Label-free detection techniques

1. Harini Chandra Affiliations Label-free detection techniques Development of reliable, sensitive and high-throughput label-free detection techniques has become imperative for proteomic studies due to drawbacks associated with label-based technologies. Label-free detection methods, which monitor inherent properties of the query molecule, promise to simplify bioassays.

2. Master Layout (Part 1) 1 This animation consists of 4 parts: Part 1 – Overview of label-free techniques Part 2 – Surface plasmon resonance (SPR) Part 3 – Surface plasmon resonance imagining (SPRi) Part 4 – Spectral reflectance imaging biosensor (SRIB) 2 Surface plasmon resonance (SPR)-based techniques Ellipsometry techniques Microcantilever LABEL-FREE DETECTION TECHNIQUES 3 Scanning Kelvin Nanoprobe (SKN) Interference-based techniques 4 Enthalpy array Electrochemical impedance spectroscopy (EIS) aptamer array Atomic Force Microscopy (AFM) 5

3. Definitions of the components:Part 1 – Overview of label-free techniques 1 1. Label-free detection: Label-free detection techniques monitor inherent properties of the query molecules such as mass, optical and dielectric properties. Unlike label-based detection methods, these techniques avoid any tagging of the query molecules thereby preventing changes in structure and function. They do not involve laborious procedures but have their own pitfalls such as sensitivity and specificity issues. 2. Surface plasmon resonance-based techniques i) Surface plasmon resonance (SPR): Detects any change in refractive index of material at the interface between metal surface and the ambient medium. ii) Surface plasmon resonance imaging (SPRi): Image reflected by polarized light at fixed angle detected. iii) Nanohole array: Light transmission of specific wavelength enhanced by coupling of surface plasmons on both sides of metal surface with periodic nanoholes. 3. Ellipsometry-based techniques i) Ellipsometry: Change in polarization state of reflected light arising due to changes in dielectric property or refractive index of surface material measured. ii) Oblique incidence reflectivity difference (OI-RD): Variation of ellipsometry that monitors harmonics of modulated photocurrents under nulling conditions. 4. Interference-based techniques: Interferometry is based on the principle of transformation of phase differences of wave fronts into readily recordable intensity fluctuations known as interference fringes. The various detection strategies that make use of this principle include: i) Spectral reflectance imaging biosensor (SRIB): Changes in optical index due to capture of molecules on the array surface detected using optical wave interference. 2 3 4 5

4. Definitions of the components:Part 1 – Overview of label-free techniques 1 ii) Biological compact disc (BioCD): Local interferometry i.e. transformation of phase differences of wave fronts into observable interference fringes, used for detection of protein capture. iii) Arrayed imaging reflectometry (AIR): Destructive interference of polarized light reflected from silicon substrate captured and used for detection. 5. Electrochemical impedance spectroscopy (EIS) -aptamer array: Aptamers are short single-stranded oligonucleotides that are capable of binding to a wide range of target biomolecules. EIS combined with aptamer arrays can offer a highly sensitive label-free detection technique. 6. Atomic force microscopy (AFM): Vertical or horizontal deflections of cantilever measured by high-resolution scanning probe microscope, thereby providing significant information about surface features. 7. Enthalpy array: Thermodynamics and kinetics of molecular interactions measured in small sample volumes without any need for immobilization or labelling of reactants. 8. Scanning Kelvin nanoprobe (SKN): A non-contact technique that does not require specialized vacuum or fluid cell, SKN detects regional variations in surface potential across the substrate of interest caused due to molecular interactions. 9. Microcantilever: These are thin, silicon-based, gold-coated surfaces that hang from a solid support. Bending of cantilever due to surface adsorption is detected either electrically by metal oxide semiconductor field effect transistors or optically by changes in angle of reflection. 2 3 4 5

