**4. Microfluidic biosensing system based on NSL and microfluidic reactor fabrication**

Recently, Song has developed a high-throughput single Ag NPs biosensing device by coupling a variety of functionalized Ag NPs fabricated by NSL into a series of microfluidic channels (Song 2009). The designed microfluidic biosensing system based on Ag single nanoparticles and nanoparticle arrays is illustrated in Figure 11. Samples were fabricated by the combination of NSL and the traditional UV-LIGA process for the microfluidic reactor fabrication (Song 2009; Song 2010; Song and Elsayed-Ali 2010). In this biosensing system, the corresponding microfluidic channels are fabricated on the designed patterns where series of single Ag NPs or arrays (Figure 11: a) have been fabricated by careful alignment. The glass cover is then connected with glass optical fiber binding on the top of the microfluidic channels after careful alignment with the desired single Ag NPs or nanoarrays. In order to alleviate the non-specific absorption in the microfluidic channels, the channels are modified by polyvinylacholol (PVA) or polyethylene glycol (PEG) solution. After that, the single Ag NPs will be surface modified by a mixture of at least two thiol compounds with one having carboxyl group or amine group as the conjugating compound (e.g. 11-mercaptoundecanoic acid: MUA), and another thiol compound without carboxyl group or amine group as spacer (e.g. 6 mercapto-1-hexanol: 6-MCH, 1-octanethiol: 1-OT). The modification reaction is shown in equation (1). The Ag NPs can then be functionalized with biomolecules, as reporter (e.g. IgG), by a conventional 1-ethyl-3-(3- dimethylaminopropyl)-carbodiimide (EDC) coupling process to form the f, as shown in equation (2) and (3) for the functionalization of Ag NPs (Figure 11: a and b).

The number per Ag NPs can be controlled by the ratio of the conjugation compounds and spacers, which can be used to calculate the number of the responding biomolecules (e.g. Protein A) that can bind with the reporters, which can be directly sensed by the LSPR peak shift. As shown in Figure 11, the solution having a specific concentration of the corresponding detected biomolecules can be delivered into the microfluidic channels (Figure 11: g). The channel widths are designed from several hundreds micro meter to ten micrometers that will play a role like a dark-field condenser for incident white light. The scattering color changes and the LSPR spectrum variations of Ag NPs (a) caused by the binding of the detected biomolecules on the reporters, (b) will be collected in the opened windows, (d) and transported into the detector and analyzer, (f) by the glass fiber, (e) after signal magnification.

Controlled Fabrication of Noble Metal Nanomaterials

functionalization process.

via Nanosphere Lithography and Their Optical Properties 521

Song has investigated the efficiency of this kind of biosensing system. Using the color change and LSPR spectra shifts based on the binding of one model biomolecule pairs (antibody: IgG is firstly functionalized on the Ag surfaces by EDC process, then the antigen: protein-A buffer solution is pumped into the microfluidic channels) as model, it can be seen that the spectra shift and the color changes from the binding of the model biomolecule pairs depend on the concentration of biomolecules and the running time. Up to now, the detection resolution of this kind of biosensors based on the scattering from single Ag NPs has reached 2 nm peak shift per 1 nM concentration change and the resolution for one single NP biosensor is calculated as 10-20 biomolecules per Ag NP(Song 2009). This result suggests another persepctive application by the combination of NSL, microfluidics and bio-

**5. Fabrication of hierarchically ordered nanowire arrays on substrates by** 

In some applications of nanomaterials, the NPs need to be arranged in some particular patterns, architectures or motifs with controlled interspacing, or conjugated with some other kinds of materials (e.g. polymers) (Chong, Zheng et al. 2006; Song , Zhang et al. 2011). The controlled arrangement and immobilization of Ag NPs on substrates will be very crucial to enable some fascinating and delicate applications, particularly in electronic circuit based electro-optical devices and long term functional composites for biological applications. Many methods have been explored for this purpose. Among them, template-assisted LIGA or structure controlled artificial fabrication methods (e.g. E-beam LIGA, NSL, PAA-LIGA) may be the most convenient techniques(Song , Zhang et al. 2011). In the NSL development, the suitability and powerful ability in the architecture and interspacing controlled fabrication of NPs and nanoarrays can be expanded extremely if the NSL can be combined with other template-assisted LIGA methods. Here we just show one example to fabricate hierarchically ordered nanowire arrays on substrates by the combination of NSL and porous

