Kelvin Physics of Protein Layers Printed in Microarray Format

Chapter 20

Kelvin Physics of Protein Layers Printed in Microarray Format Downloaded by PENNSYLVANIA STATE UNIV on August 6, 2012 | http://pubs.acs.org Publication Date: August 2, 2007 | doi: 10.1021/bk-2007-0963.ch020

*

Hong Huo, Larisa-Emilia Cheran, and Michael Thompson

Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario M 5 S 3H6, Canada

The electrical properties of protein molecules adsorbed onto a gold substrate are studied using a scanning Kelvin nanoprobe in a microarray format. The results demonstrate that this instrument can provide information on protein orientation, polarization, dimension and molecular interaction. Changes in the work function for absorbed neutravidin, bovine serum albumin and a complex of the two proteins on gold were measured. Also, the analogous changes with respect to the surface concentration of neutravidin were examined and the results indicate a saturation of work function values at a specific surface population. The results are discussed in terms of the Schottky model modified for protein semiconductive properties, such as depletion width, charge carrier density, band bending and permittivity.

312

© 2007 American Chemical Society

In New Approaches in Biomedical Spectroscopy; Kneipp, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

Downloaded by PENNSYLVANIA STATE UNIV on August 6, 2012 | http://pubs.acs.org Publication Date: August 2, 2007 | doi: 10.1021/bk-2007-0963.ch020

313

Following rapidly on the heels of genomics, proteomics has become a very prominent research field in analytical biochemistry. In this area, the protein microarray represents a highly miniaturized parallel and multiplexed solid phase assay, which allows the production of high-throughput analyses, with low consumptions of reagent samples (nL level) and attractive manufacturing costs (7). This is why the technology has become a potentially powerful tool for diagnostic and therapeutic purposes, as well as for basic research in biology and medicine. Well-established platforms, detection strategies and software tools are available for D N A microarray technology. However, this is not generally the case for protein chemistry, where there is tremendous molecular variability, coupled with complex chemical and structural properties. Moreover, significant challenges exist in detection strategies for the protein microarray, compared to the sister nucleic acid technology. Currently, labeled probe detection is the most common protocol for microarrays. Foremost amongst these is fluorescence analysis, which allows single molecule investigation. However, this method suffers from various drawbacks for protein studies, the most serious of which is the need to tag the biomolecule of interest with fluorophores or isotopes. This labeling process is both expensive and time-consuming. Additionally, the label may alter the native structure of a protein and potentially interfere with the bioaffinity interaction under study. Problems such as label stability and photo bleaching or unspecific binding of the fluorophore to the surface encountered with fluorescent reporters may also interfere with data collection and analysis. Detection using radiochemical labeling is another method of tagging, but is not widely used due to health and safety issues and long detection times (up to lOh). Label-free strategies provide an alternative route for circumventing the above-mentioned difficulties with respect to protein microarray detection. Currently, relatively few label-free detection methods such as mass spectrometry (MS) (2,3), surface plasmon resonance (SPR) (4) and atomic force microscopy ( A F M ) (5,6) are applicable to the microarray format. These label-free technologies require the use of sophisticated tools that are not available in all bioanalytical laboratories. In the present paper, we describe a scanning Kelvin nanoprobe (SKN) as an alternative label-free detection approach for work with protein microarrays (7). Based on the measurement of work function change, the S K N can measure inherent electrical properties of proteins on a solid surface, which depend on protein dimension, orientation, polarization and molecular interactions. For many decades, the Kelvin probehas been extensively applied as a surface potential technique to study monolayers at air-water interfaces(#-77). Although there are three proposed models for estimating dipole moments in surface potential measurement(P), difficulties in interpreting experimental data represent the main reason why the technique is restricted to application to the


314 air-solid interface(/2-/6). Such measurements and explanation of results based on dipole moments and Schottky models suggest that the S K N is a powerful method for the understanding of protein electrical properties offering, in turn, considerable potential for label-free microarray detection.


Experimental Reagents and materials Neutravidin biotin-binding protein was obtained from Pierce Biotechnology, Inc, Rockford, IL, U S A and used without further purification. Bovine serum albumin and biotin labeled bovine serum albumin were purchased from Sigma Aldrich, Canada. Lyophilized proteins were dissolved in D P B S buffer solutions before use. Dulbecco's phosphate buffered saline solution (DPBS) was purchased from Sigma Aldrich, Canada. Spectroscopy-grade acetone was obtained from Uniscience, Inc, Mississauga, Ont. Anhydrous ethyl alcohol and H P L C grade methanol were obtained from Commercial Alcohols Inc, Brampton, Ont., and V W R Canada (Mississauga, Ont.), respectively. A l l solvents were used as received. Optically flat silicon wafers, 300-400 μηι thick, were purchased from International Wafer Service, Canada.