5. Part 1, Step 1: 1 SPRi Nanohole array SPR Surface plasmon resonance (SPR)-based techniques Ellipsometry Ellipsometry techniques Microcantilever OI-RD 2 SRIB Scanning Kelvin Nanoprobe (SKN) Interference-based techniques AIR 3 LABEL-FREE DETECTION TECHNIQUES BioCD Enthalpy array Electrochemical impedance spectroscopy (EIS) aptamer array Atomic Force Microscopy (AFM) 4 Action Description of the action Audio Narration First show the central heading in the circle. Then show each of the arrows appearing and their respective colored boxes as shown. User must be allowed to click on any of these headings to read the definitions as given in the previous two slides. Once the user is done, he must be provided with a ‘NEXT’ option, which when clicked, must highlight the boxes indicated ‘SPR’, ‘SPRi’, ‘nanohole array’ and ‘SRIB’. Each heading must appear one at a time and the user must be allowed to click on them to understand the details. As given in the previous two slides. 5

6. Master Layout (Part 2) 1 This animation consists of 4 parts: Part 1 – Overview of label-free techniques Part 2 – Surface plasmon resonance (SPR) Part 3 – Surface plasmon resonance imagining (SPRi) Part 4 – Spectral reflectance imaging biosensor (SRIB) % Reflectivity 2 Antigen-Antibody complex Free antigen Reflection angle Flow cell system 3 Bound antibodies Gold film Glass slide Prism Incident light 4 Reflected light Change in angle of reflection 5

7. Definitions of the components:Part 2 – Surface plasmon resonance (SPR) 1 1. Flow cell system: A fluidic device that allows entry of antigens and continuously removes unbound antigens from the system. 2. Free antigen: Antigens that have not bound to their complimentary antibody are in their free state. 3. Bound antibodies: Test proteins such as antibodies that are capable of specifically capturing the desired target protein with high affinity are immobilized on to the gold-coated glass microarray slide. 4. Antigen-Antibody complex: The complex formed due to binding interaction between the free antigen and its corresponding bound antibody. 5. Glass slide: The array surface most commonly used for SPR applications. It is suitably coated with a metal film like gold or silver. 6. Gold film: A thin film of gold is used to coat the glass array surface due to its favourable electronic interband transitions which fall in the visible range. In most other metals, these transitions lie in the ultraviolet region, thereby making them unsuitable for SPR. 7. Prism: The prism placed in contact with the glass slide surface helps in reflecting the incident light from the surface. 2 3 4 5

8. Definitions of the components:Part 2 – Surface plasmon resonance (SPR) 1 8. Incident light: Light falling on the gold-coated array surface with its immobilized antibodies has a particular wavelength and is known as the incident light. 9. Reflected light: Some of the energy of the light incident on the array surface gets absorbed for molecular transitions while the remaining light of lower energy (and higher wavelength) gets reflected from the array surface at a specific angle. 10. Change in angle of reflection: Any changes in the angle of reflected light are indicative of biomolecular binding interactions on the array surface. The angle at which minimum intensity of reflected light is obtained is known as the SPR angle and serves as a quantitative measure of biomolecules binding to the array surface. 2 3 4 5

9. Part 2, Step 1: 1 % Reflectivity 2 Reflection angle Bound antibodies Gold film 3 Glass slide Prism Incident light Reflected light 4 Action Description of the action Audio Narration As shown in animation. SPR is a highly sensitive spectroscopic tool that is increasingly being used for label-free detection studies. Test proteins such as antibodies are immobilized onto the gold-coated glass array surface. Incident light striking the surface is constantly reflected at a particular angle in this state. First show appearance of grey rectangle surface followed by yellow coating with their respective labels. Then show the Y shaped object binding to the surface. Next, light beam must strike the surface and a different color beam must be reflected from it as shown followed by appearance of the graph on the right. 5

