Eric Stern

Label-free immunodetection with CMOS-compatible semiconducting nanowires

Semiconducting nanowires have the potential to function as highly sensitive and selective sensors for the label-free detection of low concentrations of pathogenic microorganisms. Successful solution-phase nanowire sensing has been demonstrated for ions, small molecules, proteins, DNA and viruses; however, ‘bottom-up’ nanowires (or similarly configured carbon nanotubes) used for these demonstrations require hybrid fabrication schemes, which result in severe integration issues that have hindered widespread application. Alternative ‘top-down’ fabrication methods of nanowire-like devices produce disappointing performance because of process-induced material and device degradation. Here we report an approach that uses complementary metal oxide semiconductor (CMOS) field effect transistor compatible technology and hence demonstrate the specific label-free detection of below 100 femtomolar concentrations of antibodies as well as real-time monitoring of the cellular immune response. This approach eliminates the need for hybrid methods and enables system-scale integration of these sensors with signal processing and information systems. Additionally, the ability to monitor antibody binding and sense the cellular immune response in real time with readily available technology should facilitate widespread diagnostic applications.

Label-free biomarker detection from whole blood.

Label-free nanosensors can detect disease markers to provide point-of-care diagnosis that is low-cost, rapid, specific and sensitive. However, detecting these biomarkers in physiological fluid samples is difficult because of ionic screening. Here, we overcome this limitation by using distinct components within the sensor to perform purification and detection.1 A microfluidic purification chip captures multiple biomarkers simultaneously from blood samples and releases them, after washing, into purified buffer for sensing by a silicon nanoribbon detector. This two-stage approach isolates the detector from the complex environment of whole blood, and reduces its minimum required sensitivity by effectively pre-concentrating the biomarkers. We show specific and quantitative detection of two model cancer antigens from a 10 uL sample of whole blood in less than 20 minutes.

Combination delivery of TGF-β inhibitor and IL-2 by nanoscale liposomal polymeric gels enhances tumour immunotherapy

The tumour microenvironment thwarts conventional immunotherapy through multiple immunologic mechanisms, such as the secretion of the transforming growth factor-β (TGF-β), which stunts local tumour immune responses. Therefore, high doses of interleukin-2 (IL-2), a conventional cytokine for metastatic melanoma, induces only limited responses. To overcome the immunoinhibitory nature of the tumour microenvironment, we developed nanoscale liposomal polymeric gels (nanolipogels; nLGs) of drug-complexed cyclodextrins and cytokine-encapsulating biodegradable polymers that can deliver small hydrophobic molecular inhibitors and water-soluble protein cytokines in a sustained fashion to the tumour microenvironment. nLGs releasing TGF-β inhibitor and IL-2 significantly delayed tumour growth, increased survival of tumour-bearing mice, and increased the activity of natural killer cells and of intratumoral-activated CD8(+) T-cell infiltration. We demonstrate that the efficacy of nLGs in tumour immunotherapy results from a crucial mechanism involving activation of both innate and adaptive immune responses.

Role of sustained antigen release from nanoparticle vaccines in shaping the T cell memory phenotype.

Particulate vaccines are emerging promising technologies for the creation of tunable prophylactics against a wide variety of conditions. Vesicular and solid biodegradable polymer platforms, exemplified by liposomes and polyesters, respectively, are two of the most ubiquitous platforms in vaccine delivery studies. Here we directly compared the efficacy of each in a long-term immunization study and in protection against a model bacterial antigen. Immunization with poly(lactide-co-glycolide) (PLGA) nanoparticles elicited prolonged antibody titers compared to liposomes and alum. The magnitude of the cellular immune response was also highest in mice vaccinated with PLGA, which also showed a higher frequency of effector-like memory T cell phenotype, leading to an effective clearance of intracellular bacteria. The difference in performance of these two common particulate platforms is shown not to be due to material differences but appears to be connected to the kinetics of antigen delivery. Thus, this study highlights the importance of sustained antigen release mediated by particulate platforms and its role in the long-term appearance of effector memory cellular response.

Semiconducting Nanowire Field-Effect Transistor Biomolecular Sensors

Recent studies have demonstrated the ability of semiconducting nanowire (NW) field-effect transistors (FETs) to serve as highly sensitive label-free sensors for biochemicals, including small molecules, proteins, and nucleic acids. The nanoscale confinement of the channel current in concert with the large-surface area-to-volume ratio enables charged molecules bound to the surface to effectively gate the device. Functionalization of the NW surface with specific receptors therefore enables direct electronic detection of particular molecules of interest. The original work in the field relied on NWs grown by the chemical vapor deposition method, which require hybrid bottom-up fabrication processes for device realization. The lack of reproducibility with these techniques and the associated inability to leverage the central advantage of complementary MOSFETs, namely, very large scale integration, have recently led a number of groups to create NW sensors using only traditional top-down fabrication techniques. In this paper, we focus primarily on these most recent studies and discuss necessary future studies as dictated by experimental and theoretical considerations.

