There is certainly a lack of experimental tools with which to discern the orientation of a protein immobilized on a Qdot surface- For specific targeting, it is highly desirable that the delivery proteins (e.g. antibody) are properly oriented and fully functional- Therefore, Qdot bioconjugation step is extremely important in obtaining success in bioimaing

2.4.2.2 Gold Nanoparticles• For over 30 years, nanometer-sized gold particles have been used to stain cells and tissue samples for electron microscopy• The basic principle of interactions between gold particles and biomolecules, like proteins, has been well studied for immunocytochemical staining applications• Although nanosize metals like gold and silver do not fluorescence they can effectively scatter light due to the collective oscillation of the conduction electrons induced by the incident electric field (light)- This is known as “surface plasmon resonance”- Thus, colloidal gold particles exhibit a range of intense colors in the visible and NIR spectral regions• Surface plasmons, also known as surface plasmon polaritons, are surface electromagnetic waves that propagate in a direction parallel to the metal/dielectric (or metal/vacuum) interface- Since the wave is on the boundary of the metal and the external medium (air or water for example), these oscillations are very sensitive to any change of this boundary, such as the adsorption of molecules to the metal surface

• The excitation of surface plasmons by light is denoted as a surface plasmon resonance (SPR) for planar surfaces or localized surface plasmon resonance (LSPR) for nanometer-sized metallic structures• This phenomenon is the basis of many standard tools for measuring adsorption of material onto planar metal (typically gold and silver) surfaces or onto the surface of metal nanoparticles. It is behind many color based biosensor applications and different lab-on-a-chip sensors.

• In the Otto setup, the light is shone on the wall of a glass block, typically a prism, and totally reflected. A thin metal (for example gold) film is positioned close enough, that the evanescent waves can interact with the plasma waves on the surface and excite the plasmons

• In the Kretschmann configuration, the metal film is evaporated onto the glass block. The light is again illuminating from the glass, and an evanescent wave penetrates through the metal film. The plasmons are excited at the outer side of the film. This configuration is used in most practical applications.

• Surface plasmons have been used to enhance the surface sensitivity of several spectroscopic measurements including fluorescence, Raman scattering, and second harmonic generation.• However, in their simplest form, SPR reflectivity measurements can be used to detect DNA or proteins by the changes in the local index of refraction upon adsorption of the target molecule to the metal surface.

• Gold nanoparticle have high absorption and scattering cross section- For example, the absorption cross section of a 5 nm diameter gold particle is about 3 nm at a wavelength of 514 nm, which is about two orders of magnitude higher than that of organic fluorescent molecules at room temperature• Scattering cross section of gold nanoparticle is much larger than polymeric spherical particles of similar size, especially in the red region of the spectrum, i.e. red to NIR range, having potential in deep tissue imaging- For example, using composite core-shell gold (dielectric silica core and gold shell) particles, it is possible to tune the scattering from 600 to 1200 nm• Due to excellent biocompatibility, gold nanoparticles have been widely used in immunohistochemistry (gold-based staining) and in ultra-sensitive DNA detection assays• However, a few literature reports are available on gold nanoparticle-based cancer imaging

• Gold nanoparticles, because of their strong SPR properties, have attracted considerable attention in bioimaging in recent years• The SPR signal originates from the collective oscillation of conduction electrons upon interaction with absorption photons• SPR frequency depends on various factors, particle size, shape, dielectric properties, aggregate morphology, surface functionalization, the refractive index of the surrounding medium

(a) Gold Nanoparticles Synthesis• Various methods have been reported for the synthesis of gold nanoparticles• There are two general approaches (so-called “top-down” and “bottom-up”) that primarily categorize most reported synthesis strategies• Synthesis of gold nanoparticles by employing laser ablation technique is an example of a “top-down” approach, where the embryonic (nascent) partcles are formed from the ionized gold atoms via nucleation and growth processes• The challenge still remains how to stabilize particles in the solution phase- Using a surfactant-based capping agent (sodium dodecylsulfate, an ionic surfactant), Kondow et al. have successfully stabilized ultrafine particles- The capping agents, in general, control the particle size and size distribution, prevent aggregation, and stabilize particle solution (such as in aqueous-based medium)