10. Part 2, Step 2: 1 Free antigen 2 Flow cell system Bound antibodies Gold film 3 Glass slide Prism Incident light Reflected light 4 Action Description of the action Audio Narration The green shapes must enter the grey box slowly from the side. Show the grey rectangle appearing followed by the arrows and the label ‘flow cell system’. Then, show the green freeform shapes appearing and entering the grey rectangle area slowly from the side. Their movement must not be sharp, point-to-point but more of a meandering and slow entry. Unlabelled free antigens or other query proteins enter via the flow cell and move towards the immobilized antibodies or other test proteins. There is no change in reflected light upon entering into the system. 5

11. Part 2, Step 3: 1 Antigen-Antibody complex % Reflectivity 2 Reflection angle Flow cell system Glass slide Gold film 3 Prism Reflected light Incident light 4 Change in angle of reflection Action Description of the action Audio Narration The green shapes must bind to the green Y shaped objects and must continuously enter and leave the system. Binding of antigen to antibody immediately brings about a change in the angle of reflection of light due to changes in the refractive index of the medium. These changes can be continuously monitored to characterize biomolecular interactions in real-time. The SPR angle i.e. the angle at which minimum intensity of reflected light is obtained is indicative of the amount of biomolecule binding to the surface. The graph represents change in reflection intensity before and after antigen binding. Show the green shapes binding to the Y shaped objects. Once binding occurs, there must be a change in the reflected light beam as shown and change in reflection angle must be shown followed by appearance of new pink curve in the graph. In the background, the green shapes must continue to enter and leave the system if not bound by the Y shaped objects. 5

12. Master Layout (Part 3) 1 This animation consists of 4 parts: Part 1 – Overview of label-free techniques Part 2 – Surface plasmon resonance (SPR) Part 3 – Surface plasmon resonance imagining (SPRi) Part 4 – Spectral reflectance imaging biosensor (SRIB) % Reflectivity Antigen-antibody complex 2 Immobilized antibodies Reflection angle Gold coated array surface 3 CCD camera Scanner mirror 4 Light source SPRi image 5 Lokate, A. M., Beusink, J. B., Besselink, G. A., Pruijn, G. J., Schasfoort, R. B., Biomolecular interaction monitoring of autoantibodies by scanning surface plasmon resonance microarray imaging. J. Am. Chem. Soc. 2007, 129, 14013–14018.

13. Definitions of the components:Part 3 – Surface plasmon resonance imaging (SPRi) 1 1. Light source: A broad beam, monochromatic, polarized light is used to illuminate the entire array surface at the same time. 2. Scanner mirror: A mirror which can reflect the light from the light source on to the biochip surface. 3. Gold-coated array surface: Similar to SPR, a gold-coated glass array surface or sometimes a gold-coated hydrogel array are used as the biochip for immobilization of the capture molecule of interest. 4. Immobilized antibodies: Test proteins such as antibodies that are capable of specifically capturing the desired target protein from a mixture are immobilized on to the gold-coated microarray slide. 5. Antigen-antibody complex: The complex formed due to specific binding interactions between the antigens and their corresponding immobilized anitbodies. 6. CCD camera: A charge-coupled device (CCD) camera continuously monitors any changes that occur on the array surface and is capable of providing real-time kinetic data. This digital imaging technology is widely used for scientific and medical applications where high quality image data is required. 7. SPRi image: Reflected light from a spot will reach a minimal value when the spot meets the optimal SPR conditions, thereby resulting in a dark spot. In this way, the SPRi image is formed for the multiple spots across the array surface. 2 3 4 5

14. Part 3, Step 1: 1 2 Immobilized antibodies 3 Gold coated array surface 4 Action Description of the action Audio Narration The brown colored Y shaped objects must bind to the yellow surface as shown. A gold coated glass array surface is used for immobilization of antibodies complimentary to the target protein of interest. First show appearance of only the yellow surface with its label. Then show the brown colored Y shaped objects binding to this surface as depicted in the animation. 5

