An implantable Teflon chip holding lithium naphthalocyanine microcrystals for secure, safe, and repeated measurements of pO2 in tissues.

Lithium naphthalocyanine (LiNc) is a crystalline material that has significant potential as a probe for EPR (electron paramagnetic resonance)-based biological oximetry (Pandian et al. J. Mater. Chem. 19:4138-4147, 2009a). However, implantation of LiNc crystals in tissues in raw or neat form is undesirable since dispersion of crystals in tissue may lead to loss of EPR signal, while also exacerbating biocompatibility concerns due to tissue exposure. To overcome these concerns, we have encapsulated LiNc crystals in an oxygen-permeable polymer, Teflon AF 2400 (TAF). Fabrication of TAF films incorporating LiNc particles (denoted as LiNc:TAF chip) was carried out using solvent-evaporation techniques. The EPR linewidth of LiNc:TAF chip was linearly dependent on oxygen-partial pressure (pO(2)) and did not change significantly relative to neat LiNc crystals. LiNc:TAF chip responded to changes in pO(2) reproducibly, enabling dynamic measurements of oxygenation in real time. The LiNc:TAF chips were stable in tissues for more than 2 months and were capable of providing repeated measurements of tissue oxygenation for extended periods of time. The results demonstrated that the newly fabricated, highly oxygen-sensitive LiNc:TAF chip will enhance the applicability of EPR oximetry for long-term and clinical applications.

Polymer coating of paramagnetic particulates for in vivo oxygen-sensing applications.

Crystalline lithium phthalocyanine (LiPc) can be used to sense oxygen. To enhance biocompatibility/stability of LiPc, we encapsulated LiPc in Teflon AF (TAF), cellulose acetate (CA), and polyvinyl acetate (PVAc) (TAF, previously used to encapsulate LiPc, was a comparator). We identified water-miscible solvents that don't dissolve LiPc crystals, but are solvents for the polymers, and encapsulated crystals by solvent evaporation. Oxygen sensitivity of films was characterized in vitro and in vivo. Encapsulation did not change LiPc oximetry properties in vitro at anoxic conditions or varying partial pressures of oxygen (pO2). EPR linewidth of encapsulated particles was linear with pO2, responding to pO2 changes quickly and reproducibly for dynamic measurements. Encapsulated LiPc was unaffected by biological oxidoreductants, stable in vivo for four weeks. Oximetry, stability and biocompatibility properties of LiPc films were comparable, but both CA and PVAc films are cheaper, and easier to fabricate and handle than TAF films, making them superior.

Fabrication and physical evaluation of a polymer-encapsulated paramagnetic probe for biomedical oximetry.

Lithium octa-n-butoxynaphthalocyanine (LiNc-BuO) is a promising probe for biological electron paramagnetic resonance (EPR) oximetry and is being developed for clinical use. However, clinical applicability of LiNc-BuO may be hindered by potential limitations associated with biocompatibility, biodegradation, and migration of individual crystals in tissue. To overcome these limitations, we have encapsulated LiNc-BuO crystals in polydimethyl siloxane (PDMS), an oxygen-permeable and bioinert polymer, to fabricate conveniently implantable and retrievable oxygen-sensing chips. Encapsulation was performed by a simple cast-molding process, giving appreciable control over size, shape, thickness and spin density of chips. The in vitro oxygen response of the chip was linear, reproducible, and not significantly different from that of unencapsulated crystals. Cast-molding of the structurally-flexible PDMS enabled the fabrication of chips with tailored spin densities, and ensured non-exposure of embedded LiNc-BuO, mitigating potential biocompatibility/toxicological concerns. Our results establish PDMS-encapsulated LiNc-BuO as a promising candidate for further biological evaluation and potential clinical application.

Oxygen sensitivity and biocompatibility of an implantable paramagnetic probe for repeated measurements of tissue oxygenation.

