Clifton Kwang-Fu Shen

Highly Efficient Capture of Circulating Tumor Cells by Using Nanostructured Silicon Substrates with Integrated Chaotic Micromixers

Metastases are the most common cause of cancer-related death in patients with solid tumors.[1–4] A considerable body of evidence indicates that tumor cells are shed from a primary tumor mass at the earliest stages of malignant progression[5–7]. These ‘break-away’ circulating tumor cells (CTCs)[8–11] enter the blood stream and travel to different tissues of the body, as a critical route for cancer metastasis. The current gold standard for determining tumor status requires invasive biopsy and subsequent genetic and proteomic analysis of biopsy samples. Alternatively, CTC measurement and analysis can be regarded as a “liquid biopsy” of the tumor, providing insight into tumor biology in the critical window where intervention could actually make a difference. However, detection and characterization of CTCs has been technically challenging due to their extremely low number in the bloodstream. CTCs are often found in the blood of patients with metastatic cancer (only up to hundreds of cells/mL) whereas common blood cells exist in high numbers (>109 cells/mL). Over the past decade, a diverse suite of technologies[8, 12–17] have been evolving to meet the challenge of counting and isolating CTCs from patient blood samples. Many employ different enrichment mechanisms such as immunomagnetic separation based on capture agent-labeled magnetic beads,[8, 16] microfluidics-based technologies[12, 14, 17] that enhance cell-surface contacts, and microfilter devices[13] that isolate CTCs based on size difference. The sensitivity of these emerging technologies, which is critical to their clinical utility for detecting early cancer progression (e.g., tumor invasion of vascular systems), relies on the degree of enrichment of CTCs.

A rapid pathway toward a superb gene delivery system: programming structural and functional diversity into a supramolecular nanoparticle library.

Nanoparticles are regarded as promising transfection reagents for effective and safe delivery of nucleic acids into a specific type of cells or tissues providing an alternative manipulation/therapy strategy to viral gene delivery. However, the current process of searching novel delivery materials is limited due to conventional low-throughput and time-consuming multistep synthetic approaches. Additionally, conventional approaches are frequently accompanied with unpredictability and continual optimization refinements, impeding flexible generation of material diversity creating a major obstacle to achieving high transfection performance. Here we have demonstrated a rapid developmental pathway toward highly efficient gene delivery systems by leveraging the powers of a supramolecular synthetic approach and a custom-designed digital microreactor. Using the digital microreactor, broad structural/functional diversity can be programmed into a library of DNA-encapsulated supramolecular nanoparticles (DNA⊂SNPs) by systematically altering the mixing ratios of molecular building blocks and a DNA plasmid. In vitro transfection studies with DNA⊂SNPs library identified the DNA⊂SNPs with the highest gene transfection efficiency, which can be attributed to cooperative effects of structures and surface chemistry of DNA⊂SNPs. We envision such a rapid developmental pathway can be adopted for generating nanoparticle-based vectors for delivery of a variety of loads.

An integrated microfluidic device for large-scale in situ click chemistry screening

An integrated microfluidic device has been developed to perform 1024 in situ click chemistry reactions in parallel using the bovine carbonic anhydrous II (bCAII) click chemistry system as a proof-of-concept study and a rapid hit identification approach using SPE purification and electrospray-ionization mass spectrometry, multiple reaction monitoring (MRM) analysis, all of which improves the sensitivity and throughput of the downstream analysis.

Cerenkov radiation imaging as a method for quantitative measurements of beta particles in a microfluidic chip

This work proposes a novel method for quantitative imaging of radioactivity on microfluidic chips by using visible light emission from Čerenkov radiation. Čerenkov radiation is generated when charged particles travel through an optically transparent material with a velocity greater than that of light in that material. It has been observed at UCLA that microfluidic chips used for 18F-related radio-synthesis studies have shown unidentified visible light emissions. In this study, the origin of the light was investigated and its feasibility as a quantitative imaging source was tested.

