Microfluidic for human health: a versatile tool for new progress in cancer diagnosis and contaminants detection

In the last ten years, interest in manipulating droplets in microchannels has emerged from two important motivations. The first arise from the desire to produce well controlled droplets for material science applications, for example in the pharmaceutical or food industries. In this context, microfluidics allows for producing such droplets in a controlled and reproducible manner, also allowing complex combinations to be designed and explored. A second motivation originates with the advent of the -omics era, which has a much need for being able to carry out experiments at the smallest possible scale (if possible single cells or molecules), on a massively parallel platform and with high throughput. In this case, droplets are viewed as micro-reactor in which samples are confined, and which offer a way to manipulate small volumes. Droplet microfluidics is the most powerful microfluidic technology used to produce and manipulate monodisperse droplets. This technique addresses the need for lower costs, shorter times, and higher sensitivities to compartmentalize reactions into picolitre volume, instead of the microlitre volumes commonly used with standard methods. Droplets can provide a well-defined environment into which individual cells can be compartmentalized in a controlled way. This coupled with the advantages of droplet microfluidics has allowed the development of several methods for single-cells analysis. In this work a microfluidics label-free approach for circulating tumor cells (CTCs) detection is presented. In the last decades, CTCs have received enormous attention as a new biomarkers for cancer study, for this reason their capture and retain represents a major challenge in cancer research. Many issues regarding the detection and characterization of CTCs are owing to their extremely rarity (one CTCs for 5 x 10^9 erythrocytes/mL and for 7 x 10^6 leucocytes /mL) and their heterogeneous nature (there is no unique biologic marker for CTCs identification). Although much promising progress has been made in CTCs detection, the robustness in distinguishing between healthy cells and CTCs, and the isolation of live CTCs need to be improved further. The method developed in this work exploits the so-called Warburg effect (WE): even in the presence of oxygen cancer cells limit largely their metabolism to glycolysis leading to increased production of lactate. Using droplet microfluidics, cancer cells are compartmentalized into a picolitre droplets and lactate secreted by cells are retained in the droplet. The secretion of lactate leads to a rapid increase in the concentration of acid in cell-containing droplets. CTCs are thus detected by monitoring the pH of the droplet using a pH- sensitive dye, without the need for surface-antigen labeling. A suspension of tumor cells (A549) mixed with white blood cells were emulsified in picoliter droplets, and it was observed a clear fraction of droplets with a reduced pH, leading to a distinct population of droplets containing a cancer cell from empty or WBC containing drops. With this method a very few number (up to 10) of tumor cells in a background of 200,000 white blood cells are detected, with average detection rates in the range of 60%. To demonstrate that this is a general method for detection of cancer cells, several cancer cell lines were tested, including ovarian TOV21G, breast MDA-MB 453, glioblastoma U231, colorectal HT-29, breast MCF-7 and MDA-MB-231 and all showed acidification of droplets. With the method established, samples based on blood cancer patients with confirmed metastatic disease were tested. The results show clearly that numerous positive droplets are detected in the sample of metastatic patients. Moreover, this method is capable of retrieving live cells, opening up routes for further large scale investigation of the nature of CTCs. Another interesting area where droplet-based microfluidics is playing an increasingly important role is the synthesis of functional polymeric microparticles or microgels. They have been suggested as diagnostic tools for the rapid multiplexed screening of biomolecules, because of their advantages in detection and quantification. In the second part of this thesis, the synthesis of polymeric microparticles, functionalized with peptides, through droplet microfluidics is presented. Peptide was efficiently encapsulated into the polymeric microparticles in order to create a functional microparticles for selective protein detection in complex fluids. Protein binding occurred with higher affinity (K D 0.1-0.4 µM) than the conventional detection methods (K D 70 µM). Current work demonstrate easy and fast realization of functionalized monodisperse microgels using droplet microfluidic and how the inclusion of small molecules within polymeric network improve both the affinity and the specificity of protein capture. This work provides advances in gel particle functionalization and opens new possibilities for direct molecules detection in complex fluids. A possible application of this method was for label-free aflatoxin M 1 (AFM 1 ) detection. AFM 1 is the most toxic, carcinogenic, teratogenic and mutagenic class of aflatoxins (AFs) and can be present due to <fungal contamination> in a wide range of food and feed commodities, such as milk and dairy products, representing an important issue especially for developing countries. Currently, the detection methods used to quantify AFM 1 require complex and laborious sample pretreatments, expensive instruments and skilled operators, thus limiting their application. Driven by the need of overcoming some of these limits, poly(ethylene glycol) dyacrilate (PEGDA) functional microparticles were produced using microfluidics. Two novel peptides were synthesized for specific aflatoxin detection and encapsulated in PEGDA microparticles for AFM 1 detection. AFM 1 -binding peptides occurred with high affinity (K D 3.66-6.57 pM, respectively for the two sequences) and detection was achieved measuring AFM 1 innate fluorescence. The detection limit of this technique for AFM 1 was estimated to be 1.64 ng/Kg, with a dynamic detection range between 3.28 ng/Kg and 70 ng/Kg, which meets present legislative limits of 50 ng/Kg for AFM 1 in milk. Therefore, the developed systems provides a promising approach for rapid screening of food contaminates because it resulted to be simple, sensitive, specific, and with not need multiple separation steps, overcoming the limits of the traditional AFM 1 detection methods, which are expensive and time consuming. The use of microfluidics has allowed development of robust, label-free, sensitive and high-throughput platforms which may be used in the near future to improve the quality of life.