The use of oxygen-sensing water-insoluble paramagnetic probes, such as lithium octa-n-butoxynaphthalocyanine (LiNc-BuO), enables repeated measurements of pO(2) from the same location in tissue by electron paramagnetic resonance (EPR) spectroscopy. In order to facilitate direct in vivo application, and hence eventual clinical applicability, of LiNc-BuO, we encapsulated LiNc-BuO microcrystals in polydimethylsiloxane (PDMS), an oxygen-permeable and bioinert polymer, and developed an implantable chip. In vitro evaluation of the chip, performed under conditions of sterilization, high-energy irradiation, and exposure to cultured cells, revealed that it is biostable and biocompatible. Implantation of the chip in the gastrocnemius muscle tissue of mice showed that it is capable of repeated and real-time measurements of tissue oxygenation for an extended period. Functional evaluation using a murine tumor model established the suitability and applicability of the chip for monitoring tumor oxygenation. This study establishes PDMS-encapsulated LiNc-BuO as a promising choice of probe for clinical EPR oximetry.

Tiny medicine: nanomaterial-based biosensors.

Tiny medicine refers to the development of small easy to use devices that can help in the early diagnosis and treatment of disease. Early diagnosis is the key to successfully treating many diseases. Nanomaterial-based biosensors utilize the unique properties of biological and physical nanomaterials to recognize a target molecule and effect transduction of an electronic signal. In general, the advantages of nanomaterial-based biosensors are fast response, small size, high sensitivity, and portability compared to existing large electrodes and sensors. Systems integration is the core technology that enables tiny medicine. Integration of nanomaterials, microfluidics, automatic samplers, and transduction devices on a single chip provides many advantages for point of care devices such as biosensors. Biosensors are also being used as new analytical tools to study medicine. Thus this paper reviews how nanomaterials can be used to build biosensors and how these biosensors can help now and in the future to detect disease and monitor therapies.

Engineering functional protein interfaces for immunologically modified field effect transistor (ImmunoFET) by molecular genetic means.

The attachment and interactions of analyte receptor biomolecules at solid-liquid interfaces are critical to development of hybrid biological-synthetic sensor devices across all size regimes. We use protein engineering approaches to engineer the sensing interface of biochemically modified field effect transistor sensors (BioFET). To date, we have deposited analyte receptor proteins on FET sensing channels by direct adsorption, used self-assembled monolayers to tether receptor proteins to planar FET SiO2 sensing gates and demonstrated interface biochemical function and electrical function of the corresponding sensors. We have also used phage display to identify short peptides that recognize thermally grown SiO2. Our interest in these peptides is as affinity domains that can be inserted as translational fusions into receptor proteins (antibody fragments or other molecules) to drive oriented interaction with FET sensing surfaces. We have also identified single-chain fragment variables (scFvs, antibody fragments) that recognize an analyte of interest as potential sensor receptors. In addition, we have developed a protein engineering technology (scanning circular permutagenesis) that allows us to alter protein topography to manipulate the position of functional domains of the protein relative to the BioFET sensing surface.

Selection and characteristics of peptides that bind thermally grown silicon dioxide films.

Dodecapeptides with affinity for thermally grown silicon dioxide were isolated by phage display. Selectants had high histidine content, though distributions of histidine are distinct from reported silica particle-precipitating peptides. Our peptides will have utility as nanoscale affinity domains when inserted into proteins intended for deposition on thermal oxide surfaces/interfaces in micro/nanodevices.

Direct label-free electrical immunodetection of transplant rejection protein biomarker in physiological buffer using floating gate AlGaN/GaN high electron mobility transistors.

Monokine induced by interferon gamma (MIG/CXCL9) is used as an immune biomarker for early monitoring of transplant or allograft rejection. This paper demonstrates a direct electrical, label-free detection method of recombinant human MIG with anti-MIG IgG molecules in physiologically relevant buffer environment. The sensor platform used is a biologically modified GaN-based high electron mobility transistor (HEMT) device. Biomolecular recognition capability was provided by using high affinity anti-MIG monoclonal antibody to form molecular affinity interface receptors on short N-hydroxysuccinimide-ester functionalized disulphide (DSP) self-assembled monolayers (SAMs) on the gold sensing gate of the HEMT device. A floating gate configuration has been adopted to eliminate the influences of external gate voltage. Preliminary test results with the proposed chemically treated GaN HEMT biosensor show that MIG can be detected for a wide range of concentration varying from 5 ng/mL to 500 ng/mL.

Isolation of scFv fragments specific for monokine induced by interferon-gamma (MIG) using phage display.

