Liaohai Chen

Bridging nonliving and living matter

Assembling non-biological materials (geomaterials) into a proto-organism constitutes a bridge between nonliving and living matter. In this article we present a simple step-by-step route to assemble a proto-organism. Many pictures have been proposed to describe this transition within the origins-of-life and artificial life communities, and more recently alternative pictures have been emerging from advances in nanoscience and biotechnology. The proposed proto-organism lends itself to both traditions and defines a new picture based on a simple idea: Given a set of required functionalities, minimize the physicochemical structures that support these functionalities, and make sure that all structures self-assemble and mutually enhance each others existence. The result is the first concrete, rational design of a simple physicochemical system that integrates the key functionalities in a thermodynamically favorable manner as a lipid aggregate integrates proto-genes and a proto-metabolism. Under external pumping of free energy, the metabolic processes produce the required building blocks, and only specific gene sequences enhance the metabolic kinetics sufficiently for the whole system to survive. We propose an experimental implementation of the proto-organism with a discussion of our experimental results, together with relevant results produced by other experimental groups, and we specify what is still missing experimentally. Identifying the missing steps is just as important as providing the road map for the transition. We derive the kinetic and thermodynamic conditions of each of the proto-organism subsystems together with relevant theoretical and computational results about these subsystems. We present and discuss detailed 3D simulations of the lipid aggregation processes. From the reaction kinetics we derive analytical aggregate size distributions, and derive key properties of the metabolic efficiency and stability. Thermodynamics and kinetics of the ligation directed self-replication of the proto-genes is discussed, and we summarize the full life cycle of the proto-organism by comparing size, replication time, and energy with the biomass efficiency of contemporary unicells. Finally, we also compare our proto-organism picture with existing origins-of-life and protocell pictures. By assembling one possible bridge between nonliving and living matter we hope to provide a piece in the ancient puzzle about who we are and where we come from.

Hole-Enhanced Raman Scattering

Stokes and anti-Stokes non-resonant hole-enhanced Raman scattering (HERS) spectra with high signal-to-noise ratio (S/N) are reported for the first time for aqueous phase R6G molecules adsorbed onto random nanoholes in thin gold films. Compared to conventional surface-enhanced Raman scattering from nanometric gold colloid particles, HERS exhibits higher strength gain, exceptional reproducibility, simple and reliable substrate preparation, and excellent mechanical stability. By correlating the hole density with Raman scattering gain, we determined optimum HERS gain for 50 nm diameter holes at ∼100 holes/μm2. Providing a Raman substrate with uniform “hot spots”, we expect that HERS will make the quantitative Raman analysis of biological molecules possible.

Proto-Organism Kinetics: Evolutionary Dynamics of Lipid Aggregates with Genes and Metabolism

A synthetic proto-organism could be self-assembled by integrating a lipid proto-container with a proto-metabolic subsystem and a proto-genetic subsystem. This three-component system can use energy and nutrients by means of either redox or photo-chemical reactions, evolve its proto-genome by means of template directed replication, and ultimately die. The evolutionary dynamics of the proto-organism depends crucially on the chemical kinetics of its sub-systems and on their interplay. In this work the template replication kinetics is investigated and it is found that the product inhibition inherent in the ligation-like replication process allows for coexistence of unrelated self-replicating proto-genes in the lipid surface layer. The combined catalytic effects from the proto-genes on the metabolic production rates determine the fate of the strain protocell.

Enhanced Raman scattering from focused surface plasmons

Surface plasmon polaritons launched at concentric arcs can be focused into a subwavelength wide focal spot of high near-field light intensity. The focused plasmons give rise to enhanced Raman scattering from R6G molecules placed in the focal area. By exploiting the polarization dependence of the focusing the authors establish an enhancement of the Raman signal by a factor of ∼6. The results show that focusing of propagating surface plasmons on flat metal surfaces may be an alternative to localized plasmons on metal nanostructures for achieving enhanced Raman scattering. In particular, a flat metal substrate enables better control over the local electric fields and the placement of analyte molecules, and, therefore, ultimately better fidelity of Raman spectra.

Protein delivery with nanoscale precision

A novel assay of protein delivery to a surface with nanoscale precision was established. This was achieved by combining recent advancements in atomic force microscopy (AFM) and bioconjugation. We utilized a heterobifunctional photocleavable cross linker to functionalize an AFM tip with proteins. Upon irradiation, the proteins were released from the tip due to a photolytic reaction of the cross linker. These proteins bound tightly to their binding partners on a substrate. When tip functionalization is carefully controlled, proteins can be locally delivered to a desired area. Importantly, the result of protein delivery can be examined immediately by high-resolution imaging in the same area using the protein-free tip. Successful protein delivery was also confirmed by fluorescence imaging and was proved to be reproducible. The approach allows protein delivery and subsequent imaging to be performed in the same local area with the same AFM tip, thus opening up the possibility of monitoring protein functions in living cells in real time.

Loss of Pluripotency in Human Embryonic Stem Cells Directly Correlates with an Increase in Nuclear Zinc

The pluripotency of human embryonic stem cells (hESCs) is important to investigations of early development and to cell replacement therapy, but the mechanism behind pluripotency is incompletely understood. Zinc has been shown to play a key role in differentiation of non-pluripotent cell types, but here its role in hESCs is directly examined. By mapping the distribution of metals in hESCs at high resolution by x-ray fluorescence microprobe (XFM) and by analyzing subcellular metal content, we have found evidence that loss of pluripotency is directly correlated with an increase in nuclear zinc. Zinc elevation not only redefines our understanding of the mechanisms that support pluripotency, but also may act as a biomarker and an intervention point for stem cell differentiation.