• ablation of a metal surface immersed in liquid could produce nanoparticles of the metal in the liquid• gas-phase metal clusters are most conviently prepared by laser ablation (In liquid phase, metal atoms are just ejected from metal surface)• Metal atoms are ablated from a metal rod by laser irradiation and are aggregated into metal clusters with a sufficiently larger sizes• Self-aggregation of the nanoparticles suspended in the liquid should be prevented by hindering direct contact of the nanoparticles • For example, Takami and co-workers have prepared small metal clusters by laser ablation of a metal plate in liquid helium, where the clusters are encapsulated in helium bubble

• Preparation of stable nanoparticles in a solution containing a surfactant by use of laser ablation on a metal plate immersed in the solution-The surfactant which surrounds each nanoparticles prevents direct contact of the nanoparticles- Sodium dodecyl sulfate

SDS

210C, 12 h 350C, 5 min 210C, 24 h

Color Red

II. Particle Growth and Aging:

+Micelles

CTAB

Reduction

+ NaOHAging Particle growth

Ag+

Ag+

Ag+

Ag+ Au3+

Au3+

Ascorbic Acid

Seeding

Color Green

+

+

+

+

+

+

+

+

+

+

+

+

+

++

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

++

+

+

+

+

+

+

++

+

+

++

+

++

+

+

+

+

++

+

+

++

+

++

+

+

+

+

++

+

+

++

+

++

• In the “bottom up” approach, gold nanoparticles and gold nanocomposites (composite of gold and silica) have been chemically synthesized by reducing gold precusors. Various reduction methods have been reported. The major synthesis is as followsi) Reduction of gold precusors (e.g. hydrochloroauric acid HAuCl4) using appropriate reducing agents, such as citrate, sodium borohydride, ascorbic acid.• The citrate reduction of gold (III) ions has been widely used• While sodium citrate reduces (AuCl4)- ions in hot aqueous solution, it forms a colloid• The reported average particle size is about 20 nm

EFTEM image

Preparation of Spherical Nanopaerticle:Preparation of Spherical Nanopaerticle:

• In conjunction with citrate ions, amphiphilic surfactants have also been used that allowed particle size tunning upon varying the gold/stabilizer ratio

+ Micelles

CTAB

Reduction

+AgingParticle growth

Ag+Ag+

Ag+Ag+ Ag+

Ag+Ascorbic Acid

Seeding

Color Green

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+NaOH

• Both the citrate ions and the oxidation (e.g. acetone dicarboxylate) act as capping agents.

- reduced gold is not stable - sulfur or oxygen capping such as citrate anion stabilize the reduced gold by providing electrons to the reduced gold

ii)Two-phase synthesis of gold nanoparticles has been reported in which a phase-transfer agent is used to transfer [AuCl4]- ions from an aqueous phase to an organic phase (toluene) containing alkanethiol stabilizer• The Au(III) in organic phase is reduced by the addition of aqueous sodium borohydride.- Resulting Au clusters are then capped immediately by alkanethiols- This method (Brust-Schiffrin) produces monodisperse particles (approx 1.4 nm) in the diameter range 1.5-5.2 nm

(b) Surface Functionalization and Bioconjugation• gold nanoparticles have been widely used as immunostaining agents for labeling cell, tissue section, blots, etc.• In general, protein conjugated gold nanoparticles are mostly used as labeling probes• Although the actual mechanism of macromoleculs (e.g. protein) binding to gold particles is poorly understand, some of accepted mechanism are- Protein binding via electrostatic (ionic) interaction. Negatively charged gold nanoparticles can bind to positively charged protein domains via electrostatic interaction- Protein binding via hydrophobic interaction. Hydrophobic domains present in the protein structure can interact with the metal surface of the particle- Protein binding via chemical interaction. Protein molecules containing sulfohydryl (-S-H-) can chemically interact with the gold atoms. This is also called dative binding• Other biomolecules, such as protein A, antibodies, lectins, avidins (or streptavidins), etc. have also been conjugated to gold nanoparticles to be used as sensitive probes.

