Micro-Nano Technology for Genomics and Proteomics BioMEMs - Ozkan

FIGURE 6.15. Future electronic pick and place system for the heterogeneous integration of nanostructures into higher order devices?

positioned above the lower electrode, and a DC electric field is applied which is just strong enough to orient the nanospheres, but does not de-hybridize them from the surface. While Figure 6.14 shows the functionalized nanospheres in a polar orientation, in terms of the

(B) and (C) sequences; the relative positioning of the electrodes can produce electric fields which yield other angles for the relative position of the (B) and (C) DNA sequences. When the nanospheres are in their correct alignment, they can be completely hybridized (A’-C/C’), by lowering the temperature, and then exposed to UV irradiation to crosslink the (A/A’) sequences. Upon de-hybridization, this process can produce nanospheres with (B) and (C) DNA sequences in relatively polar (north and south) positions. Repeating the process two more times should allow the production of nanospheres with specific (B), (C), (D), and (E) DNA sequences in polar/equatorial, tetrahedral or hexagonal coordinate positions. Thus, the electric field orientation process offers considerable promise for the manufacture of precision nanocomponents (quantum dots, photonic crystals, organic/metallic/semiconductor nanoparticles, and nanotubes), which can subsequently be used for further self-assembly into viable higher order heterogeneous structures and devices. Figure 6.15 shows a representation of a future electronic pick and place system for the heterogeneous integration of nanostructures into higher order devices.

6.4. INTEGRATION OF OPTICAL TWEEZERS FOR MANUPILATION OF LIVE CELLS

Custom designed miniature optical tweezers with the use of Vertical Cavity Surface Emitting Lasers (VCSEL) are integrated with the microelectrode array for the delivery or manipulation of live cells. For this individually addressable 2 × 2 arrays of VCSEL is focused through a series of lenses and directed onto the microarray chamber after a magnification objective. Figure 6.16 depicts the optical setup. Since the microarray chamber is

Translation

stage

Microscope Objective

100x, 1.25 N.A.

VCSEL

Array Dichroic

Beamsplitter

CCD

IR filter

Rat hepatocytes

10 m Laguerre VCSEL

FIGURE 6.16. Schematic drawing of inverted VCSEL driven optical multi-beams setup. In-situ observations are made through a CCD camera.

fabricated with a transparent material (indium tin oxide), penetration of the optical beam is enabled. After optical trapping of the cells or other objects such as microspheres, precision controlled translation stage offers transport or manipulation of these objects without jeopardizing the sterility of the environment [41–43]. This remains to be the major issue with the mechanical manipulators.

For biological applications, mouse 10 µm diameter 3T3 fibroblast cells are manipulated with single Laguerre-Gaussian gradient VCSEL driven optical micro beam. The cells are continuously monitored for a week after they were exposed to the laser beam. There was no superficial evidence of cell damage from the laser beam, though we did not explicitly look at stress response genes. When cultured, cells attached, spread and underwent mitosis. As compared to microspheres made of polystyrene live fibroblast cell are held less strongly in the trap. We speculate that this is due to its lower dielectric constant and irregular shape. A 5 µm cell is transported with a speed of 2 µm/sec, which is about 4 times slower compared to the same size polystyrene sphere. The trapping force on the cell is estimated as 0.1 pN. In this case since the refractive index of the surrounding medium, nm, is close to the refractive index of cell, n, there is very limited force acting on the cell. Alternatively, when n is greater than nm (polystyrene (1.58)-water (1.33) system), the sphere is always pushed out of the laser beam, which is also observed experimentally. During velocity measurements the glass

Trapped rat hepatocyte

20 m

B F

C G

D H

FIGURE 6.17. Primary rat hepatocyte in an electrically pre-patterned array is manipulated with 850 nm diode laser and transported to the next neighbor cell within the array.

substrate surface is pre-treated with a non-adhesion-promoting chemical to prevent possible measurement errors due to probable cell-substrate adhesion.

Since the index mismatch between cellular solution and the cover slip is more compared to the index mismatch between deionized water and the cover slip, the losses in the trapping force is more due to enhanced spherical aberration in the former case. This effect also contributes to the reason why trapping force for cells is predicted as less than the polystyrene model system with the same size.

Towards the practice of integrated system, electrically a pre-arrayed rat hepatocyte is manipulated with 850 nm single VCSEL beam and is transported next to a neighbor cell within the array. This experiment demonstrates ability of individual manipulation of cells within the array and also depicts the possibility of sample retrieval after a chemical or biological treatment in parallel. This experiment is summarized in Figure 6.17. In addition, mouse fibroblasts that are initially fluorescent tagged green and red are optically manipulated after patterning electrophoretically [44]. Red fluorescent labeled fibroblast is transported to the next die by using Laguerre mode VCSEL at 850 nm. This experiment demonstrates the first essential steps towards microscopic monitoring and micromanipulation of many

live cells in real time in chip-based biosystems. This technology may find applications in cell-based functional genomics, high throughput phenotyping.