Iterative affinity selection procedures were used to isolate a number of single chain Fv (scFv) antibody fragment clones from naïve Tomlinson I+J phage display libraries that specifically recognize and bind a chemokine, monokine induced by interferon-gamma (MIG/CXCL9). MIG is an important transplant rejection/biology chemokine protein. ELISA-based affinity characterization results indicate that selectants preferentially bind to MIG in the presence of key biopanning component materials and closely related chemokine proteins. These novel antibody fragments may find utility as molecular affinity interface receptors in various electrochemical biosensor platforms to provide specific MIG binding capability with potential applications in transplant rejection monitoring, and other biomedical applications where detection of MIG level is important.

A paramagnetic implant containing lithium naphthalocyanine microcrystals for high-resolution biological oximetry.

Lithium naphthalocyanine (LiNc) is a microcrystalline EPR oximetry probe with high sensitivity to oxygen [R.P. Pandian, M. Dolgos, C. Marginean, P.M. Woodward, P.C. Hammel, P.T. Manoharan, P. Kuppusamy, Molecular packing and magnetic properties of lithium naphthalocyanine crystal: hollow channels enabling permeability and paramagnetic sensitivity to molecular oxygen J. Mater. Chem. 19 (2009) 4138-4147]. However, direct implantation of the crystals in the tissue for in vivo oxygen measurements may be hindered by concerns associated with their direct contact with the tissue/cells and loss of EPR signal due to particle migration in the tissue. In order to address these concerns, we have developed encapsulations (chips) of LiNc microcrystals in polydimethyl siloxane (PDMS), an oxygen-permeable, bioinert polymer. Oximetry evaluation of the fabricated chips revealed that the oxygen sensitivity of the crystals was unaffected by encapsulation in PDMS. Chips were stable against sterilization procedures or treatment with common biological oxidoreductants. In vivo oxygen measurements established the ability of the chips to provide reliable and repeated measurements of tissue oxygenation. This study establishes PDMS-encapsulated LiNc as a potential probe for long-term and repeated measurements of tissue oxygenation.

A molecular paramagnetic spin-doped biopolymeric oxygen sensor.

Electron paramagnetic resonance (EPR) oximetry is a powerful technique capable of providing accurate, reliable, and repeated measurements of tissue oxygenation, which is crucial to the diagnosis and treatment of several pathophysiological conditions. Measurement of tissue pO(2) by EPR involves the use of paramagnetic, oxygen-sensitive probes, which can be either soluble (molecular) in nature or insoluble paramagnetic materials. Development of innovative strategies to enhance the biocompatibility and in vivo application of these oxygen-sensing probes is crucial to the growth and clinical applicability of EPR oximetry. Recent research efforts have aimed at encapsulating particulate probes in bioinert polymers for the development of biocompatible EPR probes. In this study, we have developed novel EPR oximetry probes, called perchlorotriphenylmethyl triester (PTM-TE):polydimethyl siloxane (PDMS) chips, by dissolving and incorporating the soluble (molecular) EPR probe, PTM-TE, in an oxygen-permeable polymer matrix, PDMS. We demonstrate that such incorporation (doping) of PTM-TE in PDMS enhanced its oxygen sensitivity several fold. The cast-molding method of fabricating chips enabled them to be made with increasing amounts of PTM-TE (spin density). Characterization of the spin distribution within the PDMS matrix, using EPR micro-imaging, revealed potential inhomogeneties, albeit with no adverse effect on the oxygen-sensing characteristics of PTM-TE:PDMS. The chips were resistant to autoclaving or in vitro oxidoreductant treatment, thus exhibiting excellent in vitro biostability. Our results establish PTM-TE:PDMS as a viable probe for biological oxygen-sensing, and also validate the incorporation of soluble probes in polymer matrices as an innovative approach to the development of novel probes for EPR oximetry.

Microfluidic ELISA: on-chip fluorescence imaging.

Fluorescent reactions of a heterogeneous sandwich enzyme-linked immunoassay (ELISA) in an all-PDMS [poly (dimethylsiloxane)] microfluidic device were detected using a cooled charge coupled device (CCD) camera interfaced with an epifluorescence microscope. The study represents preliminary efforts to integrate biochemical reactions and detection on-chip using the "hybrid" detection approach. In initial experiments, the PDMS chip microsensor was successfully used to quantify a model analyte (sheep IgM) with sensitivity down to 17nM. Thus, we demonstrate here the extension of this hybrid integrated technique to on-chip imaging and quantification of light emission from a biochemical immunoassay in PDMS chip.