(c) Protein-A Gold Conjugate• Protein A-gold conjugates are generally prepared by adsorbing protein-A onto the gold surface• Following a similar method, many other immunoglobulin binding proteins can also be attached to gold nanoparticles (Protein A’s ability to bind immunoglobulins)- Protein A is a 40-60 kDa MSCRAMM surface protein originally found in the cell wall of the bacteria Staphylococcus aureus- It has found use in biochemical research because of its ability to bind immunoglobulins- It binds proteins from many of mammalian species, most notably IgG’s

- It binds with the Fc region of immunoglobulins through interaction with the heavy chain

• These probes have been used as “universal” probes for labeling cells, tissue sections and various blots• In a typical tissue labeling experiment, primary antibodies are specifically targeted to the tissue antigens - In the following step, protein A-gold conjugates bind to the antibodies - The advantage of this labeling technique is that the same protein A-gold conjugate can be used for various immunochemical procedures

(d) Antibody-Gold Conjugate • Antibody-gold conjugated probes are prepared by coating antibodies directly onto the gold nanoparticle surface.• These probes have been successfully used for the detection, localization and quantificaiton of antigens on the target specimens• This is powerful technique for detection of pathogens, intracellular foreign substances, monitoring cellular metabolic processes, etc.

(e) Lectin-Gold Conjugate• Lectin-coated gold nanoparticle probes have been used for the detection of sugar-binding receptors that are expressed on the cell membranes- Lectins are sugar-binding proteins which are highly specific for their sugar moieties- They typically play a role in biological recognition phenomena involving cells and proteins

• Lectin molecules have specific carbohydrate binding sites.• In this assay, a specific carbohydrate molecule is sandwiched between the lectin molecule and the cellular receptor• The objective of this assay is to localize glycoproteins (proteins that contain oligosaccharide chains), glycolipids (carbohydrate-attached lipids), etc. on cell surfaces

(f)Avidin (or streptavidin)-Gold Conjugate• Avidin-gold conjugated probes have been used to localize, detect and quantify biotin molecules

lectin

Gold lectin

Gold

lectin

Gold

lectin

Gold

Sugar bindingreceptor

Sugar

Surface Plasmon Resonance Scattering and Absorption of anti-EGFR Antibody

Conjugated Gold Nanoparticles in Cancer Diagnostics: Applications in Oral

Cancer

Ivan H. El-Sayed, Xiaohua Huang,

Mostafa A. El-Sayed

Nano Lett., 5 (5), 829 -834, 2005.

AbstractSPR scattering imaging or absorption spectroscopy generated from antibody conjugated gold nanoparticles can be useful in molecular biosensor techniques for the diagnosis and investigation of oral epithelial living cancer cells in vivo and in vitro.

Comparisons of cells without gold nanoparticles, with colloidal gold nanoparticles, With gold nanoparticles conjugated to monoclonal anti-epidermal growth factor receptor (anti-EGFR) antibodies

Colloidal gold nanoparticles are found in dispersed and aggregated forms and provide anatomic labeling information, but their uptake is nonspecific for malignant cells.

The anti-EGFR antibody conjugated nanoparticles specifically and homogeneously bind to the surface of the cancer type cells with 600% greater affinity than to the noncancerous cells.

IntroductionQuantum dots are widely used and studied due to their unique size-dependent fluorescence properties. (potential human toxicity and cytotoxicity of the semiconductor material) Alternative consideration: Colloidal gold nanoparticles

The ability of gold nanoparticles : resonantly scatter visible and near-infrared light upon the excitation of their surface plasmon oscillation. The scattering light intensity is extremely sensitive to the size and aggregation state of the particles.

They scatter light intensely, much brighter than chemical fluorophores. They do not photobleach and they can be easily detected in as low as 10-16 M concentration.

Introduction

The advantage of gold nanoparticles ease of preparation ready bioconjugation potential noncytotoxicity

Scattering images and the absorption spectra recorded from anti-EGFR antibody conjugated gold nanoparticles incubated with cancerous and noncancerous cells are very different and offer potential techniques for cancer diagnostics.

Method The preparation of Gold NPs : the citrate reduction of chloroauric acid.

The preparations of the anti-EGFR/gold conjugates :

Dilution of the gold NPs in 20 mM HEPES buffer (pH 7.4) to a final concentration with optical density of 0.8 at 529 nm. .

Adding of 40 uL anti-EGFR monoclonal antibodies (host mouse) to 960 uL of the same HEPES buffer to form 1 mL dilute solution.

Mixing of 10 mL of the gold solution with the dilute antibody solution for 20 min.

Adding of 0.5 mL of 1% poly(ethylene glycol) to the mixture to prevent aggregation

Centrifugation

Redispersion of the anti-EGFR/gold pellet in PBS buffer (pH = 7.4)

Method * Incubation of gold nanoparticlesThe cover slips were coated with collagen type I in advance for optimum cell growth.

