses identified morphologic features as the best predictors of disease (area under the curve < or =0.93) individually, whereas a combination of all features further enhanced discrimination of oral cancer and precancerous conditions (area under the curve, 0.94) with high sensitivity and specificity. Further clinical trials are necessary to validate the regression model and evaluate other potential biomarkers, but this pilot study supports the NBC sensor technique as a promising new diagnostic tool for early detection of oral cancer, which could enhance patient care and survival.

Oligonucleotide-gold nanoparticle networks for detection of Cryptosporidium parvum heat shock protein 70 mRNA.

We report on a novel strategy for the detection of mRNA targets derived from Cryptosporidium parvum oocysts by the use of oligonucleotide-gold nanoparticles. Gold nanoparticles are functionalized with oligonucleotides which are complementary to unique sequences present on the heat shock protein 70 (HSP70) DNA/RNA target. The results indicate that the presence of HPS70 targets of increasing complexity causes the formation of oligonucleotide-gold nanoparticle networks which can be visually monitored via a simple colorimetric readout measured by a total internal reflection imaging setup. Furthermore, the induced expression of HSP70 mRNA in Cryptosporidium parvum oocysts via a simple heat shock process provides nonenzymatic amplification such that the HSP70 mRNA derived from as few as 5 x 10(3) purified C. parvum oocysts was successfully detected. Taken together, these results support the use of oligonucleotide-gold nanoparticles for the molecular diagnosis of cryptosporidiosis, offering new opportunities for the further development of point-of-care diagnostic assays with low-cost, robust reagents and simple colorimetric detection.

Cell-based sensor for analysis of EGFR biomarker expression in oral cancer.

Oral cancer is the sixth most common cancer worldwide and has been marked by high morbidity and poor survival rates that have changed little over the past few decades. Beyond prevention, early detection is the most crucial determinant for successful treatment and survival of cancer. Yet current methodologies for cancer diagnosis based upon pathological examination alone are insufficient for detecting early tumor progression and molecular transformation. To address this clinical need, we have developed a cell-based sensor to detect oral cancer biomarkers, such as the epidermal growth factor receptor (EGFR) whose over-expression is associated with early oral tumorigenesis and aggressive cancer phenotypes. The lab-on-a-chip (LOC) sensor utilizes an embedded track-etched membrane, which functions as a micro-sieve, to capture and enrich cells from complex biological fluids or biopsy suspensions. Once captured, "on-membrane" immunofluorescent assays reveal the presence and isotype of interrogated cells via automated microscopy and fluorescent image analysis. Using the LOC sensor system, with integrated capture and staining technique, EGFR assays were completed in less than 10 minutes with staining intensity, homogeneity, and cellular localization patterns comparable to conventional labeling methods. Further examination of EGFR expression in three oral cancer cell lines revealed a significant increase (p < 0.05) above control cells with EGFR expression similar to normal squamous epithelium. Results obtained in the microfluidic sensor system correlated well with flow cytometry (r(2) = 0.98), the "gold standard" in quantitative protein expression analysis. In addition, the LOC sensor detected significant differences between two of the oral cancer cell lines (p < 0.01), accounting for disparity of approximately 34 000 EGFR per cell according to quantitative flow cytometry. Taken together, these results support the LOC sensor system as a suitable platform for rapid detection of oral cancer biomarkers and characterization of EGFR over-expression in oral malignancies. Application of this technique may be clinically useful in cancer diagnostics for early detection, prognostic evaluation, and therapeutic selection. Having demonstrated the functionality of this integrated microfluidic sensor system, further studies using clinical samples from oral cancer patients are now warranted.

GFAP and nuclear lamins share an epitope recognized by monoclonal antibody J1-31.

