**1. Introduction**

Biosensing is a scientific and technological hot topic, given its potential in the field of medical diagnosis [1], healthcare [2], environment [3], defense [4], and food security [5].

Nowadays, the healthcare and pharmaceutical sectors continuously demand more powerful analytical and diagnostic tools for the identification of disease, the development of new medicines, and better diagnostic tests. In these fields, the specific and sensitive detection of targets in short-time analyses plays a key role. While a number of commercial bioassay kits are already on the market, there still remains a major challenge to develop novel biodetec‐ tion methods to meet the ever-increasing request. High-capability optical biosensing sys‐ tems are actually emerging as a way to reach this aim [6].

Optical biosensing is a powerful analytical tool used to detect optical changes upon the interaction between an analyte of interest and its ligand, previously immobilized on the sensing device as biological probe; the intensity of obtained signal can be measured, and its value is a function of the analyte concentration in the sample [7, 8]. Generally, optical biosensors use two different detection protocols: direct detection of the analyte (label-free detection) or indirect detection through optically labeled probes (label-based detection). In label-based detection, fluorescent, enzymatic, or radioactive tags, linked on target or on probe molecules, are used; the intensity of the signal indicates the presence of analyte and the interaction with the recognition molecule. This type of detection is extremely sensitive, since itis possible to detect up a single molecule [9, 10]; nevertheless, complex procedures are needed for labeling, and they may interfere with the functionality of the target molecule. In contrast, in label-free detection, target molecules are not labeled or altered and are free to interact in their natural forms: Recent progresses in this field are showing promising results [10]. This strategy is relatively easy and cheap to perform and allows for quantitative and kinetic measurement of molecular interactions. These advantages, with low detection limit, nondestructive approach, and with the ability to recognize a wide variety of analytes or multi‐ ple analytes at the same time with fast signal monitoring and analysis [11], make label-free optical detection one of the leading recognition methods in the biosensor field.

Label-free biosensors are very versatile platforms, since they can be developed in different materials, such as silicon or its compounds, glasses, metals, or polymers, and they offer different detection modes and configurations that can be combined [12]. In perspective, optical label-free biosensors are expected to replace fluorescent biosensors in DNA micro-arrays and lab-on-a-chip (LOC) applications [13–15].

The use of silicon-based technology—the same developed for microelectronics—could allow the integration of microfluidic circuits for analyte handling, sensing elements, and control/ reading electronics into a single chip. This could pave the way to the mass production of micro total analysis systems and LOCs capable to provide rapid, sensitive, and multiplexed measurements in any place at any time.

The selection of the biological element to immobilize on the sensor surface is dictated by the application and must be chosen to be highly specific for the target molecule and stable enough

to be immobilized without losing functionality. Several types of routes can be used to biofunctionalize the sensor surface [16], and the choice of an appropriate immobilization procedure has become a key factor in the biosensor area. An ideal immobilization procedure should guarantee an efficient coverage by the molecular probes of the sensing layer without interfering with their properties (structure, biological activity, affinity, specificity). In addi‐ tion, the possibility to preserve their stability in normal storage conditions and also for regeneration could be useful in the case of integration in portable LOC. Despite enormous research efforts have been made to find novel strategies according to the application, a universally valid procedure has not yet been developed and the realization of cheap hand‐ held platforms is almost limited. For this reason, the choice of the most effective strategy of immobilization represents the critical step that turns a sensing device into an applicable analytical tool with the required quality standards. Currently, a lot of biomolecules can be used as bioreceptors (antibodies, nucleic acid, peptides, enzymes, cell receptors, and many others). Among these, artificial peptides provide an opportunity to develop the desired molecular biosensor due to their desirable properties such as diversified structure, high affinity to ligands, matured synthesis protocol, and modified approach [17].