One nonmalignant epithelial cell line HaCaT (human keratinocytes) two malignant epithelial cell lines HOC 313 clone 8 and HSC 3 (human oral squamous cell carcinoma) were cultured on glass cover slips in DMEM plus 5% FBS at 37 C under 5% CO2.

For the incubation of colloidal gold, nanoparticles (~ 0.3 nM) were added into the medium and the cells were grown for 48 h. The cells on the cover slips were rinsed with PBS buffer and fixed with 1.6% paraformaldehyde and sealed.

For the incubation of conjugated nanoparticles, the cells were grown on the cover slips for 48 h and then the cell monolayer was immersed into the conjugated nanoparticle solution for 40 min, rinsed with PBS buffer, fixed with paraformaldehyde, and sealed.

MethodThe light scattering images were taken using an inverted Olympus IX70 microscope. When the light beam direction is optimized, the center illumination light beam does not enter the light collection cone of the microscope objective, and only the scattered light of the side beam by the sample is collected.

Gold nanoparticles are introduced into cells by the endocytosis process during cell differentiation and proliferation processes.

Smaller nanoparticles cross the cytoplasmic membrane more easily, but their scattering light cross-section is smaller than larger nanoparticles. They also give more greenish scattered color which cannot be easily resolved from the scattered green light from the cellular organelles. Larger nanoparticles have higher scattering cross-section but have smaller labeling efficiency, possibly due to steric hindrance.

After experimental determination of the particle uptake efficiency, the cellular labeling efficiency, and the light scattering intensities of the nanoparticles, gold nanoparticles with the average sizes of 35 nm was selected.

Results

Figure 1. Light scattering images of HaCaT noncancerous cells, HOC cancerous cells , and HSC cancerous cells without gold nanoparticles. Dim greenish light : This green light is due to autofluorescence and scattered lightfrom the cell organelles in cell cytoplasm and membrane.

The weak greenish scattered light from the cells shows large difference in the sizes and shapes of the three different types of cells.

For the size :

HOC cancer cells

are almost four times

larger than HaCaT or HSC.

For the shape : HaCaT and HSC cells show almost homogeneous diamond shapes while HOC cells have other shapes for some cells.

Diamond shape

Diamond shape

The incorporated gold nanoparticles scatter strong yellowish light and make individual cells easily identifiable. The images show that the particles are inside the cells in the cytoplasm region but do not seem to adsorb strongly on the nuclei of the cells. In most HaCaT noncancerous cells the gold nanoparticles demonstrate a spotted pattern inside the cytoplasm, while the nanoparticles are homogeneously distributed in the cytoplasm of HOC and HSC cancerous cells.

The HSC specimens give the strongest scattering light due to the large amount of

accumulated gold nanoparticles.

Figure 2 Light scattering images of one noncancerous two cell cancerous cells after incubation with unconjugated colloidal gold nanoparticles.

• The NPs inside all cells have a major peak around 545 nm, characteristic of the surface plasmon absorption of the individual nanoparticles inside the cytoplasm of the cells that are red shifted by 16 nm compared to the colloid nanoparticle suspension at 529 nm. • This suggests that the nanoparticle surface has a different dielectric environment when present inside the cells. The broad long wavelength tails: the presence of aggregates, which is likely induced by the salts

548 nm 548 nm 548 nm

Absorption maximum around 548 nm

Figure 2. The absorption spectra of one noncancerous two cell cancerous cells after incubation with unconjugated colloidal gold nanoparticles.• Nanoparticles have an SPR absorption maximum around 548 nm, independent of the cell type.

SPR absorption : red : larger particle: aggregation blue: smaller particle

• The capping material could also be dissolved inside cells and thus leads to aggregation of the resulting metallic nanostructures. • In HSC cells, the aggregates have the absorption maximum around 715 nm. In HaCaT cells, the size of these large aggregates is smaller as concluded from the shorter wavelength surface plasmon absorption maximum. • The absorption of the aggregates inside HOC is not as resolved due to the shorter wavelength (679 nm), which is close to the absorption maximum of the surface plasmon absorption of the individual nanoparticles. • The different sizes of the aggregates inside different kind of cells may reflect the difference in the cell cytoplasm medium or differences in the intracellular processing of the nanoparticles by the cells. • The ability to resolve aggregates within cells by SPRA spectroscopy suggests that different capping agents could be utilized to monitor intracellular processes as aggregates are formed.

* Absorption maximum around 548 nm

548 nm

548 nm 548 nm

Figure 2. The absorption spectra : nanoparticles have an SPR absorption maximum around 548 nm, independent of the cell type.

