Biosensors for bioengineering

Drug discovery pathway relies heavily on in vivo animal models and in vitro cell mediums. In the case of animal models we have not only some ethical problems but also the ability to extrapolate data to human conditions is limited and in vitro platforms often do not simulate the complex cell–cell and cell–matrix interactions crucial for regulating cell behaviour.  

Pancreas islet stained in blue for nuclei and red for actin inside a microporous microfibrillated cellulose-gelatin scaffold stained with green.

Biosensors for bioengineering group is focused in a new line of research that has become of extreme importance in the last years. The idea is to integrate biosensor technology and nanotechnology with stem cell research and with tissue engineering. Engineered tissues are integrated with biosensing technology to obtain microdevices for detecting cellular responses to external stimuli, monitoring the quality of the microenvironment (e.g., metabolites, nutrients), and supporting diverse cellular requirements. This research on 3D-functional engineered tissues is expected to develop knowledge of tissue construction and their functions and relation with some human diseases. Integration of fully functional tissues with microscale biosensor technology allowed us to obtain “organs-on-a-chip”. These chips could be used in pharmaceutical assays and could be a step toward the ultimate goal of producing in vitro drug testing systems crucial to the medicine and pharmaceutical industry. 

Engineered skeletal muscle from patients’ cells with myotonic dystrophy disease.

Despite the increasing number of organs-on-a-chip that have been developed in the past decade, limited efforts have been made to integrate a sensing system for in situ continual measurements of biomarkers from three-dimensional (3D) tissues. Here, we present a custom-made integrated platform for muscle cell stimulation under fluidic conditions connected with a multiplexed high-sensitivity electrochemical sensing system for in situ monitoring. To demonstrate this, we use our system to measure the release levels and release time of interleukin 6 and tumor necrosis factor alpha in vitro by 3D muscle microtissue under electrical and biological stimulations. Our experimental design has enabled us to perform multiple time point measurements using functionalized screen-printed gold electrodes with sensitivity in the ng mL−1 range. This affordable setup is uniquely suited for monitoring factors released by 3D single cell types upon external stimulation for metabolic studies.

Concentrations down to 3 nM of the rhS100A4 protein, associated with human tumor development, have been detected in undiluted urine using an integrated sensor based on microring resonators in the emerging Al2O3 photonic platform. The fabricated microrings were designed for operation in the C-band (λ = 1565 nm) and exhibited a high-quality factor in air of 3.2 × 105. The bulk refractive index sensitivity of the devices was ~100 nm/RIU (for TM polarization) with a limit of detection of ~10−6 RIU. A surface functionalization protocol was developed to allow for the selective binding of the monoclonal antibodies designed to capture the target biomarker to the surface of the Al2O3 microrings. The detection of rhS100A4 proteins at clinically relevant concentrations in urine is a big milestone towards the use of biosensors for the screening and early diagnosis of different cancers. Biosensors based on this microring technology can lead to portable, multiplexed and easy-to-use point of care devices.

Keywords: Distributed feedback lasers, Effective refractive index, Laser coupling, Polarization maintaining fibers, Refractive index, Scanning electron microscopy

Understanding the protein-secretion dynamics from single, specific tissues is critical toward the advancement of disease detection and treatments. However, such secretion dynamics remain difficult to measure in vivo due to the uncontrolled contributions from other tissue populations. Here, we describe an integrated platform designed for the reliable, near real-time measurements of cytokines secreted from an in vitro single-tissue model. In our setup, we grow 3D biomimetic tissues to discretize cytokine source, and we separate them from a magnetic microbead-based biosensing system using a Transwell insert. This design integrates physiochemically controlled biological activity, high-sensitivity protein detection (LOD < 20 pg mL−1), and rapid protein diffusion to enable non-invasive, near real-time measurements. To showcase the specificity and sensitivity of the system, we use our setup to probe the inflammatory process related to the protein Interleukine 6 (IL-6) and to the Tumor Necrosis Factor (TNF-α). We show that our setup can monitor the time-dependence profile of IL-6 and TNF-α secretion that results from the electrical and chemical stimulation of 3D skeletal muscle tissues. We demonstrate a novel and affordable methodology for discretizing the secretion kinetics of specific tissues for advancing metabolic-disorder studies and drug-screening applications.