Monoclonal antibody J1-31 was raised against plaque materials taken from brains of patients who had suffered from multiple sclerosis (MS). Preliminary characterization of the antigen revealed it to be a protein of M(w) 68-70 kDa with both a cytoplasmic and nuclear localization. Here we report the results of isolation and peptide sequencing of the antigen from human brains, and immunocytochemical analysis of the antigen in F98 glioma cells. Purification and peptide sequencing indicate that the antibody recognizes a form of glial fibrillary acidic protein, possibly a phosphorylated variant. However, confocal immunocytochemistry and western analysis of F98 glioma cells raise the possibility that it also recognizes a phosphorylated epitope found in nuclear lamins. Analysis of the expression of the J1-31 epitope in F98 cells with respect to time in culture, cell density, and DNA synthesis showed a developmental relationship: cells that were engaged in rapid growth and DNA synthesis exhibited strong J1-31 staining in nuclei, whereas quiescent cells did not. We conclude that mAB J1-31 remains a useful antibody for studying multiple sclerosis, and is likely to prove useful in studies of the dynamics of nuclear lamins, particularly in models for wound-healing.

Discrete nuclear structures in actively growing neuroblastoma cells are revealed by antibodies raised against phosphorylated neurofilament proteins.

BACKGROUND: Nuclear objects that have in common the property of being recognized by monoclonal antibodies specific for phosphoprotein epitopes and cytoplasmic intermediate filaments (in particular, SMI-31 and RT-97) have been reported in glial and neuronal cells, in situ and in vitro. Since neurofilament and glial filaments are generally considered to be restricted to the cytoplasm, we were interested in exploring the identity of the structures labeled in the nucleus as well as the conditions under which they could be found there. RESULTS: Using confocal microscopy and western analysis techniques, we determined 1) the immunolabeled structures are truly within the nucleus; 2) the phosphoepitope labeled by SMI-31 and RT-97 is not specific to neurofilaments (NFs) and it can be identified on other intermediate filament proteins (IFs) in other cell types; and 3) there is a close relationship between DNA synthesis and the amount of nuclear staining by these antibodies thought to be specific for cytoplasmic proteins. Searches of protein data bases for putative phosphorylation motifs revealed that lamins, NF-H, and GFAP each contain a single tyrosine phosphorylation motif with nearly identical amino acid sequence. CONCLUSION: We therefore suggest that this sequence may be the epitope recognized by SMI-31 and RT-97 mABs, and that the nuclear structures previously reported and shown here are likely phosphorylated lamin intermediate filaments, while the cytoplasmic labeling revealed by the same mABs indicates phosphorylated NFs in neurons or GFAP in glia.

Functional melatonin receptors and metabolic coupling in cultured chick astrocytes.

Melatonin is an important hormone regulating circadian clocks in birds, but the specific cellular sites of action are not completely known. The present study was designed to determine whether astrocytes derived from chick brain contained functional melatonin receptors. Primary cell cultures of diencephalon astrocytes that express glial fibrillary acidic protein (GFAP), but not neuron-specific enolase (NSE) immunoreactivity, were employed to determine the cellular distribution and physiological role for the three known receptor subtypes. Saturation and Scatchard analysis of 2-[(125)I]iodomelatonin binding demonstrated melatonin receptor binding with a high affinity and a pharmacological profile similar to that obtained from brain. In situ hybridization for receptor subtypes revealed Mel(1A) and Mel(1C) receptor mRNA, but not Mel(1B). Administration of pharmacological levels of melatonin acutely inhibited forskolin-stimulated 2-deoxyglucose (2DG) uptake, while rhythmic administration of physiological levels of melatonin gradually imposed a rhythm in 2DG uptake and of the release of both lactate and pyruvate into the medium. These results indicate that (1) there are functional Mel(1A) and Mel(1C) melatonin receptors in astrocyte-rich cultures, and (2) rhythmic administration of melatonin plays an important role in the regulation of astrocytic metabolic activity. Together, the data suggest that the circadian secretion of melatonin probably plays a role in the global metabolic economy of the avian brain through rhythmic regulation of metabolism in astrocytes.