15. Part 3, Step 2: 1 Immobilized antibodies 2 Gold coated array surface CCD camera 3 Scanner mirror Light source SPRi image 4 Action Description of the action Audio Narration As shown in animation. A broad beam, monochromatic, polarized light originating from a suitable light source is used to illuminate the entire biochip surface with the help of mirrors placed at suitable angles that will reflect the light onto the surface. Reflected light from each spot on the array surface is captured by means of a CCD camera and used to generate the SPRi image. First show the purple can, the grey ‘mirror’, the two orange ovals and the green ‘camera’. Then show the yellow light rays moving as shown in the animation until the grey surface below is reached. 5

16. Part 3, Step 3: 1 Antigens % Reflectivity Immobilized antibodies 2 Gold coated array surface Reflection angle CCD camera 3 Scanner mirror Light source SPRi image 4 Action Description of the action Audio Narration The green dots must bind to the brown Y shaped objects and spots must appear on the grey surface below. Binding of target antigen to the antibody is detected in real-time due to changes in intensity of reflected light from every spot on the array surface. Multiple biomolecular interactions can be studied simultaneously in a HT manner and changes occurring on the array surface can provide kinetic data about the interactions. Show the green dots binding to the brown Y shaped objects. Once binding occurs, there must be a change in the color of reflected light beam as shown and spots must appear on the grey surface shown below as depicted in the animation. 5

17. Master Layout (Part 4) 1 This animation consists of 4 parts: Part 1 – Overview of label-free techniques Part 2 – Surface plasmon resonance (SPR) Part 3 – Surface plasmon resonance imagining (SPRi) Part 4 – Spectral reflectance imaging biosensor (SRIB) Surface Height relative to surface (nm) CCD camera 2 Position on sample (mm) Illumination 3 SiO2 coating Silicon surface 4 Change in OPD 5 Ozkumur, E., Needham, J. W., Bergstein, D. A., Gonzalez, R. et al., label-free and dynamic detection of biomolecular interactions for high-throughput microarray applications. Proc. Natl. Acad. Sci. USA 2008, 105, 7988–7992.

18. Definitions of the components:Part 4 – Spectral reflectance imaging biosensor (SRIB) 1 1. Illumination: A tunable laser beam of a specific wavelength that has been made spatially incoherent by passing the beam through spinning ground-glass disks is used to illuminate the array surface immobilized with biomolecules. 2. Silicon surface: A silicon wafer having a thermally grown surface coating of silicon oxide (SiO2) is used as the solid support for immobilization of biomolecules. 3. SiO2 coating: The thermally grown and polished SiO2 layer can be used as the reflecting surface instead of conventional glass microscopic slides due to its superior uniformity and smoothness. Reproducible functionalization of these surfaces is also easily achievable due to the known chemical composition and surface chemistry. 4. Change in OPD: Light incident on the SiO2 surface gets reflected at a specific wavelength, the magnitude of which depends on the optical path length difference (OPD) between the top of the surface and the buried SiO2 -Si interface. Any binding of biomolecules to the top surface results in a further increase in OPD which exhibits itself as a characteristic shift in spectral reflectivity and as an intensity difference at a particular wavelength. 5. CCD camera:: A charge-coupled device (CCD) camera continuously monitors any changes that occur on the SiO2 array surface and is capable of providing high throughput, real-time kinetic data. This digital imaging technology is widely used for scientific and medical applications where high quality image data is required. 2 3 4 5

19. Part 4, Step 1: 1 Surface Height relative to surface (nm) CCD camera 2 Position on sample (mm) 3 Illumination Change in OPD SiO2 coating Silicon surface 4 Action Description of the action Audio Narration As shown in animation. First show appearance of brown surface, blue coating layer and then the orange light beam. Show the first set of curved green arrows and then the pink camera on top capturing it with appearance of 1st two grey blocks. Then show the first part of graph on the right. Next show a blue layer appearing and the second green arrows on the surface, the next two cameras and grey blocks. Then show the second part of graph. Finally show the last two blue blocks and the third set of green arrows and last 2 cameras and 3 grey blocks. Finally show the last part of the graph. A SiO2 coated Si surface is functionalized with the biomolecule of interest. The magnitude of total reflected light at a particular wavelength depends entirely on the OPD between the top surface and the SiO2-Si interface. Binding of the target to the immobilized biomolecule further increases the OPD and is seen as a shift in the spectral reflectivity. SRIB therefore serves as a useful tool for HT, real-time detection of biomolecular interactions. 5

