Saman Sadeghi

Micro-chemical synthesis of molecular probes on an electronic microfluidic device

We have developed an all-electronic digital microfluidic device for microscale chemical synthesis in organic solvents, operated by electrowetting-on-dielectric (EWOD). As an example of the principles, we demonstrate the multistep synthesis of [18F]FDG, the most common radiotracer for positron emission tomography (PET), with high and reliable radio-fluorination efficiency of [18F]FTAG (88 ± 7%, n = 11) and quantitative hydrolysis to [18F]FDG (> 95%, n = 11). We furthermore show that batches of purified [18F]FDG can successfully be used for PET imaging in mice and that they pass typical quality control requirements for human use (including radiochemical purity, residual solvents, Kryptofix, chemical purity, and pH). We report statistical repeatability of the radiosynthesis rather than best-case results, demonstrating the robustness of the EWOD microfluidic platform. Exhibiting high compatibility with organic solvents and the ability to carry out sophisticated actuation and sensing of reaction droplets, EWOD is a unique platform for performing diverse microscale chemical syntheses in small volumes, including multistep processes with intermediate solvent-exchange steps.

On Chip Droplet Characterization: A Practical, High-Sensitivity Measurement of Droplet Impedance in Digital Microfluidics

We demonstrate a new approach to impedance measurement on digital microfluidics chips for the purpose of simple, sensitive, and accurate volume and liquid composition measurement. Adding only a single series resistor to existing AC droplet actuation circuits, the platform is simple to implement and has negligible effect on actuation voltage. To accurately measure the complex voltage across the resistor (and hence current through the device and droplet), the designed system is based on software-implemented lock-in amplification detection of the voltage drop across the resistor which filters out noise, enabling high-resolution and low-limit signal recovery. We observe picoliter sensitivity with linear correlation of voltage to volume extending to the microliter volumes that can be handled by digital microfluidic devices. Due to the minimal hardware, the system is robust and measurements are highly repeatable. The detection technique provides both phase and magnitude information of the real-time current flowing through the droplet for a full impedance measurement. The sensitivity and resolution of this platform enables it to distinguish between various liquids which, as demonstrated in this paper, could potentially be extended to quantify solute concentrations, liquid mixtures, and presence of analytes.

Accurate dispensing of volatile reagents on demand for chemical reactions in EWOD chips

Digital microfluidic chips provide a new platform for manipulating chemicals for multi-step chemical synthesis or assays at the microscale. The organic solvents and reagents needed for these applications are often volatile, sensitive to contamination, and wetting, i.e. have contact angles of <90° even on the highly hydrophobic surfaces (e.g., Teflon® or Cytop®) typically used on digital microfluidic chips. Furthermore, often the applications dictate that the processes are performed in a gas environment, not allowing the use of a filler liquid (e.g., oil). These properties pose challenges for delivering controlled volumes of liquid to the chip. An automated, simple, accurate and reliable method of delivering reagents from sealed, off-chip reservoirs is presented here. This platform overcomes the issues of evaporative losses of volatile solvents, cross-contamination, and flooding of the chip by combining a syringe pump, a simple on-chip liquid detector and a robust interface design. The impedance-based liquid detection requires only minimal added hardware to provide a feedback signal to ensure accurate volumes of volatile solvents are introduced to the chip, independent of time delays between dispensing operations. On-demand dispensing of multiple droplets of acetonitrile, a frequently used but difficult to handle solvent due to its wetting properties and volatility, was demonstrated and used to synthesize the positron emission tomography (PET) probe [(18)F]FDG reliably.

Scanning Kelvin nanoprobe detection in materials science and biochemical analysis

The Kelvin nanoprobe is an extremely sensitive instrument capable of discerning subtle molecular interactions using vibrating electromagnetic and acoustic fields. It is based on the measurement of a fundamental material property, the work function. Modulation of this substrate parameter is caused by the adsorption or desorption of molecules, oxidation, corrosion, contamination, mechanical stress, illumination, temperature changes, electrostatic charging, surface treatment, attached dipolar structures and/or the immobilization of biomolecules. The present article explains the general principles of the method and offers an indication of the wide range of possible applications, with an emphasis on potential use in the biotechnological arena.