The Therapeutic Efficacy of Camptothecin-Encapsulated Supramolecular Nanoparticles

Nanomaterials have been increasingly employed as drug(s)-incorporated vectors for drug delivery due to their potential of maximizing therapeutic efficacy while minimizing systemic side effects. However, there have been two main challenges for these vectors: (i) the existing synthetic approaches are cumbersome and incapable of achieving precise control of their structural properties, which will affect their biodistribution and therapeutic efficacies, and (ii) lack of an early checkpoint to quickly predict which drug(s)-incorporated vectors exhibit optimal therapeutic outcomes. In this work, we utilized a new rational developmental approach to rapidly screen nanoparticle (NP)-based cancer therapeutic agents containing a built-in companion diagnostic utility for optimal therapeutic efficacy. The approach leverages the advantages of a self-assembly synthetic method for preparation of two different sizes of drug-incorporated supramolecular nanoparticles (SNPs), and a positron emission tomography (PET) imaging-based biodistribution study to quickly evaluate the accumulation of SNPs at a tumor site in vivo and select the favorable SNPs for in vivo therapeutic study. Finally, the enhanced in vivo anti-tumor efficacy of the selected SNPs was validated by tumor reduction/inhibition studies. We foresee our rational developmental approach providing a general strategy in the search of optimal therapeutic agents among the diversity of NP-based therapeutic agents.

Microfluidic-based 18F-labeling of biomolecules for immuno-positron emission tomography.

Methods for tagging biomolecules with fluorine 18 as immuno–positron emission tomography (immunoPET) tracers require tedious optimization of radiolabeling conditions and can consume large amounts of scarce biomolecules. We describe an improved method using a digital microfluidic droplet generation (DMDG) chip, which provides computer-controlled metering and mixing of 18F tag, biomolecule, and buffer in defined ratios, allowing rapid scouting of reaction conditions in nanoliter volumes. The identified optimized conditions were then translated to bench-scale 18F labeling of a cancer-specific engineered antibody fragments, enabling microPET imaging of tumors in xenografted mice at 0.5 to 4 hours postinjection.

A Hydrodynamically Focused Stream as a Dynamic Template for Site-Specific Electrochemical Micropatterning of Conducting Polymers

Micropatterning technology[1–3] has been a major driving force behind the development of organic microelectronic devices. There have been significant efforts devoted to exploring this technology for the fabrication of conducting polymer (CP)-based devices in particular, because CPs[4–6] exhibit the unique advantages of tunable conductance, chemical specificity, flexible modification, and low fabrication cost. In general, most of the existing micropatterning approaches,[1–3] for example, the embossing method,[7] imprint lithography,[8] capillary molding,[9, 10] and microcontact printing,[11, 12] require the use of prefabricated solid molds or templates to determine the features and dimensions of the micropatterns. These molds and templates are normally fabricated by lithographic means, so their embedded features are very much fixed and it is unlikely that they would be reprogrammed for different micropattern features.

