Optothermal biophysics:
Light driven molecule traps, Microscale Thermophoresis,
Imaging of kinetics and Autonomous evolution
Prof. Dieter Braun
Systems Biophysics, Center for Nanoscience, Ludwig Maximilians University Munich, Germany

Light driven molecule traps. Trapping single ions under vacuum allows for precise spectroscopy inatomic physics. However the confinement of biological molecules in bulk water is hindered by the lack ofcomparably strong forces. Molecules have been immobilized to surfaces, often with detrimental effects
on their function.
Here, we optically trap molecules by creating the microscale analog of a conveyor belt: a bidirectional
flow is combined with a perpendicular thermophoretic molecule drift [1]. Arranged in a toroidal geometry,the conveyor accumulates a hundredfold excess of 5-base DNA within seconds. The concentrations ofthe trapped DNA scale exponentially with length, reaching trapping potential depths of 14kT for 50bases.
The mechanism does not require microfluidics, electrodes or surface modifications. It uses the previously
found light driven microfluidics of thermoviscous expansion [2][3]. As a result, the trap can be
dynamically relocated. The optical conveyor can be used to enhance diffusion-limited surface reactions,redirect cellular signaling, observe individual biomolecules over a prolonged time or approach singlemolecule chemistry in bulk water.

Biomolecule Binding quantified with Thermophoresis. Methods to measure biomolecule binding areessential for medicine, biology and the pharmaceutical industry. We use thermophoresis, the directedmovement of molecules in a temperature gradient to quantify binding in titration experiments. By
combining highly defined microfluidics with all-optical heating and detection, the thermophoretic
depletion is measured with an error of <10^-3 down to nanomolar concentrations.
We fluorescently label one binder either specifically or unspecifically and track the changes ofthermophoretic depletion under the titration of a binding target. The results are quantitative binding
curves over a wide range of binding affinities. Advantages of the method are low volume consumption,
fast response time and surface-free detection. However the measurement in various physiologicalbuffers is the hallmark of the approach. Binding can be for example quantified in untreated serum.
Although thermophoresis is performed in bulk fluid, significant background signals from surface binding
are commonly detected. Competing surface-based methods such as ELISA or surface plasmon
resonance (SPR, e.g. Biacore) have to measure on unreliable surfaces. As result, they cannot measurebinding quantitatively in untreated or undiluted physiological solutions.

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Imaging kinetics with TOOL. Molecules in a cell are subjected to significant crowding from their sistermolecules. While measurements of anomalous diffusion inside cells point towards a marked effect ofmolecular crowding, its impact on the rate of reactions are hard to assess.
We have developed a novel technique to image kinetics in living cells using an optical lock-in
approach[4]. The reaction time constant is resolved in frequency space with optical resolution under amoderate temperature oscillation and sinusoidal illumination.
DNA hybridization kinetics in living cells is strongly length selective: 16 base pair DNA has a seven-foldfaster on-rate as compared to the in vitro situation, whereas 12bp DNA has a five-fold slower on-rate invivo as compared to in vitro. Evidence points towards a catalytic acceleration for longer DNA and aslowing down by DNA binding proteins.
Above result is not expected from molecular crowding. We assessed molecular crowding with Dextranand Ficoll at high concentrations [20% (w/v)] and find no significant changes in the hybridization kinetics,
indicating a minor role of molecular crowding for bi-molecular DNA hybridization.

Replication Trap. The hallmark of living matter is the replication of genetic molecules and their active
storage against diffusion. We have argued in the past that thermal convection can host the million-foldaccumulation even of single nucleotides and at the same time trigger exponential replication [5].
Accumulation is driven by thermophoresis and convection in elongated chambers, replication by theinherent temperature cycling in convection. Only now we could experimentally combine both and show
replication and trapping of the replicated DNA in the same chamber [6]. As we are missing a solid
chemistry of prebiotic replication, we used as a proxy reaction for to replication the polymerase chain
reaction.
Convective flow both drives the DNA replicating polymerase chain reaction (PCR) while concurrent
thermophoresis accumulates the replicated 143 base pair DNA in bulk solution. The time constant for
accumulation is 92 s while DNA is doubled every 50 s. The length of the amplified DNA is checked withthermophoresis. Finite element simulations confirm the findings. The experiments explore conditions inpores of hydrothermal rock which can serve as a model environment for the origin of life and has

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prospects towards the first autonomous evolution, hosting the Darwin process by molecular selection
using the thermophoretic trap. On the other side, the implemented continuous evolution will be able tobreed well specified DNA or RNA molecules in the future.

References[1] An Optical Conveyor for Molecules, Franz M. Weinert and Dieter Braun,
Nano Letters 9, 4264-4267 (2009)[2] Microscale Fluid Flow Induced by Thermoviscous Expansion Along a Traveling Wave,
Weinert, Kraus, Franosch and Braun, Physical Review Letters 100, 164501 (2008)[3] Optically driven fluid flow along arbitrary microscale patterns using thermoviscous expansion,Weinert and Braun, Journal of Applied Physics 104, 104701 (2008)[4] Hybridization Kinetics is Different Inside Cells, Schoen, Krammer and Braun, PNAS, 106: 2164921654 (2009)[5] Extreme Accumulation of Nucleotides in Simulated Hydrothermal Pore Systems, Baaske, Weinert,Duhr, Lemke, Russell, Braun, PNAS 104, 9346–9351 (2007)[6] A Thermal Trap for DNA Replication, Mast and Braun, Physics Review Letters, in press (2010)

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