695 nm 679 nm

715 nm

• The light scattering pattern of gold nanoparticles is significantly different when anti-EGFR antibodies were conjugated to gold nanoparticles before incubation with the cells. • The HaCaT noncancerous cells are poorly labeled by the nanoparticles and the cells could not be identified individually. The nanoparticles are also found on the HaCaT noncancerous cells due to part of the specific binding, but mostly due to the nonspecific interactions between the antibodies and the proteins on the cell surface, and thus the nanoparticles are randomly distributed.

• When the conjugates are incubated with HOC and HSC cancerous cells for the same amount of time, the nanoparticles are found on the surface of the cells, especially on the cytoplasm membranes for HSC cancer cells. - This contrast difference is due to the specific binding of overexpressed EGFR on the cancer cells with the anti-EGFR antibodies on the gold surface. The nonspecific interaction between the anti-EGFR antibodies and the collagen matrix also exists, which is shown as the reddish scattering light of the gold nanoparticles on the collagen background.

Figure 3. Light scattering images of cells after incubation with anti-EGFR antibody conjugated gold nanoparticles. The conjugated nanoparticles bind specifically with high concentrations to the surface of the cancer cells.

Figure 3. Absorption spectra of cells with anti-EGFR antibody conjugated gold nanoparticles.Conjugated nanoparticles did not show aggregation tendency (no long wavelength broad tail is observed).

• The nanoparticles bound to HOC and HSC cancer cells have similar absorption maxima at around 545 nm, which is 9 nm red shifted compared to the isolated anti-EGFR/Au solutions at 536 nm. • Red shift: the specific binding of the anti-EGFR antibodies on the gold surface to EGFR on the cell surface : the interparticle interaction resulting from the arrangement of the conjugates on the cell surface in two dimensions. • For HaCaT noncanerous cells, the particles with maximum at 545 nm are found to have a maximum absorption of 0.01.- The rest of the nanoparticles have their maximum at 552 nm. This red shift indicates that these nanoparticles are nonspecifically bound. • The maximum absorbance of the conjugated particles to cancer cells is 0.06 for HOC cells and 0.07 for HSC cells. Binding ability of the anti-EGFR antibody conjugated nanoparticles to HOC and HSC cancerous cells is 600% and 700%, respectively, over the HaCaT noncancerous cells.

552nm

545 nm

543 nm 545 nm

0.01

0.06

0.08

ConclusionsThere is a distinct difference in the distribution of anti-epidermal growth factor receptor antibody conjugated nanoparticles when incubated with cancerous and noncancerous cells.

Conjugated nanoparticles bind homogeneously and specifically to the surface of the cancer cells with an absorption maximum at 545 nm.

Both SPR scattering imaging and SPR absorption spectroscopy from anti-EGFR antibodies conjugated gold nanoparticles are found to distinguish between cancerous and noncancerous cells. This makes either technique potentially useful in cancer diagnostics.

2.4.2.3 Dye-doped Silica Nanoparticles• Amorphous silica nanopartcles that are produced via stober’s sol-gel or microemulsion technique• found in application in bioimaging• Unlike Qdots or gold nanoparticles, silica does not have inherent strong fluorescence that can be exploited for sensitive imaging applications• However, silica nanoparticles can be made fluorescence by incorporating fluorescent dye molecules inside the silica matrix (dye-doping)• Another approach could be attaching fluorescent dye molecules (via covalent binding) on the silica surface• For bioimaing applications, it is preferable that dye molecules remain encapsulated by the silica matrix for the following reasons- Silica-based nanoparticles exhibit several attractive features, e.g. silica is water dispersible and is resistant to microbial attack- The size of silica particles remains unchanged by changing solvent polarity (i.e., resistant to swelling) and therefore, silica porosity remains unaltered in a wide selection of solvents, including aqueous-based neutral and acidic solutions.- A silica matrix is optically transparent, allowing excitation and emission light to pass through efficiently- Fluorescent dyes can be effectively entrapped inside the silica particles.- spectral characteristics of dye molecules remain almost intact- silica encapsulation provides a protective layer around dye molecules, reducing oxygen molecule penetration (which causes photodegradation of dye molecules) both in air and in aqueous medium. As a result, photostability of dye molecules increases substantially compared with bare dyes in solution