### **2. Biosensing application in cancer**

**1. Introduction**

180 Lab-on-a-Chip Fabrication and Application

Biosensing is a scientific and technological hot topic, given its potential in the field of medical

Nowadays, the healthcare and pharmaceutical sectors continuously demand more powerful analytical and diagnostic tools for the identification of disease, the development of new medicines, and better diagnostic tests. In these fields, the specific and sensitive detection of targets in short-time analyses plays a key role. While a number of commercial bioassay kits are already on the market, there still remains a major challenge to develop novel biodetec‐ tion methods to meet the ever-increasing request. High-capability optical biosensing sys‐

Optical biosensing is a powerful analytical tool used to detect optical changes upon the interaction between an analyte of interest and its ligand, previously immobilized on the sensing device as biological probe; the intensity of obtained signal can be measured, and its value is a function of the analyte concentration in the sample [7, 8]. Generally, optical biosensors use two different detection protocols: direct detection of the analyte (label-free detection) or indirect detection through optically labeled probes (label-based detection). In label-based detection, fluorescent, enzymatic, or radioactive tags, linked on target or on probe molecules, are used; the intensity of the signal indicates the presence of analyte and the interaction with the recognition molecule. This type of detection is extremely sensitive, since itis possible to detect up a single molecule [9, 10]; nevertheless, complex procedures are needed for labeling, and they may interfere with the functionality of the target molecule. In contrast, in label-free detection, target molecules are not labeled or altered and are free to interact in their natural forms: Recent progresses in this field are showing promising results [10]. This strategy is relatively easy and cheap to perform and allows for quantitative and kinetic measurement of molecular interactions. These advantages, with low detection limit, nondestructive approach, and with the ability to recognize a wide variety of analytes or multi‐ ple analytes at the same time with fast signal monitoring and analysis [11], make label-free

diagnosis [1], healthcare [2], environment [3], defense [4], and food security [5].

optical detection one of the leading recognition methods in the biosensor field.

lab-on-a-chip (LOC) applications [13–15].

measurements in any place at any time.

Label-free biosensors are very versatile platforms, since they can be developed in different materials, such as silicon or its compounds, glasses, metals, or polymers, and they offer different detection modes and configurations that can be combined [12]. In perspective, optical label-free biosensors are expected to replace fluorescent biosensors in DNA micro-arrays and

The use of silicon-based technology—the same developed for microelectronics—could allow the integration of microfluidic circuits for analyte handling, sensing elements, and control/ reading electronics into a single chip. This could pave the way to the mass production of micro total analysis systems and LOCs capable to provide rapid, sensitive, and multiplexed

The selection of the biological element to immobilize on the sensor surface is dictated by the application and must be chosen to be highly specific for the target molecule and stable enough

tems are actually emerging as a way to reach this aim [6].

Cancer is one of the main causes of death worldwide. Early diagnosis is the key to enhance the success of medical treatment. In the last few years, in parallel with a growing interest in detecting cancer cells, a wide variety of techniques were developed for detection at the cellular level [18–21]; nevertheless, most of these modalities are expensive and time-consuming, and they are often associated to risks deriving from radioactive tracers.

At this point, despite some considerable achievements, the realization of simple, rapid, nondestructive, and low cost methods for early detection of cancer and minimal residual disease, important for diagnosis and reduction in mortality for certain cancers, still remains an unfulfilled goal [22, 23]. To meet these specific requirements, biosensors have attracted increasing attention since biosensing technology, taking advantage of the properties of biological systems combined to functional advanced materials, is providing rapid, reprodu‐ cible, and highly sensitive cell detection.

Among the various types of cancer, lymphoma is the most common blood cancer, which incidence is recently increasing. This malignancy is a clonal expansion of neoplastic cells that may result in fatal outcomes [24]. Despite the great progress that has been made over the last several decades in the treatment of lymphoma, the prognosis for patients with particular subtypes of disease remains quite poor. Lymphomas are a heterogeneous group of tumors arising in the reticuloendothelial and lymphatic systems. The major types are Hodgkin lymphoma and non-Hodgkin lymphoma. Great progresses in the use of monoclonal antibodies, chemo‐ therapy, radioimmunotherapy, and peripheral blood stem cell transplants have achieved significant responses in the treatment of these diseases and also markedly improved the outcome of the cure among elected sub-populations of patients, since not all the patients or

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‐ tered and the approaches we adopted for their overcoming.