Self-assembly of pH-responsive fluorinated dendrimer-based particulates for drug delivery and noninvasive imaging

Dendrimers are nanoscale macromolecules with well-defined branching chemical structures. Control over the architecture and function of these structures has enabled many advances in materials science and biomedical applications. Though dendrimers are directly synthesized by iteration of simple repetitive steps, generation of the larger, more complex structures required for many biomedical applications by covalent synthetic methods has been challenging. Here we demonstrate a spontaneous self-assembly of poly(amidoamine) dendrimers into complex nanoscopic and microscopic particulates following partial fluorination of the constituent dendrimer subunits. These dense particulates exhibit a stimulus-induced response to low external pH that causes their disassembly over time, enabling controlled release of encapsulated agents. In addition, we show that these assemblies offer a sufficiently high density of fluorine spins to enable detection of their site-specific accumulation in vivo by (19)F magnetic resonance imaging ((19)F MRI). Fluorinated dendrimer-based particulates present new features and capabilities important for a wide variety of emerging biomedical applications.

Electrical characterization of single GaN nanowires

In this paper a statistically significant study of 1096 individual GaN nanowire (NW) devices is presented. We have correlated the effects of changing growth parameters for hot-wall chemically-vapour-deposited (HW-CVD) NW sf abricated via the vapour–liquid–solid mechanism. We first describe an optical lithographic method for creating Ohmic contacts to NW field effect transistors with both top and bottom electrostatic gates to characterize carrier density and mobility. Multiprobe measurements show that carrier modulation occurs in the channel and is not a contact effect. We then show that NW fabrication runs with nominally identical growth parameters yield similar electrical results across sample populations of >50 devices. By systematically altering th eg rowth parameters we were able to decrease the average carrier concentration for these as-grown GaN NWs ∼10-fold, from 2.29 × 10 20 to 2.45 × 10 19 cm −3 ,a nd successfully elucidate the parameters that exert the strongest influence on wire quality. Furthermore, this study shows that nitrogen vacancies, and not oxygen impurities, are the dominant intrinsic dopant in HW-CVD GaN NWs.

Enhanced cellular activation with single walled carbon nanotube bundles presenting antibody stimuli.

Efficient immunotherapy can be accomplished by expanding T cells outside the body using single walled carbon nanotube (SWNT) bundles presenting antibody stimuli. Owing to the large surface area of these bundles, which can reach 1560 m (2)/g, T cell stimulating antibodies such as anti-CD3, can be presented at high local concentrations inducing potent activation of T cells. We show that anti-CD3 adsorbed onto SWNT bundles stimulate cells more effectively than equivalent concentrations of soluble anti-CD3. Stimulation by antibody adsorbed onto SWNT is significantly higher than other high surface area materials (activated carbon, polystyrene, and C60 nanoparticles), suggesting unique properties of SWNT bundles for stimuli presentation. We demonstrate the surface area tunability of these bundles by chemical treatment and its effect on antibody adsorption and subsequent T cell activation. In addition, the T cell response varied with the concentration of SWNT in a concentration dependent manner. Antibody stimuli adsorbed onto SWNT bundles represent a novel paradigm for efficient activation of lymphocytes, useful for basic science applications and clinical immunotherapy.

Nanogel-based delivery of mycophenolic acid ameliorates systemic lupus erythematosus in mice.

The ability to selectively inactivate immune cells with immunosuppressants is a much sought-after modality for the treatment of systemic lupus erythematosus and autoimmunity in general. Here, we designed and tested a novel nanogel drug delivery vehicle for the immunosuppressant mycophenolic acid (MPA). Treatment with MPA-loaded nanogels increased the median survival time (MST) of lupus-prone NZB/W F1 mice by 3 months with prophylactic use (MST was 50 weeks versus 38 weeks without treatment), and by 2 months when administered after the development of severe renal damage (MST after proteinuria onset was 12.5 weeks versus 4 weeks without treatment). Equivalent and greater doses of MPA administered in buffer were not efficacious. Nanogels had enhanced biodistribution to organs and association with immune cells. CD4-targeted nanogels yielded similar therapeutic results compared with nontargeted formulations, with protection from glomerulonephritis and decreases in IFN-γ-positive CD4 T cells. DCs that internalized nanogels helped mediate immunosuppression, as they had reduced production of inflammatory cytokines such as IFN-γ and IL-12. Our results demonstrate efficacy of nanogel-based lupus therapy and implicate a mechanism by which immunosuppression is enhanced, in part, by the targeting of antigen-presenting cells.

Label-free electronic detection of the antigen-specific T-cell immune response.

Detection of antigen-specific T-cells is critical for diagnostic assessment and design of therapeutic strategies for many disease states. Effective monitoring of these cells requires technologies that assess their numbers as well as functional response. Current detection of antigen-specific T-cells involves flow cytometry and functional assays and requires fluorescently labeled, soluble forms of peptide-loaded major histocompatability complexes (MHC). We demonstrate that nanoscale solid-state complementary metal-oxide-semiconductor (CMOS) technology can be employed to allow direct, label-free electronic detection of antigen-specific T-cell responses within seconds after stimulation. Our approach relies on detection of extracellular acidification arising from a small number of T-cells (as few as approximately 200), whose activation is induced by triggering the T-cell antigen receptor. We show that T-cell triggering by a nonspecific anti-CD3 stimulus can be detected within 10 s after exposure to the stimulus. In contrast, antigen-specific T-cell responses are slower with response times greater than 40 s after exposure to peptide/MHC agonists. The speed and sensitivity of this technique has the potential to elucidate new understandings of the kinetics of activation-induced T-cell responses. This combined with its ease of integration into conventional electronics potentially enable rapid clinical testing and high-throughput epitope and drug screening.