Reusable electrochemical cell for rapid separation of [18F]fluoride from [18O]water for flow-through synthesis of 18F-labeled tracers

A brass-platinum electrochemical micro-flow cell was developed to extract [(18)F]fluoride from an aqueous solution and release it into an organic-based solution, suitable for subsequent radio-synthesis, in a fast and reliable manner. This cell does not suffer electrode erosion and is thus reusable while operating faster by enabling increased voltages. By optimizing temperature, trapping and release potentials, flow rates, and electrode materials, an overall [(18)F]fluoride trapping and release efficiency of 84 ± 5% (n=7) was achieved. X-ray photoelectron spectroscopy (XPS) was used to analyze electrode surfaces of various metal-metal systems and the findings were correlated with the performance of the electrochemical cell. To demonstrate the reactivity of the released [(18)F]fluoride, the cell was coupled to a flow-through reactor and automated synthesis of [(18)F]FDG with a repeatable decay-corrected yield of 56 ± 4% (n=4) was completed in < 15 min. A multi-human dose of 5.92GBq [(18)F]FDG was also demonstrated.

On-demand droplet loading for automated organic chemistry on digital microfluidics

Organic chemistry applications on digital microfluidic devices often involve reagents that are volatile or sensitive and must be introduced to the chip immediately before use. We present a new technique for automated, on-demand loading of ~1 μL droplets from large (~1 mL), sealed, off-chip reservoirs to a digital microfluidic chip in order to address this challenge. Unlike aqueous liquids which generally are non-wetting to the hydrophobic surface and must be actively drawn into the electrowetting-on-dielectric (EWOD) chip by electrode activation, organic liquids tend to be wetting and can spontaneously flood the chip, and hence require a retracting force for controlled liquid delivery. Using a combination of compressed inert gas and gravity to exert driving and retracting forces on the liquid, the simple loading technique enables precise loading of droplets of both wetting and non-wetting liquids in a reliable manner. A key feature from a practical point of view is that all of the wetted parts are inexpensive and potentially disposable, thus avoiding cross-contamination in chemical and biochemical applications. We provide a theoretical treatment of the underlying physics, discuss the effect of geometry and liquid properties on its performance, and show repeatable reagent loading using the technique. Its versatility is demonstrated with the loading of several aqueous and non-aqueous liquids on an EWOD digital microfluidic device.

Work-function measurement by high-resolution scanning Kelvin nanoprobe

Nanoscience promises to transform todays world in the same way that integrated semiconductor devices transformed the world of electronics and computation. In the post-genomic era, the greatest challenge is to make connections between the structures and functions of biomolecules at the nanometre-scale level in order to underpin the understanding of larger scale systems in the fields of human biology and physiology. To achieve this, instruments with new capabilities need to be researched and developed, with particular emphasis on new levels of sensitivity, precision and resolution for biomolecular analysis. This paper describes an instrument able to analyse structures that range from tenths of a nanometre (proteins, DNA) to micron-scale structures (living cells), which can be investigated non-destructively in their normal state and subsequently in chemical- or biochemical-modified conditions. The high-resolution scanning Kelvin nanoprobe (SKN) measures the work-function changes at molecular level, instigated by local charge reconfiguration due to translational motion of mobile charges, dipolar relaxation of bound charges, interfacial polarization and structural and conformational modifications. In addition to detecting surface electrical properties, the instrument offers, in parallel, the surface topographic image, with nanometre resolution. The instrument can also be used to investigate subtle work function/topography variations which occur in, for example, corrosion, contamination, adsorption and desorption of molecules, crystallographic studies, mechanical stress studies, surface photovoltaic studies, material science, biocompatibility studies, microelectronic characterization in semiconductor technology, oxide and thin films, surface processing and treatments, surfaces and interfaces characterization. This paper presents the design and development of the instrument, the basic principles of the method and the challenges involved to achieve nanometric resolution and sub-millivolt sensitivity, for both the topographic imaging of surface micromorphology and surface potential and work-function determination.

