1. Development of novel nanoprobes with multiple applications in biology and medicine Kathleen M. Beckingham and R. Bruce Weisman Depts. Biochemistry/Cell Biology and Chemistry, Rice University

2. Antibodies: some of the most powerful analytical tools known in biosciences Antibodies can be readily raised against any protein or peptide. Antibodies can also be raised against other chemical entities- DNA, steroid type molecules, etc. By adding a detectable “tag” to the antibody, a tool to probe for the protein/chemical in question can be generated.

3. Uses of tagged antibodies in biomedicine/biotechology Monitoring of hormones - insulin, thyroid hormones Detection of tumor associated antigens - PSA test Screening newborns for genetic diseases - phenylketonurea galactosemia Pregnancy tests - chorionic gonadotropin Detection of pathogens - bacteria, toxins, viruses

4. Fluorescently tagged antibodies - widely used tools for diagnostic and biotechnological applications The most widely used tags on antibodies are fluorophores - small organic molecules, eg, the Alexafluor series from Molecular Bioprobes - quantum dots, luminescent semiconductor nanocrystals coated with zinc sulfide and amphiphilic polymer

5. Fluorescently-labeled antibodies allow for simultaneous detection of multiple components In many diagnostic/research or biotechnical applications it is desirable to probe for several proteins/entities simultaneously on a single sample By using fluorescent tags with different emission spectra on the relevant antibodies, simultaneous detection of this type is possible. Multiplexed analysis of this type is already in widespread and new applications continue to be developed.

6. Examples of increasing usage of multiplexed analysis with fluorescently labeled antibodies 1. In 2005, 36 million newborns were screened for four pathologies by just one of the commercial multiplex analysis systems 2. In suspected cancer, simultaneous analysis for multiple cancer-associated proteins on hard-to-obtain tissue samples (biopsies) increases the accuracy of diagnosis. Multiplex analysis for multiple tumor-associated proteins is continuously being developed.

7. Limitations to multiplexing analysis with the current fluorescent tags MAJOR PROBLEM - SPECTRAL CROWDING The commonly used fluorescent tags fluoresce in the visible spectrum (400-800 nm) and have relatively broad emission spectra. In practical terms, this means that typically only three/four fluorophores (antibodies) can be detected simultaneously.

8. A new class of fluorophores, opens up the near infra-red spectrum for multiplex analysis Unmodified single walled carbon nanotubes (SWCNTs) fluoresce in a completely different region of the spectrum - the near infra-red (900 nm-1600 nm). Many different species of SWCNTs are available each with its own distinct fluorescent emission spectrum. Using different SWCNT species as tags for antibodies will increase the power of multiplex analysis permitting simultaneous analysis for many more entities on a single sample. As such it will increase diagnostic certainty and increase efficiency in testing both in terms of cost and time.

9. Why would this be useful to NASA? 1. Monitoring of astronaut health is a high priority. during space missions. Multiplexed analyses on blood or urine will expand the repertoire of analyses that can be performed while the same time decreasing equipment load, sample size and frequency. 2.Detecting microbial contamination in space is a high priority. Again the repertoire of tests, efficiency of testing and equipment load will be improved. 3.Monitoring all life forms aboard space vehicles (food plants, research organisms, etc) would be enhanced.

10. Our goals 1.Efficient purification of individual SWCNT species for use as antibody tags. 2. Generation of methods to tag antibodies with individual SWCNT species while maintaining SWCNT near-IR fluorescence. 3. Development of suitable near-IR instrumentation for detection. 4. Demonstration that SWCNT tagged antibodies can be used for multiplex detection.

11. GOAL 1 Purification of individual SWCNT species So what are single wall carbon nanotubes?

12. Single-walled Carbon Nanotube (SWCNT) SWCNTs are related to C 60

13. taken from http://www.photon.t.u-tokyo.ac.jp/~maruyama/wrapping.files/frame.html Rolling up Graphene makes a SWCNT

14. armchair (  = 30°) zigzag (  = 0°) intermediate (0    30°) zigzag Many SWCNT Structures Exist ( different diameters and angles )

15. Prof. C. Lieber, Harvard Univ. STM Image of a Single-Walled Carbon Nanotube

16. Constructing Nanotubes from a Graphene Sheet Roll-up vector Chiral angle Nanotube axis

17. Each SWCNT species has a unique excitation and emission spectrum

18. Purification of individual SWCNT species Current commercial preparations (HiPco preps) of SWCNTs are heterogeneous mixtures of multiple SWCNT species The Weisman lab is at the forefront of efforts to purify individual SWCNT species, using ultra-centrifugation in iodixanol (density) gradients. Ten SWCNT species have been separated by this technique. GOAL 1 - purification of individual SWCNT fluorophores for conjugation to antibodies - is well-advanced.