CONCLUSIONS

Active microelectronic array technology provides a number of distinct advantages for DNA hybridization diagnostics, DNA/protein/cell based affinity assays in molecular biology research and for potential nanofabrication applications. Microelectronic arrays have been designed and fabricated with 25 to 10,000 microscopic test sites. The higher density devices have CMOS elements incorporated into the underlying silicon structure that provide on-board control of current and voltage to each of the test sites on the device. Microelectronic chips are incorporated into a cartridge type device so as to be conveniently used with a probe loading station and fluorescent detection system. Active microelectronic arrays are differentiated from other DNA chip or array technologies by a number of important attributes. Active microelectronic arrays allow DNA molecules (genomic DNA, RNA, oligonucleotide probes, PCR amplicons), proteins, nanostructures, cells and microscale devices to be rapidly transported and selectively addressed (spotted) to any of the test sites on the microelectronic array surface. Microelectronic array devices have considerable potential for nanofabrication applications, including the directed self-assembly of molecular, nanoscale and microscale components into higher order mechanisms, structures, and devices. Electric field assisted self-assembly using active microelectronic arrays is a type of “Pick and Place” heterogeneous Integration process for the fabrication of 3D structures within defined perimeters of larger silicon or semiconductor structures. This technology provides the best aspects of a top-down and bottom-up process, and has the inherent hierarchical logic of allowing one to control the organization and assembly of components from molecular level —> to the nanoscale level —> to micro/macro scale 3D integrated structures and devices.

ABBREVIATIONS

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7

Peptide Arrays in Proteomics

and Drug Discovery

Ulrich Reineke1, Jens Schneider-Mergener1,2, and Mike Schutkowski1

1Jerini AG, Invalidenstr. 130, 10115 Berlin, Germany

2Institut fur¨ Medizinische Immunologie, Universitatsklinikum¨ Charite,´ Humboldt Universitat¨ zu Berlin, Schumannstr. 20–21, 10117 Berlin, Germany

7.1. INTRODUCTION

Array technology has become a powerful tool for today’s high throughput approaches in biology and chemistry. These large-scale technologies emerged mainly from the genomics field driven by the human genome project and other species sequencing efforts. The associated development of many new technologies led to the initiation of several additional “omics” fields such as proteomics, lipidomics, glycomics and others. Scientists in these fields subsequently demanded tools that allow rapid and reliable characterization of large numbers of molecules of very different natures including nucleic acids, proteins, carbohydrates, peptides, and small molecules with very small amounts of biological or synthetic material.

A paramount principle was the array technology emerging in the late 1980s with DNA arrays. Initial developments involving peptide arrays began almost simultaneously. Protein arrays arose later because several critical factors such as stability, native folding and activity of the immobilized proteins had to be addressed. The main array technology characteristics are spatially addressable immobilization of large numbers of different molecules (libraries), simultaneous analysis with one or more purified or crude biological samples used to probe the array, and a general tendency towards miniaturization, automated read out and integrated data analysis. The benefits are an unprecedented number of data points, rapid generation of data, and the extremely small biological sample volumes required per data point, providing opportunities for completely new insights within life sciences. This is reflected by an

increasing market with an estimated market size for non-DNA biochips of about 200 million US$ and an annual growth rate of 36%.

In 1991 two different technologies for the preparation of peptide arrays were published (Section 7.2.3.1.). (1) Light-directed, spatially addressable parallel chemical synthesis [151] is a synthesis technology permitting extreme miniaturization of array formats but, conversely, involves sophisticated and rather tedious synthesis cycles. (2) The SPOT synthesis concept developed by Ronald Frank is the stepwise synthesis of peptides on planar supports (originally cellulose membranes) applying standard peptide chemistry [154, 155]. SPOT synthesis is technically very simple and flexible and does not require any expensive laboratory automation or synthesis hardware. In contrast, the degree of miniaturization is significantly lower. However, due to the simplicity of the technology more applications of the SPOT concept by far were published since 1991 compared to light-directed, spatially addressable parallel chemical synthesis (Section 7.6). In addition to these pioneering and meanwhile well established technologies, the last few years have seen the introduction of several other concepts for peptide array preparation accessing several new developments in microarray technologies inspired by DNA as well as protein array approaches.

In Section 7.2 the technologies for peptide array generation are described in detail, including coherent surfaces and surface modification, microstructured surfaces, peptide array preparation and printing techniques for peptide array production. Thereafter, the different types of peptide libraries such as protein sequence-derived and de novo approaches are described in Section 7.3. Section 7.4 summarizes assay principles for peptide arrays. This Section is divided into the subsections “screening” and “read-out” addressing either the molecular recognition event such as ligand binding or enzymatic conversion, or how one observes which peptide was bound and/or converted by an interaction partner or enzyme (chemoluminescence, fluorescence, radioactivity, chromogenic or label-free read-out). The different applications of peptide arrays such as antibody epitope and paratope mapping, protein-protein interaction mapping, identification of enzyme substrates and inhibitors, DNA and metal ion binding, chemical transformations, cell binding, and peptidomimetic alterations, are covered in Section 7.5. Finally, in Section 7.6 we present an extensive bibliography of publications describing peptide array applications (Table 7.4).

The scope of this review article is peptide arrays on planar supports. Thus, we do not consider the following topics: peptide synthesis on polymeric pins, surfaces modified with only one peptide rather than a peptide collection as often employed in material sciences, read-out methods such as surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOFMS) with peptides immobilized on the surface and surface plasmon resonance with immobilized peptides in standard throughput configurations (Biacore) as well as small molecule and protein arrays.

7.2. GENERATION OF PEPTIDE ARRAYS

Materials used for the preparation of peptides arrays are flexible porous planar supports such as cellulose [127, 153, 155], cotton [128, 492], polymeric films [34], disks [208, 337] and membranes [97, 578, 592], or rigid, non-porous materials, such as glass, gold-coated surfaces, titanium, aluminum oxide, silicon, and modified polymers such as polypropylene, polyethylene or polyurethane.