[18F]CFA as a clinically translatable probe for PET imaging of deoxycytidine kinase activity

Significance Deoxycytidine kinase (dCK) is required for the activation of multiple nucleoside analog prodrugs used in cancer therapy and is a potential new therapeutic target in hematological malignancies. Here, we identify [18F]Clofarabine; 2-chloro-2′-deoxy-2′-[18F]fluoro-9-β-d-arabinofuranosyl-adenine ([18F]CFA) as a new candidate PET probe for dCK, with superior specificity and biodistribution in humans compared with existing probes. [18F]CFA PET may provide a useful companion biomarker for therapeutic interventions against cancer that include nucleoside analog prodrugs, dCK inhibitors, and immunotherapies. Deoxycytidine kinase (dCK), a rate-limiting enzyme in the cytosolic deoxyribonucleoside (dN) salvage pathway, is an important therapeutic and positron emission tomography (PET) imaging target in cancer. PET probes for dCK have been developed and are effective in mice but have suboptimal specificity and sensitivity in humans. To identify a more suitable probe for clinical dCK PET imaging, we compared the selectivity of two candidate compounds—[18F]Clofarabine; 2-chloro-2′-deoxy-2′-[18F]fluoro-9-β-d-arabinofuranosyl-adenine ([18F]CFA) and 2′-deoxy-2′-[18F]fluoro-9-β-d-arabinofuranosyl-guanine ([18F]F-AraG)—for dCK and deoxyguanosine kinase (dGK), a dCK-related mitochondrial enzyme. We demonstrate that, in the tracer concentration range used for PET imaging, [18F]CFA is primarily a substrate for dCK, with minimal cross-reactivity. In contrast, [18F]F-AraG is a better substrate for dGK than for dCK. [18F]CFA accumulation in leukemia cells correlated with dCK expression and was abrogated by treatment with a dCK inhibitor. Although [18F]CFA uptake was reduced by deoxycytidine (dC) competition, this inhibition required high dC concentrations present in murine, but not human, plasma. Expression of cytidine deaminase, a dC-catabolizing enzyme, in leukemia cells both in cell culture and in mice reduced the competition between dC and [18F]CFA, leading to increased dCK-dependent probe accumulation. First-in-human, to our knowledge, [18F]CFA PET/CT studies showed probe accumulation in tissues with high dCK expression: e.g., hematopoietic bone marrow and secondary lymphoid organs. The selectivity of [18F]CFA for dCK and its favorable biodistribution in humans justify further studies to validate [18F]CFA PET as a new cancer biomarker for treatment stratification and monitoring.

Co-targeting of convergent nucleotide biosynthetic pathways for leukemia eradication

Co-targeting of both de novo and salvage pathways for dCTP biosynthesis shows efficacy in T-ALL and B-ALL.

INDs for PET Molecular Imaging Probes—Approach by an Academic Institution

We have developed an efficient, streamlined, cost-effective approach to obtain Investigational New Drug (IND) approvals from the Food and Drug Administration (FDA) for positron emission tomography (PET) imaging probes (while the FDA uses the terminology PET drugs, we are using “PET imaging probes,” “PET probes,” or “probes” as the descriptive terms). The required application and supporting data for the INDs were collected in a collaborative effort involving appropriate scientific disciplines. This path to INDs was successfully used to translate three [18 F]fluoro-arabinofuranosylcytosine (FAC) analog PET probes to phase 1 clinical trials. In doing this, a mechanism has been established to fulfill the FDA regulatory requirements for translating promising PET imaging probes from preclinical research into human clinical trials in an efficient and cost-effective manner.