James S. Brennan

Microfluidic immunoassays as rapid saliva-based clinical diagnostics

At present, point-of-care (POC) diagnostics typically provide a binary indication of health status (e.g., home pregnancy test strip). Before anticipatory use of diagnostics for assessment of complex diseases becomes widespread, development of sophisticated bioassays capable of quantitatively measuring disease biomarkers is necessary. Successful translation of new bioassays into clinical settings demands the ability to monitor both the onset and progression of disease. Here we report on a clinical POC diagnostic that enables rapid quantitation of an oral disease biomarker in human saliva by using a monolithic disposable cartridge designed to operate in a compact analytical instrument. Our microfluidic method facilitates hands-free saliva analysis by integrating sample pretreatment (filtering, enrichment, mixing) with electrophoretic immunoassays to quickly measure analyte concentrations in minimally pretreated saliva samples. Using 20 μl of saliva, we demonstrate rapid (<10 min) measurement of the collagen-cleaving enzyme matrix metalloproteinase-8 (MMP-8) in saliva from healthy and periodontally diseased subjects. In addition to physiologically measurable indicators of periodontal disease, conventional measurements of salivary MMP-8 were used to validate the microfluidic assays described in this proof-of-principle study. The microchip-based POC diagnostic demonstrated is applicable to rapid, reliable measurement of proteinaceous disease biomarkers in biological fluids.

Integrated microfluidic platform for oral diagnostics.

Abstract:  While many point‐of‐care (POC) diagnostic methods have been developed for blood‐borne analytes, development of saliva‐based POC diagnostics is in its infancy. We have developed a portable microfluidic device for detection of potential biomarkers of periodontal disease in saliva. The device performs rapid microfluidic chip‐based immunoassays (<3–10 min) with low sample volume requirements (10 μL) and appreciable sensitivity (nM–pM). Our microfluidic method facilitates hands‐free saliva analysis by integrating sample pretreatment (filtering, enrichment, mixing) with electrophoretic immunoassays to quickly measure analyte concentrations in minimally pretreated saliva samples. The microfluidic chip has been integrated with miniaturized electronics, optical elements, such as diode lasers, fluid‐handling components, and data acquisition software to develop a portable, self‐contained device. The device and methods are being tested by detecting potential biomarkers in saliva samples from patients diagnosed with periodontal disease. Our microchip‐based analysis can readily be extended to detection of biomarkers of other diseases, both oral and systemic, in saliva and other oral fluids.

Results With the Neutron Scatter Camera

We describe the design, calibration, and measurements made with the neutron scatter camera. Neutron scatter camera design allows for the determination of the direction and energy of incident neutrons by measuring the position, recoil energy, and time-of-flight (TOF) between elastic scatters in two liquid scintillator cells. The detector response and sensitive energy range (0.5-10 MeV) has been determined by detailed calibrations using a 252Cf neutron source over its field of view (FOV). We present results from several recent deployments. In a laboratory study we detected a 252Cf neutron source at a stand off distance of 30 m. A hidden neutron source was detected inside a large ocean tanker. We measured the integral flux density, differential energy distribution and angular distribution of cosmic neutron background in the fission energy range 0.5-10 MeV at Alameda, CA (sea level), Livermore, CA (174 m), Albuquerque, NM (1615 m) and Fenton Hill, NM (2630 m). The neutron backgrounds are relatively low, and non-isotropic. The camera has been ruggedized, deployed to various locations and has performed various measurements successfully. Our results show fast neutron imaging could be a useful tool for the detection of special nuclear material (SNM).

Fully Integrated Microfluidic Platform Enabling Automated Phosphoprofiling of Macrophage Response

The ability to monitor cell signaling events is crucial to the understanding of immune defense against invading pathogens. Conventional analytical techniques such as flow cytometry, microscopy, and Western blot are powerful tools for signaling studies. Nevertheless, each approach is currently stand-alone and limited by multiple time-consuming and labor-intensive steps. In addition, these techniques do not provide correlated signaling information on total intracellular protein abundance and subcellular protein localization. We report on a novel phosphoFlow Chip (pFC) that relies on monolithic microfluidic technology to rapidly conduct signaling studies. The pFC platform integrates cell stimulation and preparation, microscopy, and subsequent flow cytometry. pFC allows host-pathogen phosphoprofiling in 30 min with an order of magnitude reduction in the consumption of reagents. For pFC validation, we monitor the mitogen-activated protein kinases ERK1/2 and p38 in response to Escherichia coli lipopolysaccharide (LPS) stimulation of murine macrophage cells (RAW 264.7). pFC permits ERK1/2 phosphorylation monitoring starting at 5 s after LPS stimulation, with phosphorylation observed at 5 min. In addition, ERK1/2 phosphorylation is correlated with subsequent recruitment into the nucleus, as observed from fluorescence microscopy performed on cells upstream of flow cytometric analysis. The fully integrated cell handling has the added advantage of reduced cell aggregation and cell loss, with no detectable cell activation. The pFC approach is a step toward unified, automated infrastructure for high-throughput systems biology.