Highly efficient energy and charge transfer in thin self-assembled multilayered polymer films

We report the synthesis and characterization of multilayer self-assembled polymer films made from a water-soluble conjugated polymer, poly(2,5-methoxy-propyloxy sulfonate phenylene vinylene) (MPS-PPV). We observe a red shift of both the absorption and fluorescence spectra with increasing numbers of active MPS-PPV layers. We attribute this red shift to changing polymer conformation and efficient energy transfer. Upon adding a water-soluble C 60 or C 60 -VBA copolymer top layer, the luminescence spectrum is strongly quenched due to charge transfer. The estimated charge transfer quantum efficiency from PL quenching is ∼95%. We discuss in detail the unidirectional energy transfer followed by charge transfer in the self-assembled multilayered films.

Photoinduced electron transfer double fragmentation: An oxygen-mediated radical chain process in the co-fragmentation of substituted pinacol donors with carbon tetrachloride

Abstract Investigations of photoinduced electron transfer processes have led to the observation of several ion radical fragmentation reactions in which strong covalent bonds in the neutral (or starting) molecules rapidly cleave in the one-electron redox products. Donors that undergo rapid bond breaking reactions on one-electron oxidation include 1,2-diarylethanes, pinacols, diamines, and aminoalcohols. One-electron reduction of acceptors, such as ethers, esters and organic halides, can also result in bond cleavage. The efficiency of these reactions is determined by the competition between back electron transfer (k−et), fragmentation (kr) and separation of the ion radical pair (ksep). The quantum yields are generally low since this competition is dominated by back electron transfer (k−et ≈ 109−1011 s−1). One strategy to increase the fragmentation efficiency is to utilize very rapid co-fragmentations of both donor and acceptor thus allowing return electron transfer to be minimized. Cleavage reactions of organic halides are potentially useful in this regard because electrochemical studies and thermochemical calculations suggest that, for certain reduced halides, the electron transfer is dissociative. In this paper, we report the excited state reactivity of several amino-and methoxy-substituted pinacols with the halogenated acceptor carbon tetrachloride. Low to moderate quantum efficiencies (approximately 0.04–0.6) are observed for these reactions when irradiations are carried out under degassed conditions. However, for some pinacols, irradiation in the presence of O2 results in significantly larger quantum yields (approximately 1–10) suggesting a chain mechanism. Data from nuclear magnetic resonance (NMR), electron spin resonance (ESR) and time-resolved absorption spectroscopy suggest that the primary halide radical formed from dissociation is captured by O2 to give peroxyl radical which then propagates the chain reaction.

Metabolic photofragmentation kinetics for a minimal protocell: Rate-limiting factors, efficiency, and implications for evolution

A key requirement of an autonomous self-replicating molecular machine, a protocell, is the ability to digest resources and turn them into building blocks. Thus a protocell needs a set of metabolic processes fueled by external free energy in the form of available chemical redox potential or light. We introduce and investigate a minimal photodriven metabolic system, which is based on photofragmentation of resource molecules catalyzed by genetic molecules. We represent and analyze the full metabolic set of reaction-kinetic equations and, through a set of approximations, simplify the reaction kinetics so that analytical expressions can be obtained for the building block production. The analytical approximations are compared with the full equation set and with corresponding experimental results to the extent they are available. It should be noted, however, that the proposed metabolic system has not been experimentally implemented, so this investigation is conducted to obtain a deeper understanding of its dynamics and perhaps to anticipate its limitations. We demonstrate that this type of minimal photodriven metabolic scheme is typically rate-limited by the front-end photoexcitation process, while its yield is determined by the genetic catalysis. We further predict that gene-catalyzed metabolic reactions can undergo evolutionary selection only for certain combinations of the involved reaction rates due to their intricate interactions. We finally discuss how the expected range of metabolic rates likely affects other key protocellular processes such as container growth and division as well as gene replication.

Target-Specific Copper Hybrid T7 Phage Particles

Target-specific nanoparticles have attracted significant attention recently, and have greatly impacted life and physical sciences as new agents for imaging, diagnosis, and therapy, as well as building blocks for the assembly of novel complex materials. While most of these particles are synthesized by chemical conjugation of an affinity reagent to polymer or inorganic nanoparticles, we are promoting the use of phage particles as a carrier to host organic or inorganic functional components, as well as to display the affinity reagent on the phage surface, taking advantage of the fact that some phages host well-established vectors for protein expression. An affinity reagent can be structured in a desired geometry on the surface of phage particles, and more importantly, the number of the affinity reagent molecules per phage particle can be precisely controlled. We previously have reported the use of the T7 phage capsid as a template for synthesizing target-specific metal nanoparticles. In this study herein, we reported the synthesis of nanoparticles using an intact T7 phage as a scaffold from which to extend 415 copies of a peptide that contains a hexahistidine (6His) motif for capture of copper ions and staging the conversion of copper ions to copper metal, and a cyclic Arginine-Glycine-Aspartic Acid (RGD4C) motif for targeting integrin and cancer cells. We demonstrated that the recombinant phage could load copper ions under low bulk copper concentrations without interfering with its target specificity. Further reduction of copper ions to copper metal rendered a very stable copper hybrid T7 phage, which prevents the detachment of copper from phage particles and maintains the phage structural integrity even under harsh conditions. Cancer cells (MCF-7) can selectively uptake copper hybrid T7 phage particles through ligand-mediated transmembrane transportation, whereas normal control cells (MCF-12F) uptake 1000-fold less. We further demonstrated that copper hybrid T7 phage could be endocytosed by cancer cells in culture.