19. DGU spatially separates HiPco sample into (n,m) species

20. Separated fractions contain robust near-IR fluorophores with distinct emission peaks Ghosh, Bachilo, and Weisman, Nature Nanotechnology, published online May 9, 2010

21. GOAL 2 Development of instrumentation for near-IR detection Instrumentation will vary with the application. We have focused on building a near-IR microscope system for detecting multiplexed SWCNTs in tissue preparations. A functional system has been developed and is in use. It combines bright field and near-IR images. An instrument model that detects near-IR and visible fluorescence is planned.

22. Apparatus for near-IR fluorescence microscopy Modified from Tsyboulski, et al. Nano Lett. 5, 975 (2005)

23. Detection of SWCNTs (red) in the major blood vessel (green) of a fruit fly larva Bright field and near-IR images from near-IR microscope merged with conventional fluorescence image for Green Fluorescent Protein in the blood vessel wall. Use of near-IR microscope system to detect SWCNTs in biological samples

24. Individual Nanotubes are Imaged in Biological Tissues and Show Distinct Emission Spectra

25. GOAL 3 Methods for linking SWCNTs to antibodies ISSUES FOR CONSIDERATION 1. SWCNTs are hydrophobic - any linking protocol has to include a step to solubilize the SWCNTs. SOLUTION: SWCNTs are solubilized with short DNA oligos - they wrap tightly around the nanotubes and don’t come off upon on dilution.

26. GOAL 3 Methods for linking SWCNTs to antibodies cont. ISSUES FOR CONSIDERATION 2.Most covalent modifications of SWCNTs destroy the near IR of the nanotubes SOLUTION: Link the antibody to the DNA oligo used to solubilize the nanotubes.

27. Approaches to Oligo-Antibody linkage for SWCNT antibody tagging APPROACH 1 1. Solubilize nanotubes with oligos 2.Glutaraldehyde cross-link the antibody to the oligos on the nanotubes 3.Purify modified antibody and associated oligos/SWCNTs on Protein A sepharose RESULT : Low conjugation of SWCNTs to antibody but antibody retained its specificity

28. Model studies with Drosophila PS1 antibody that stains a particular compartment in wing discs ABC Unstained wing disc Conventional fluorescent detection shows the antibody does not stain the ventral compartment of the wing disc

29. PS1 antibody retains its specificity but shows very low SWCNT conjugation using Approach 1 Conventional fluorescence detection shows PS1 retains its specificity for the dorsal compartment Analysis for SWCNTs shows a very low level of SWCNT conjugation to the PS1 antibody

30. Approaches to Oligo-Antibody linkage for SWCNT antibody tagging (cont.) APPROACH 2 Crosslink sugar residues on Fc portion of antibody to a 5’amino group engineered onto end of oligo. ADVANTAGES: 1. No interference with oligo-SWNT interaction 2. No interference with antigen binding sites of antibody

31. Initial attempts at oligo-antibody linkage via sugar OH - 5’amine crosslinking Conjugation of (GT)20 oligo to the PS1 antibody. Size separating denaturing gel electrophoresis shows additional bands in lane 3 (blue arrows) corresponding to one or more oligo molecules covalently linked to either the light or heavy antibody chain. Lane 1- unconjugated antibody showing heavy and light chains Lane 2 – antibody after addition of crosslinking moiety Lane 3 – antibody after conjugation to the (GT)20 oligo.

32. Summary 1.Efforts on all aspects of the project are advanced. 2.SWCNT-based near-IR fluorophores will revolutionize multiplexed biodetection systems. 3.Multiple applications of this new technology are relevant to NASA.

33. Acknowledgements Weisman lab Sergei Bachilo Saunab Ghosh Dmitri Tsyboulski Paul Cherukuri Tonya Leeuw Beckingham lab Rebecca Simonette R. Michelle Reith Rebecca Divers Jeffery Chen