• surface of a silica nanoparticle can be easily modified to attach biomolecules such as protein… using conventional silane based chemistry-For exapmple, carboxylated silica nanoparticle can be covalently attached to the amine groups of proteins, antibodies etc. through the formation of stable amide bond

Silicon oxide surface

APTES Oligoglutaraldehyde

NH2

(CH2)3

Si

O O O

NH2

(CH2)3

Si

O O O

NH

(CH2)3

Si

O O O

Oligoglutaraldehyde

C o

Anti-E.coli O157:H7 antibody ( -NH2 )

Amide bond

• general synthesis strategy of fluorescent silica nanoparticles is the incorporation of organic of organic or metalloorganic dye molecules inside the silica matrix-Metalloorganic dye, tri(2,2’-bipyridyl)dichlororuthenium (II) (Rubpy), has been entrapped inside silica nanoparticles using a reverse microemulsion-based synthesis approach where positively charged Rubpy moleculecs were electrostatically bound to the negatively charged silica matrix

(a) Synthesis• Sol-gel, reverse microemulsioni) Stober’s method• In a typical stober’s method, Alkoxysilane compounds, tetraethylorthosilicate (TEOS) , tetramethylorthosilicate (TMOS), or various TEOS or TMOS derivatives, etc undergo base-catalyzed hydrolysis and condensation in an ammonia-ethanol-water mixture• Following Stober’s protocol with a slight modification, fairly monodisperse organic dye doped fluorescent silica nanoparticles have been synthesized- Since organic dyes are normally hydrophobic, doping them inside the hydrophilic silica matix is not straightforward.- typically, a reactive derivative of organic dye(e.g. amine-reactive fluorescein isothiocyannate FITC, carboxyl group) is first reacted with an amine-containing silane compound (APTS)- Then FITC conjugated APTS and TEOS are allowed ot hydrolyze and condense to form FITC conjugated silica nanoparticle- Note that particles so formed will have some amount of bare dye molecules on the particle surface that is covalently attached- These bare dyes, due to their hydrophobic nature, will somewhat compromise the overall particle aqueous dispersibility and also they will be prone to photobleaching- therefore, an additional coating with pure silica is usually applied around the dye-conjugated silica nanoparticle

Si

OR

NH2

(CH2)3 N C (CH2)3 COH

OR

Si N

H

H

O

O

(a)

(b)

N

H

H

R= Avidin or biotinylated oligo

R

(CH2)3 N C (CH2)3 C N R

H

(c)

H H

Glutaraldehyde

APTESR= -CH2CH3

Sili

con

waf

er

Si xN

y

Si xN

yOz

Sili

con

waf

er

Si xN

y

Si xN

yOz

Sili

con

waf

er

Si xN

y

Si xN

yOz

Sili

con

waf

er

Si xN

y

Si xN

yOz

Oligonucleotide immobilization on plasma-cleaned silicon nitride surface Oligonucleotide immobilization on plasma-cleaned silicon nitride surface

Surface treatment of silicon chips with APTES-glutaraldehyde Surface treatment of silicon chips with APTES-glutaraldehyde

c. Reaction between the amine group on the

protein and the aldehyde group on the

surface attached are

c. Reaction between the amine group on the

protein and the aldehyde group on the

surface attached are

a . APTES reacts with silica leaving a primary

amine group on the surface

a . APTES reacts with silica leaving a primary

amine group on the surface

b. Glutaraldehyde treatment yields an

aldehyde that can form an imine linkage

with the primary amines on the protein

b. Glutaraldehyde treatment yields an

aldehyde that can form an imine linkage

with the primary amines on the protein

Si

OR

OR

Si

(a)

(b)

N

H

H

R= Avidin or biotinylated oligo

R

R

(c)

GOPSR= -CH3

Sili

con

waf

er

Si xN

y

Si xN

yOz

Sili

con

waf

er

Si xN

y

Si xN

yOz

Sili

con

waf

er

Si xN

y

Si xN

yOz

Sili

con

waf

er

Si xN

y

Si xN

yOz

Sili

con

waf

er

Si xN

y

Si xN

yOz

Surface treatment of silicon chips with GOPS-CDISurface treatment of silicon chips with GOPS-CDI

a) Treatment with GOPS forms an epoxy layera) Treatment with GOPS forms an epoxy layer

Residual imidazole carbamate groups can be

hydrolyzed to hydroxyl groups with water or water

solutions at higher pH value.