20. Interactivity option 1:Step No: 1 1 SPR imaging has been used for serum proteomics studies to characterize the antigens present in patients with hepatocellular carcinoma (HCC) (Lausted et al., 2008). Antibodies specific to liver protein targets were arrayed on a gold-coated surface and ten probed with human serum samples from HCC as well as non-HCC patients. The authors detected 39 significant protein changes in this study, 10 of which were already known including the commonly used liver cancer marker a-fetoprotein. 2 Antigens from human serum samples Arrange the optics system for the experiment in their correct positions. Then click on the light source to view the SPRi image. Antibodies against liver-specific proteins 3 4 Gold coated array surface Results Interacativity Type Options Boundary/limits Once the user places the shapes in their correct positions, he must be allowed to click on the purple cylinder (light source) which must result in the emission of the light rays as shown followed by appearance of the final grey surface at the bottom. User must drag and drop the shapes shown in the next slide in their correct positions indicated by their dotted outlines. Drag and drop. 5 Lausted, C., Hu, Z., Hood, L. Quantitative Serum Proteomics from Surface Plasmon Resonance Imaging. Mol. Cell Proteomics 2008, 7, 2464-2474.

21. Interactivity option 1:Step No: 2 1 2 Antigen-antibody binding interaction Optics system for SPRi Antibodies against liver-specific proteins 3 Scanner mirror Gold coated array surface 4 CCD camera 5 Light source SPRi image

22. Questionnaire 1 1. Which of the following label-free techniques relies on thermodynamic changes occurring due to molecular interactions? Answers: a) SPR b) SRIB c) Enthalpy array d)‏ SKN 2. Which of the following is not an interference-based detection technique? Answers: a) SRIB b) SKN c) AIR d)‏ BioCD 3. Surface features of an object can be studied in detail using which of the techniques below? Answers: a) AIR b) SPRi c) Nanohole array d)‏ AFM 4. A change in the optical path length difference upon binding of target to the immobilized biomolecule occurs in which technique? Answers:a) SRIB b) SPR c) AFM d)‏ Ellipsometry 5. Surface plasmon resonance detects changes in which of the following properties? Answers: a) Electrical conductivity b) Phase difference c) Temperature d)‏ Refractive index 2 3 4 5

23. Links for further reading Research papers: • Ray, S., Mehta, G., Srivastava, S. Label-free detection techniques for protein microarrays: Prospects, merits and challenges. Proteomics 2010, 10, 731-748. • Ramachandran, N., Larson, D. N., Stark, P. R., Hainsworth, E., LaBaer, J., Emerging tools for real-time label-free detection of interactions on functional protein microarrays. FEBS J. 2005, 272, 5412–5425. • Yu, X., Xu, D., Cheng, Q., Label-free detection methods for protein microarrays. Proteomics 2006, 6, 5493–5503. • Lee, H. J., Nedelkov, D., Corn, R. M., Surface plasmon resonance imaging measurements of antibody arrays for the multiplexed detection of low molecular weight protein biomarkers. Anal. Chem. 2006, 78, 6504–6510. • Yuk, J. S., Kim, H. S., Jung, J. W., Jung, S. H. et al., Analysis of protein interactions on protein arrays by a novel spectral surface plasmon resonance imaging. Biosens. Bioelectron. 2006, 21, 1521–1528. • de Boer, R. A., Hokke, C. H., Deelder, A. M., Wuhrer, M., Serum antibody screening by surface plasmon resonance using a natural glycan microarray. Glycoconj. J. 2008, 25, 75–84.