Bill Trogler

UCSD

Current Biosensing Cannot Readily Detect Current Biosensing Cannot Readily Detect Individual Cancer Cells in the Body Individual Cancer Cells in the Body

11

10001000

1 million1 million

1 billion1 billion

1 trillion or 40 generations1 trillion or 40 generations

Number of cancer cellsNumber of cancer cells

Polymer template

Polymer template

polyamine

Si(OH)4

silica shell

calcination

dissolution

functionalization

amine, thiol, alkylhollow silica spheres

5-10nm

40-1000 nm

Hollow Nanoparticles A Biomimetic Synthetic Analogue of a Virus for Cell Delivery and Sensing

200 nm SiO2 500 nm SiO2 200 nm TiO2

Silica like virus particles readily enter living cellswith Davorka Messmer’s group

Uptake of FITC-labeled 100 nmSiO2 NPs by primary human dendritic cells (40x)

45nm-FITC nanoparticles are taken up by dendritic cells and traffic to the nucleus (Confocal microscopy 60x)

Targeted Delivery to Endothelial Cellswith Carson and Esener groups

100 nm amine modified silica hollow spheres with CGKRK targeting peptide uptake by HUVEC cells vs. nontargeting CREKA control

Hollow Nanosphere Cell Biosensorswith A. Kummel and S. Esener’s groups

SiO

O

O

OH

Si

Si

HO

HO

poly Si

OCH3

OCH3

OH

OH

Surface anchored luminescent polymer, targeting agent, PEG, ....

Interior contains pH sensitiveFluorophore, redox sensitiveFluorophore, temperature Sensing quantum dot, ….

TEM image

Figure 1. The SiCHAS platform . Arrays of microwells will be fabricated by microlithography and coated with a mixture of secretant detection antibodies. Secretant molecules bound to the antibodies on the bottom of the wells will be detected by fluorescently labeled secondary antibodies with different emission spectra. Cell surface molecules will be simultaneously detected with quantum dot labeled antibodies. The wells will be connected with narrow slits to allow the cells to communicate via secretants or bigger slits to allow physical cell-cell contact.

Arrays of microwells will be coated with a mixture of secretant detection antibodies. Secretant molecules bound to the antibodies on the bottom of the wells will be detected by fluorescently labeled secondary antibodies with different emission spectra.

Cell surface molecules will be simultaneously detected with quantum dot labeled antibodies.

The wells will be connected with narrow slits to allow the cells to communicate via secretants or bigger slits to allow physical cell-cell contact.

Note the wells will actually be at least 10x taller than the cells (100 micron) but only 20x20 microns in-plane to limit out diffusion; they are shown as short wells here just for graphical clarity.

The Single Cell Hyper Analyzer for Secretants (SiCHAS)D. Messmer, A. C. Kummel, W. C. Trogler UCSD

(TOP) Cell seeding in microwells. 50 x 50 x 50 μm wells 10μm thickwalls. 50,000 cells were spread over 1cm2 so we had less than one cells/well initially. The cells divide over time so there density increases. Green is a membrane stain.

(Bottom Left) Proof of Viability: The cells were kept in the wells sealed by a coverslip for 5 days in culture media. After 5 days, a small amount of propidium iodide (PI) was added which stains dead cells red. The image on the left shows there are almost no dead cells.

(Bottom Right) Proof of Diffusion: If a mixture of dead and live cells is used and stain with PI, many of the cells are red proving we can diffuse stain to the bottom of the wells. The wells employed for this experiment were 60x60x50 μm nl/well.

The Single Cell Hyper Analyzer for Secretants (SiCHAS)D. Messmer, A. C. Kummel, W. C. Trogler UCSD

Specific Absorption of Breast Cancer Cells

MCF-7 – with Dr. Sarah Blair, A. Kummel’s groups

Gold Coverage=8.73%Cells on Substrate=21Cells on Gold Pads=16=76.19%

Coverage Ratio: = 8.73 x random

Selective binding of breast cancer cell line tested. (Above) Bright yellow circles are cells and dim red circles are the gold pads. (Right) Cell nucleus is blue. Cell membrane is green. Au pad is red.

Gold pads represent only 9% of the surface area but 76% of the cells attached to the gold pads. This is 9x random.

Increased selectivity will be obtained by improvement of the washing step.

Grand Challenges

• Viral-like endocytosed intracellular biosensors

• Sensing cell-cell signaling in ordered cell arrays to capture biodiversity of population

• Parallel sensing equivalent of flow cytometry on ordered arrays of cells

• Cell specific preconcentration