Residual imidazole carbamate groups can be

hydrolyzed to hydroxyl groups with water or water

solutions at higher pH value.

c) Imidazole carbamate groups react easily with amine

containing ligand (such as avidin or biotin) forming a

stable carbamate linkage.

c) Imidazole carbamate groups react easily with amine

containing ligand (such as avidin or biotin) forming a

stable carbamate linkage.

b) Reaction between hydroxyl groups of epoxy and 1,

1’

carbonyldiimidazole (CDI) results in formation of

reactive imidazole carbamate groups

b) Reaction between hydroxyl groups of epoxy and 1,

1’

carbonyldiimidazole (CDI) results in formation of

reactive imidazole carbamate groups

(b) Surface Functionalization and Bioconjugation• This surface modification involves a few steps.- Firstly, the particle surface should be modified to obtain appropriate functional groups such as amines, carboxyls, thiols, etc.- secondly, using suitable coupling reagents, nanoparticles are attached to the bio-recognition molecules (such as antibodies, folate,..)- Lastly, bioconjugated particles are targeted to cancers- Note that all these steps are usually carried out in aqueous-based solutions- A few bioconjugation methods are briefly mentioned belowi) Bioconjugation with carboxylated particles.- The surface of the nanoparticle is modified to obtain carboxyl groups (-COOH) by using a carboxylated silane reagent- Biomolecules such as proteins, antibodies, etc. containing free amine functional groups are then covalently attached to the carboxyl functionalized nanoparticle, using carbodiimide-coupling chemistyii) Bioconjugation with aminated particles: Many cancer cells overexpress folate receptors.- Cancer targeting with folate-conjugated nanoparticle has been recently reported- Folates are chemically attached to aminated silica nanoparticles using carbodiimide chemistry- APTES treatment: aminated silica nanoparticle can be achieved

NH2

(CH2)3

Si

O O O

APTES

Folic acid

Silica nanoparticleAttached with folic acid

iii) Bioconjugation with avidin-biotin binding• Avidin-coated nanoparticles are typically attached to biotinylated molecules such as antibodies, proteins, etciv) Bioconjugation through disulfide bonding: • Sulfohydryl-modified nanoparticles are conjugated to disulfide-linked oligonucleotide• in this method, oligonucleotides are attached to nanoparticles through di-sulfide bond formationV) Bioconjugation using cyanogen bromide chemistry• nanoparticles with hydroxyl groups (such as silica) can be activated with cyanogen bromide to form a reactive –OCN derivative of the nanoparticles. • The OCN derivative then readily reacts with proteins (via amine groups), forming a “zero-length” bioconjugate as there is no spacer between the particle surface and the protein molecule

2.5 Optical imaging of Cancer with Nanoparticles• These ultra-sensitive and specific probes (nanoparticles) provide a viable alternative to rapidly and non-invasively image the uptake, distribution and binding of nanoparticles to tumors• To establish the widespread use, it is important to understand the delivery, interaction and recognition mechanism of these contrast agents with cancer cells• Various delivery vehicles with varying specificity have been used to target cancer tissues, mainly for drug delivery applications, some of which are folates, antibodies, lectins, growth factors, cytokines, hormones and low-density lipoproteins• most of these carriers can be similarly used for molecular imaging applications• These can be broadly classified as active and passive targeting

2.5.1 Active Targeting• this refers to the conjugation of targeting ligands to nanoparticles to provide preferential accumulation into the tumor antigents and blood vessels with high affinity and specificity• This relies on specific interactive forces between lectins-carbohydrate, ligand-receptors and antibody-antigens• Lectins can recognize and bind to glycopreteins that occur on the surface of cells. - These proteins can bind to certain carbohydrates in a specific manner. - Direct and reverse lectin targeting have made used of this specific interactions to receptors or antigens expressed by the plasma membrane•

• Folate receptor-based interactions are an excellent example of ligand-receptor based active targeting.- Folate receptors are overexpressed on the surface of various cancers like those of the brain, ovary, kidney, breast and lungs- Confocal microscopic studies have demonstrated the selective intake and receptor-medicated endocytosis of folate-conjugated nanoparticles by tumor cells

• Antibody-mediated tumor targeting has been performed for detecting the presence of antigenic moieties on the surface of cancer cells- Tumor targeting ligands like monoclonal antibodies are attached to nanoparticles to target the specific receptors.- These moieties are minimally present on the surface of normal tissues- Only certain antigens are actually tumor-specific and are referred to as tumor-specific antigens