Results with the neutron scatter camera

We present results from recent deployments with the neutron scatter camera. We successfully detected and pinpointed a hidden 252Cf neutron source in a large ocean tanker at Alameda, CA. In a lab study we detected a 252Cf neutron source at a stand off distance of about 100 ft. We measured the integral flux, differential flux and angular distribution of cosmic neutron background in the fission energy range 0.5–10MeV at Alameda, CA (sea level), Livermore, CA (570 ft), Albuquerque, NM (5300 ft) and Fenton Hill, NM (8630 ft). The neutron backgrounds are relatively low, uniform and well understood. We recently increased the camera effective area 3x. The camera has been successfully ruggedized, deployed to various locations and has performed various measurements. Our results are encouraging and suggest fast neutron imaging could be a useful tool for the detection of special nuclear material (SNM).

Advances in imaging fission neutrons with a neutron scatter camera

Special nuclear material (SNM) emits high energy radiation during active and passive interrogation. This radiation can be imaged thus allowing visualization of shielded and/or smuggled SNM. Lower backgrounds and higher penetration through hi-Z materials make neutrons the preferred detectable in many scenarios. We have developed a neutron scatter camera that directly images fast fission neutrons from SNM sources while simultaneously measuring energy spectra. We have made many significant advances in the design and implementation of such instruments leading to an over 30 fold improvement in sensitivity. We will present results from our detector including analysis techniques that we have developed for neutron imaging and particle discrimination techniques. We will discuss camera calibration and performance under realistic threat detection scenarios, and future prospects in this field.

Gene activation of SMN by selective disruption of lncRNA-mediated recruitment of PRC2 for the treatment of spinal muscular atrophy

Significance Autosomal recessive mutations or deletions of the gene Survival Motor Neuron 1 (SMN1) cause spinal muscular atrophy, a neurodegenerative disorder. Transcriptional up-regulation of a nearly identical gene, SMN2, can functionally compensate for the loss of SMN1, resulting in increased SMN protein to ameliorate the disease severity. Here we demonstrate that the repressed state of SMN2 is reversible by interrupting the recruitment of a repressive epigenetic complex in disease-relevant cell types. Using chemically modified oligonucleotides to bind at a site of interaction on a long noncoding RNA that recruits the repressive complex, SMN2 is epigenetically altered to create a transcriptionally permissive state. Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by progressive motor neuron loss and caused by mutations in SMN1 (Survival Motor Neuron 1). The disease severity inversely correlates with the copy number of SMN2, a duplicated gene that is nearly identical to SMN1. We have delineated a mechanism of transcriptional regulation in the SMN2 locus. A previously uncharacterized long noncoding RNA (lncRNA), SMN-antisense 1 (SMN-AS1), represses SMN2 expression by recruiting the Polycomb Repressive Complex 2 (PRC2) to its locus. Chemically modified oligonucleotides that disrupt the interaction between SMN-AS1 and PRC2 inhibit the recruitment of PRC2 and increase SMN2 expression in primary neuronal cultures. Our approach comprises a gene-up-regulation technology that leverages interactions between lncRNA and PRC2. Our data provide proof-of-concept that this technology can be used to treat disease caused by epigenetic silencing of specific loci.

Measurement of the Fast Neutron Energy Spectrum of an

We have measured the neutron energy spectrum of an 241Am-Be(α,n) source between 1.5 MeV and 9 MeV using a neutron scatter camera. The apparatus consists of two segmented planes each with 16 liquid scintillator cells (Eljen EJ-309), for a total of 32 elements; the neutron energy spectrum is measured using double elastic scatter events. After unfolding resolution effects using a maximum likelihood technique, the measurement is compared to reference Am-Be spectra. Further, we discuss the ability of the neutron scatter camera to distinguish between an Am-Be source and a spontaneous fission source.

^{241}{\rm Am\!-\!Be}

Because of their penetrating power, energetic neutrons and gamma rays (∼1 MeV) offer the best possibility of detecting highly shielded or distant special nuclear material (SNM). Of these, fast neutrons offer the greatest advantage due to their very low and well understood natural background. We are investigating a new approach to fast-neutron imaging — a coded aperture neutron imaging system (CANIS). Coded aperture neutron imaging should offer a highly efficient solution for improved detection speed, range, and sensitivity. We have demonstrated fast neutron and gamma ray imaging with several different configurations of coded masks patterns and detectors including an “active” mask that is composed of neutron detectors. Here we describe our prototype detector and present some initial results from laboratory tests and demonstrations.

Source Using a Neutron Scatter Camera

Because of their penetrating power, energetic neutrons and gamma rays (>~1 MeV) offer the best possibility of detecting highly shielded or distant special nuclear material (SNM). Of these, fast neutrons offer the greatest advantage due to their very low and well understood natural background. We are investigating a wholly new approach to fast-neutron imaging-an active coded-aperture system that uses a coded mask composed of neutron detectors. The only previously demonstrated method for long-range fast neutron imaging is double-scatter imaging. Active coded-aperture neutron imaging should offer a highly efficient alternative for improved detection speed, range, and sensitivity. We will describe our detector including design considerations and present initial results from a lab prototype.