**4. Chemical functionalization procedures**

subtypes of lymphoma are responsive to these "conventional" approaches. For example, tumorigenic B-cell lymphomas are sensitive to anticancer treatments, including convention‐ al chemotherapy, radiation therapy, and corticosteroids [25]. Nevertheless, the disease is associated with incomplete response to clinical treatments that result in a minimal residual disease in which a few neoplastic cells undetected *in vivo* replenish the cancer cell reservoir. This grim scenario calls for novel strategies to detect tumorigenic B cells. Random peptide libraries (RPLs) allow the selection of therapeutic peptides for tumor cell-surface receptors. Idiotypic determinants of the Ig-BCR, expressed by lymphoma cells, function as a specific tumor antigen that may be exploited for cell-specific detection or targeted therapy. Here, we present the complex different strategies that we adopted in order to construct a biosensor for the detection of tumorigenic lymphoma B cells and discuss all the difficulties that we encoun‐

Our vision was to create a highly sensitive, label-free optical biosensing system forthe targeting of patient-specific neoplastic B cells during the minimal residual disease. The material used to develop this unique toolfor biosensing include mainly silicon, since it possesses great potential because of its many unique properties, including biocompatibility, which is an important precondition for biological and biomedical applications, abundance, tailorable surface chemistry, and unique electronic, optical, photonic, and mechanical properties, among others. In addition, high surface-to-volume ratio of silicon derivatives offers exciting opportunities to design high-performance silicon-based functional devices for biomedical applications. Moreover, silicon is very abundant on earth allowing inexpensive resources forlarge-scale and low-cost preparation for practical applications. Taking advantage of these attractive features, the interest in the use of silicon is widely grown leading to its applications not only in biology

In our research activity, flat silicon devices of fixed thickness (400 μm) were obtained from highly doped p+ type, 0.003 Ω cm resistivity, (100)-oriented silicon wafers, cut into 10 × 10 mm square pieces. The wafers were cleaned by a standardRCA process [27] and thermally oxidized at 1050°C for 5 h. An electrochemical etching process of planar silicon was used to pattern

silicon is that its morphology can be tuned by modification of process parameters [28] so that the resulting structures can be adapted to obtain the best performance for chemical and biological processes that happen on their surface. Moreover, the porosity of the material coupled to the low-cost production makes porous silicon an ideal bulky model system to follow each functionalization step: The concentration of reagents and molecules in the pores allow to quantify few nanometers thick films of passivating agents, exploiting signal enhancements,

/cm3

). An advantage in the use of porous

tered and the approaches we adopted for their overcoming.

**3. The choice of appropriate support for biosensing**

but also in a lot of other fields [26].

182 Lab-on-a-Chip Fabrication and Application

porous silicon with a high specific surface (up to 500 m2

that cannot easily measured on flat supports.

The selection of an appropriate procedure for the immobilization of a biological element on the sensor surface that interacts with the desired target forthe specific recognition of an analyte has become a critical step in the biosensor area, and enormous efforts are continuously invested in order to optimize novel strategies according to the application. The biofunctionalization of chemical modified surfaces can be achieved in several manners that can be grouped in just two approaches: (i) direct adsorption and (ii) physical adsorption [29–33]: in both cases, each immobilization route presents advantages and drawbacks.

In the direct adsorption method, there is no bond formation between probe and device, reagents are not required so either structure or functionality of biomolecules is not affected. Nevertheless, the efficiency of this strategy is very low.

The bioreceptor physisorption is a quick and widely used approach to immobilize biomole‐ cules on chip surfaces based on electrostatic, hydrophobic, and covalent interactions. Despite the efficiency and the simplicity, electrostatic, and hydrophobic approaches are direct fast methods, since no linker molecules are needed, but are limited to situations that require no directional orientation of the bioprobes. Moreover, both techniques request long incubation times and the risk of folding and desorption due to changes of parameters, such as pH, ionic strength, or temperature, cannot be excluded. The covalent attachment of probe is more efficient in terms of stability and binding strength. Generally, the binding occurs between a functional chemical group of the biomolecules, whose blocking does not affect the function‐ ality, and one on the modified surface. For proteins covalent coupling, amino, carboxylic, or thiol groups are preferred, whereas in the case of nucleic acids, it is possible to take advant‐ age of the versatility of their synthesis to insert reactive groups at the end of the sequence. More difficult is the immobilization of immunoglobulins in a correct orientation, which can be achieved by controlled linkage of carbohydrates groups in the constant region or using affinity proteins (such as A or G Protein) [31]. In all physical adsorption types, a chemical modification of the platform surface is required to the extent that the material properties are tuned to accomplish the best analytical characteristics.

The drawing up of an efficient and correct immobilization procedure is a crucial point to avoid a wide variety of factors that may negatively affect the biosensor functionality. The orienta‐ tion of probe, the density of coating on the detection surface, pH, target concentration, operating conditions, and chemical environment provided by transducer must be closely explored. An efficient biofunctionalization process should take in account few important observances: The preservation of the molecular probe structure to guarantee subsequent binding of analyte; limited chemical steps and minimal consumption of reagents and sam‐ ples to make the whole procedure lean and easily reproducible; low optical adsorption at the working wavelengths; homogeneously thin layer formation compatible with evanescent field sensing; uniform surface coating; saturation of eventually free binding sites to reduce the possibility of false-positive signals; biocompatible conditions; integrability with large-scale fabrication. The exploration of these traits offers the possibility to improve biosensors features increasing the power of detection.

**Figure 1.** Types of silicon surface chemical modifications for biosensors: (a) organosilane-based, (b) phosphonatebased, and (c) glutaraldehyde-based strategies.

**Figure 1** represents the main functionalization approaches employed to construct integrated optics (IO) biosensors. Before the biofunctionalization step, a previous chemical activation of the sensor surface is always needed. To this aim, our group employed the self-assembly of organofunctional alkoxysilanes (**Figure 1a**), an easy and versatile system for organic conju‐ gating [34]. However, silicon-based surfaces require a prior activation step to oxidize the surface and to expose the silanol groups for cross-linking with the silane. The formation of a thin silane self-assembled film allows applying a great number of chemical reactions. Immediately before silanization, surfaces are cleaned with oxidant media to remove organic pollutants and to increase the hydroxyl moieties on the surface [35]. The used oxidant is piranha solution [36–39], consisting of a concentrated sulfuric acid mixed with hydrogen peroxide at 3:1 ratio. This treatment is performed by heating for 30 min only. Hundreds of different organosilanes with different structures and functionalities are nowadays commer‐ cially available, although the most commonly employed are those with short alkyl chain that present an amino, thiol, epoxy, or carboxylic group at the terminus. Among this vast variety of compounds, 3-aminopropyltriethoxysilane (APTES) was chosen for its reactivity to aldehyde, carboxylic acid, and epoxy functionalities.

The reaction between the oxidized surface and the organosilane is based on the condensa‐ tion between the Si–O–Si of the silane and the OH present on the device; generally, besides the hydroxyl groups already present on the native silicon oxide layer, a thermal oxidation is a common procedure to form a new efficient oxide film [40–42] in order to assure a plenty of silanol groups for an efficient coverage of the organic layer.

Furthermore, after silanization, APTES layer was cured at high temperature [43]. The aminosilane is more reactive, and it can be applied on a surface using pure organic solvent. The advantage of the curing is that a more controlled deposition of the compound can be obtained to create a thinner film of the aminopropyl groups on the chip. The self-assembled monolayers generally present a thickness in the range of 1–3 nm and create a nanometer-scale organic thin coat [44, 45].