Like NSL, porous anodic alumina (PAA) templates have attracted intense attention in nanodevice-oriented fabrication in recent years(Xu, Meng et al. 2009). As a welldeveloped template, PAA offers amazing simplicity and convenience for nanofabrication due to the capabilities of forming high-density, well-aligned, and hexagonally packed sub-100-nm pores, the ability to control the 3D pore structures by simply varying the anodization conditions, and the ease of selectively removing the template after fabrication(Chong, Zheng et al. 2006). As shown in Figure 12, one typical PAA-LIGA process includes(Lombardi, Cavallotti et al. 2007): (a) formation of a 300 nm thick PAA film on Al by a two step anodization process in 0.3M oxalic acid; (b) dissolution of unoxidized Al; (c) barrier layer etching in 5wt% phosphoric acid; (d) transfer of the PAA mask onto Au-coated Si followed by a thermal treatment to improve the adhesion of the films to the substrate; (e) Ag electrodeposition through the PAA pores; (f) PAA mask removal. By carefully controling the sizes and interpore spacing of the nanoholes, very uniform Ag nanorods with controlled interspacing can be fabricated by electroplating. The typical Ag nanorods prepared by this template assisted electrodeposition process can give a much uniform size and interparticle spacing distribution, with a standard size

**combination of NSL and Porous anodic alumina (PAA)** 

anodic alumina (PAA) LIGA(Chong, Zheng et al. 2006).

deviation less than 5% and a spacing deviation less than 7%.

Fig. 11. a: nanoparticles (e.g. triangular Ag NPs); b: biomolecule (e.g. antibody); c: induced light (e.g. white light from tungsten lamp); d-1 - d-n):Series of detecting windows connecting with optical microfibers in different microchannels;e-1 – e-n:glass microfibers for signal transport;f:Optical spectroscopy (e.g. micro optical fiber spectroscopy-S2000, Spectropro-150, surface enhanced Raman spectroscopy; g: the sealed microchannels). (Adapted from reference Y. Song, China Patent, Appl. No. CN200910085973.9)

HO

HO

HO

HO HO

HO

<sup>N</sup> <sup>+</sup> NH Cl-

HN<sup>+</sup> Cl-

HS OH HS COOH

OOC COO

HO HO

HO

PBS»º ³å Òº /PVA

Ag nanorpisms; IgG; HS COOH : e.g. MUA;

H <sup>N</sup> <sup>C</sup>

HN Cl <sup>+</sup> -

HO HO OH

HOOC COOH

HO HO OH

OH OH

OH OH

OC OC

d-n-1 d-n

e-n

g

e-n-1

c

HOHO OH

NH NH

OH OH

C NH N

HOOC

OOC COO

H <sup>N</sup> <sup>C</sup>

HN Cl <sup>+</sup> -

NH Cl- <sup>C</sup> NH

HOHO OH

N

HO HO

HO

HO HO

HS OH: e.g.:1-OT

c

e-1

a

b

g d-1 d-2

g g

e-2

HO

N <sup>+</sup>

OH OH

OH OH

+

HN C N

H2N

…

……

a aa

b b b

+ s-NHS

EDC

HO HO OH

+

(1)

(2)

(3)

f

Fig. 11. a: nanoparticles (e.g. triangular Ag NPs); b: biomolecule (e.g. antibody); c: induced light (e.g. white light from tungsten lamp); d-1 - d-n):Series of detecting windows

hν hν

connecting with optical microfibers in different microchannels;e-1 – e-n:glass microfibers for signal transport;f:Optical spectroscopy (e.g. micro optical fiber spectroscopy-S2000, Spectropro-150, surface enhanced Raman spectroscopy; g: the sealed microchannels). (Adapted from reference Y. Song, China Patent, Appl. No. CN200910085973.9)

Song has investigated the efficiency of this kind of biosensing system. Using the color change and LSPR spectra shifts based on the binding of one model biomolecule pairs (antibody: IgG is firstly functionalized on the Ag surfaces by EDC process, then the antigen: protein-A buffer solution is pumped into the microfluidic channels) as model, it can be seen that the spectra shift and the color changes from the binding of the model biomolecule pairs depend on the concentration of biomolecules and the running time. Up to now, the detection resolution of this kind of biosensors based on the scattering from single Ag NPs has reached 2 nm peak shift per 1 nM concentration change and the resolution for one single NP biosensor is calculated as 10-20 biomolecules per Ag NP(Song 2009). This result suggests another persepctive application by the combination of NSL, microfluidics and biofunctionalization process.