Functional antibody arrays through metal ion-affinity templates

In the post-genomic era, surface-based proteomics tools in high-throughput formats are becoming crucial for analyzing protein expression, protein–protein interactions, signal-transduction pathways, and the processes underlying cellular functions. Protein microand nanoarrays hold great promise in areas of health-related research, drug discovery, and diagnostics in which well-defined features and their spacing are important for studying surface–cellular interactions and detecting biomacromolecules. Thus far, a variety of techniques have been developed for immobilizing proteins, specifically antibodies, on surfaces. These techniques have relied primarily on antibody-binding proteins (proteins A, G, A/G, and L), 7] geneticengineering technologies to produce unnatural binding tags for directed surface attachment, electrostatically driven adsorption, covalent linking, or a combination thereof. Although these approaches have been widely used, they have drawbacks ranging from cost and complexity to inactivation of the antibody structures due to denaturation, which leads to poor antigen binding. Here, we present a novel method that utilizes metal ions to immobilize unmodified antibodies in a way that preserves their biorecognition properties in the context of microand nanoarrays. This approach is related to ones used for immobilizing antibodies with metal ions (i.e. Zn, Cu, Ni, Co) on three-dimensional chromatographic supports. Researchers have utilized direct-write techniques, such as robotic spotting and dip-pen nanolithography (DPN), in combination with metal ions as linking groups to generate oligonucleotide and virus particle micro-/nanoarrays. Metal ions as surface linking groups have the advantages of being readily accessible, robust, economical, and stable over long periods of time. Moreover, they are not susceptible to denaturation, as are many of the proteins used for antibody immobilization (i.e. proteins A, G, A/G, and L, etc.). In this report, we demonstrate how the Zn ion can be used as a versatile linker to immobilize antibodies on surfaces in an active state. This includes the assembly of a large class of unmodified polyand monoclonal antibodies (goat IgG, rabbit IgG, mouse IgG1, chicken IgY, and mouse IgM) in the context of microand nanoscale features generated by microcontact printing (m-CP) and DPN, respectively. The activity and utility of metal ion-immobilized antibody arrays are demonstrated with protein and viral antigens. Moreover, we show that one can use nanoarrays as a “litmuslike test” for evaluating the activity of surface-immobilized antibodies. The reduced spot area in a nanoarray demands efficient and relatively uniform immobilization of antibody structures in active states to exhibit uniform activity from spot to spot within the array. With larger features, such as those found in microarrays, inefficient immobilization (i.e. smaller percentage of active antibodies) still leads to apparent uniform activity from feature to feature within the array. Finally, we also demonstrate that we can use this approach to make nanoarrays of antibody structures, such as IgY and IgM, that others have claimed cannot be immobilized in active forms using the protein A/G approach.

Microfluidics for Positron Emission Tomography Probe Development

Owing to increased needs for positron emission tomography (PET), high demands for a wide variety of radiolabeled compounds will have to be met by exploiting novel radiochemistry and engineering technologies to improve the production and development of PET probes. The application of microfluidic reactors to perform radiosyntheses is currently attracting a great deal of interest because of their potential to deliver many advantages over conventional labeling systems. Microfluidics-based radiochemistry can lead to the use of smaller quantities of precursors, accelerated reaction rates, and easier purification processes with greater yield and higher specific activity of desired probes. Several proof-of-principle examples along with the basics of device architecture and operation and the potential limitations of each design are discussed. Along with the concept of radioisotope distribution from centralized cyclotron facilities to individual imaging centers and laboratories (“decentralized model”), an easy-to-use, stand-alone, flexible, fully automated, radiochemical microfluidic platform can provide simpler and more cost-effective procedures for molecular imaging using PET.

Ionic strength and solvent control over the physical structure, electronic properties and superquenching of conjugated polyelectrolytes

In this paper, we investigate the photophysical properties of the conjugated poly electrolyte poly(2-methoxy-5-propyloxy sulfonate phenylene vinylene) (MPS-PPV), dissolved in both water and DMSO as a function of the solution ionic strength. Dynamic light scattering indicates that MPS-PPV chains exist in a highly agglomerated conformation in both solvents, and that the size of the agglomerates depends on both the ionic strength and the charge of the counter-ion. Even though the degree of agglomeration is similar in the two solvents, we find that the fluorescence quantum yield of MPS-PPV in DMSO is nearly 100-times greater than that in water. Moreover, intensity-dependent femtosecond pump-probe experiments show that there is a significant degree of exciton-exciton annihilation in water but not in DMSO, suggesting that the MPS-PPV chromophores interact to form interchain electronic species that quench the emission in water. Given that the emission quenching properties depend sensitively on the chain conformation and degree of chromophore contact, we also explore the superquenching may be either enhanced or diminished in either of the solvents via addition of simple salts, and we present a molecular picture to rationalize how the conformational properties of conjugated polyelectrolytes can be tuned to enhance their emissive behavior for sensing applications.