Henrik Schiøtt Sørensen

Free-Solution, Label-Free Molecular Interactions Studied by Back-Scattering Interferometry

Free-solution, label-free molecular interactions were investigated with back-scattering interferometry in a simple optical train composed of a helium-neon laser, a microfluidic channel, and a position sensor. Molecular binding interactions between proteins, ions and protein, and small molecules and protein, were determined with high dynamic range dissociation constants (Kd spanning six decades) and unmatched sensitivity (picomolar Kds and detection limits of 10,000s of molecules). With this technique, equilibrium dissociation constants were quantified for protein A and immunoglobulin G, interleukin-2 with its monoclonal antibody, and calmodulin with calcium ion Ca2+, a small molecule inhibitor, the protein calcineurin, and the M13 peptide. The high sensitivity of back-scattering interferometry and small volumes of microfluidics allowed the entire calmodulin assay to be performed with 200 picomoles of solute.

Injection molded chips with integrated conducting polymer electrodes for electroporation of cells

We present the design-concept for an all polymer injection molded single use microfluidic device. The fabricated devices comprise integrated conducting polymer electrodes and Luer fitting ports to allow for liquid and electrical access. A case study of low voltage electroporation of biological cells in suspension is presented. The working principle of the electroporation device is based on a focusing of the electric field by means of a constriction in the flow channel for the cells. We demonstrate the use of AC voltage for electroporation by applying a 1 kHz, ±50 V square pulse train to the electrodes and show delivery of polynucleotide fluorescent dye in 46% of human acute monocytic leukemia cells passing the constriction.

Highly sensitive biosensing based on interference from light scattering in capillary tubes

Human IgG interactions with surface bound protein A are monitored label-free using microinterferometric backscatter detection. An electromagnetic wave-based model is developed and used to quantitatively describe the change in interference pattern as a consequence of the molecular interaction with the affinity layer on the fused silica capillary. Within the framework of the model it is of paramount importance to establish a valid stop criterion for the infinite summations involved in the fringe pattern computations. The high sensitivity towards surface changes, ease of changing the surface chemistry to other specific interacting layers, and simplicity of the optical sensor make this technique a powerful tool in biosensing.

Evaluation of back scatter interferometry, a method for detecting protein binding in solution.

Back Scatter Interferometry (BSI) has been proposed to be a highly sensitive and versatile refractive index sensor usable for analytical detection of biomarker and protein interactions in solution. However the existing literature on BSI lacks a physical explanation of why protein interactions in general should contribute to the BSI signal. We have established a BSI system to investigate this subject in further detail. We contribute with a thorough analysis of the robustness of the sensor including unwanted contributions to the interferometric signal caused by temperature variation and dissolved gasses. We report a limit of the effective minimum detectability of refractive index at the 10(-7) level. Long term stability was examined by simultaneously monitoring the temperature inside the capillary revealing an average drift of 2.0 × 10(-7) per hour. Finally we show that measurements on protein A incubated with immunoglobulin G do not result in a signal that can be attributed to binding affinities as otherwise claimed in literature.

Biosensing with backscattering interferometry

Backscattering interferometry (BSI)1 uses light interaction with a microfluidic channel (see Figure 1) to measure temporal changes in refractive index (RI), which can be caused by the channel’s bulk material properties or solutes (dissolved substances). It enables high-resolution investigation of unlabeled proteins, both in solution2 and adsorbed (or immobilized) to a surface.3 Upon coherent-laser illumination of a microfluidic channel, a highly modulated fringe pattern is produced perpendicular to the channel. Its bright and dark features shift position with changes in the RI of the sampled liquid, and monitoring this shift forms the basis of BSI. Using BSI in fused-silica capillary tubes enables detection4 of RI changes of order 10−9 (see Figure 2). Modeling the capillarytube optical train was found useful for performing absolute RI measurements with high accuracy.5, 6 One major step forward in BSI development was the transition from capillary tubes to microfluidic networks. The microfluidic channels can be fabricated in a number of ways including standard SU-8 (a viscous polymer) photoresist procedures. By casting and curing polydimethylsiloxane (PDMS), these microstructures can be replicated. In the PDMS flow channel, immobilization prepares the chip for biochemical-interaction experiments. Photobiotin forms a covalent bond to the oxidized PDMS surface. And, ExtrAvidin, which reacts with biotin, provides a useful surface for heterogeneous binding studies. Binding biotinylated protein A and DNA allows preparation of a surface for BSI analysis. The immobilized molecule targets will now interact with solute samples as they travel through the channels. Two of the systems we studied were protein-A binding to the Fc fragment (the crystallizable region of an antibody that interacts with cell-surface receptors) of human immunoglobulin G (IgG) and a 30-mer DNA strand for hybridization experiments. In addition, we performed BSI hybridization studies on the Figure 1. Backscattering interferometry setup. CPU: Central processing unit.