Instrumentation The scanning Kelvin nanoprobe A prototype version of the scanning Kelvin nanoprobe (SKN) constructed in our laboratory was employed for all measurements^ 7). Figure 1 presents the main modules of the S K N block-diagram. The probe (tungsten, guarded, driven by a piezo actuator from Sensor Technology Ltd., Collingwood, Ont., Canada) vibrates over the sample, which is placed on an X Y scanning table that can also be moved in the Ζ axis, using a second piezo actuator (Polytech PI, Auburn, M A , U S A ) . The probe does not touch the surface, but follows its topography at an extremely small, constant distance. The signal is amplified by an ultra-low noise charge amplifier (Electro Optical Components, Inc. Santa Rosa, C A , U S A ) and converted to a voltage, which is fed into two lock-in amplifiers (Stanford Research Systems, Sunnyvale, C A , USA). One serves as a detector of contact



Figure 1. Block diagram of the scanning Kelvin nanoprobe. An ultra-low noise amplifier detects the Kelvin current. Two lock-in amplifiers are usedfor detecting the contact potential difference and the topography signals. The tip-sample distance is monitored by capacitative control, using a frequency well above the vibrating frequency and superposing a small AC signal to generate the control current. (Reprinted by kind permission of the Royal Society of Chemistry)


316


potential difference (the difference in work function between the tip material and the sample material) and the other for derivation of the topographic signal. The latter is extracted from the feedback loop that controls the constant distance between probe and surface, which is based on a capacitative measurement using a superimposed high frequency, low amplitude voltage. The whole instrument is controlled by a LabView program with a data acquisition board (National Instruments, U S A ) . Data analysis is performed using a dedicated Origin 7 program (Origin Lab Corporation, M A , USA).

Microarrayer Printing of protein spots was achieved using a Virtek Chipwriter Professional arrayer. (Waterloo, Ont., Canada).

Procedures

Gold substrate fabrication Before coating, the silicon wafers were cut into 1mm χ 2 cm pieces and cleaned in an ultrasonic bath for 15 min in acetone, ethanol and methanol, respectively. Following removal from solution, the substrates were dried in a stream of nitrogen. The clean silicon substrates were primed with a thin layer (lOnm) of chromium, followed by the deposition of 200 nm of gold. The goldcoated silicon substrates were first rinsed with deionised water, and then sonicated in acetone, ethanol and methanol for 15 min, respectively. After sonication, the slides were extensively rinsed in deionised water again and dried under a stream of nitrogen.

Microarraying

ofproteins

A l l protein arrays were printed by contact printing, based on capillary action, using the robotic microarrayer. The micro-machined pins consistently delivered samples of approximately 1 nL onto the gold slides at designated locations. Typically, circular spots with diameters ranging from 150 to 250 μηι with pitches of about 250-300 μιη were produced. The printing was performed at a relative humidity of 65% and a temperature of 25°C. After spotting, the protein immobilized slides were kept in a humid environment (65% relative humidity,


317


25°C) for 120 min. After this time, the slides were washed with DPBS buffer and ultra pure water. The slides were then carefully inverted and gently immersed in D P B S buffer for 15 minutes to remove any unbound proteins. This was followed by rinsing with pure water, which removed any buffer salt. Finally, the arrayed slides were dried under a stream of nitrogen before being scanned with the S K N . A l l the samples were analyzed in air, at ambient temperature and pressure, using a lateral scanning step correlated with the dimensions of the investigated arrays.

Kelvin physics of gold-attached protein

Fermi levels and work function In this work, The Kelvin nanoprobe has been used under ambient atmospheric conditions to measure the difference in work function between a tungsten probe and protein molecules attached to gold-coated silicon substrates. The work function, φ, is the minimum energy required to remove an electron from the Fermi level to a point just outside the metal, where the potential of the solid is negligible, or to an infinitely large distance from the surface, where the kinetic energy is zero, the so-called vacuum level. Electrons in a metal are distributed in available states following Fermi-Dirac statistics where the probability of occupancy f( of energy state £, is given by(/#): Ei)

l + exp(£,.

(1)

-E )/kT F

The Fermi level E is defined as the energy level at which the probability of being occupied by an electron is 0.5. If two metals (such as the tungsten Kelvin probe, and a gold surface) with different work functions,

Kelvin Physics of Protein Layers Printed in Microarray Format

Recommend Documents