Keywords: Microphysiological tissues, Tissue engineering, Electrochemical, biosensors, Magnetic particles, Skeletal muscle, Electric stimulation

Abstract New biocompatible materials have enabled the direct 3D printing of complex functional living tissues, such as skeletal and cardiac muscle. Gelatinmethacryloyl (GelMA) is a photopolymerizable hydrogel composed of natural gelatin functionalized with methacrylic anhydride. However, it is difficult to obtain a single hydrogel that meets all the desirable properties for tissue engineering. In particular, GelMA hydrogels lack versatility in their mechanical properties and lasting 3D structures. In this work, a library of composite biomaterials to obtain versatile, lasting, and mechanically tunable scaffolds are presented. Two polysaccharides, alginate and carboxymethyl cellulose chemically functionalized with methacrylic anhydride, and a synthetic material, such as poly(ethylene glycol) diacrylate are combined with GelMA to obtain photopolymerizable hydrogel blends. Physical properties of the obtained composite hydrogels are screened and optimized for the growth and development of skeletal muscle fibers from C2C12 murine cells, and compared with pristine GelMA. All these composites show high resistance to degradation maintaining the 3D structure with high fidelity over several weeks. Altogether, in this study a library of biocompatible novel and totally versatile composite biomaterials are developed and characterized, with tunable mechanical properties that give structure and support myotube formation and alignment.

Observing biochemical processes within living cell is imperative for biological and medical research. Fluoresce imaging is widely used for intracellular sensing of cell membranes, nuclei, lysosomes, and pH. Electrochemical assays have been proposed as an alternative to fluorescence-based assays because of excellent analytical features of electrochemical devices. Notably, thanks to the rapid progress of micro/nanotechnologies and electrochemical techniques, intracellular electrochemical sensing is making rapid progress, leading to a successful detection of intracellular components. Such insight can provide a deep understanding of cellular biological processes and, ultimately, define the human healthy and diseased states. In this review, we present an overview of recent research progress in intracellular electrochemical sensing. We focus on two main topics, electrochemical extraction of cytosolic contents from cells and intracellular electrochemical sensing in situ.

Keywords: Micro/nanoelectrode, Analytical electrochemistry, Intracellular sensing, Cell analysis

Al2O3 microresonators were realized for sensing applications of both passive and active devices. Passive microring resonators exhibited quality factors up to 3.2×105 in air. A bulk refractive index sensitivity of 100 nm/RIU was demonstrated together with a limit of detection of 10-6 RIU. Functionalizing their surface allowed for the label-free detection of the biomarker rhS100A4 from urine with a limit of detection of 3 nM. Furthermore, single-mode Al2O3:Yb3+ microdisk lasers were realized that could operate in an aqueous environment. Upon varying the bulk refractive index their lasing wavelength could be tuned with a sensitivity of 20 nm/RIU and a LOD of 3×10-6 RIU.

The Al2O3 waveguide technology was explored for sensing applications. Passive microring resonators with a quality factor in air of 3.2×105 were developed with a bulk refractive index sensitivity of ~100 nm/RIU and limit of detection of ~10-6 RIU. These were functionalized to detect the biomarker rhS100A4 from urine down to concentrations of 3 nM. Furthermore, Al2O3:Yb3+ microdisk lasers were realized that exhibited single mode lasing operation in water. Their lasing wavelength was tuned by varying the bulk refractive index and a bulk refractive index sensitivity of ~20 nm/RIU with a LOD of ~3×10-6 was achieved.

Animal models have been the main resources for drug discovery and prediction of drugs’ pharmacokinetic responses in the body. However, noticeable drawbacks associated with animal models include high cost, low reproducibility, low physiological similarity to humans, and ethical problems. Engineered tissue models have recently emerged as an alternative or substitute for animal models in drug discovery and testing and disease modeling. In this review, we focus on skeletal muscle and cardiac muscle tissues by first describing their characterization and physiology. Major fabrication technologies (i.e., electrospinning, bioprinting, dielectrophoresis, textile technology, and microfluidics) to make functional muscle tissues are then described. Finally, currently used muscle tissue models in drug screening are reviewed and discussed.

Keywords: Cardiac muscle, Drug screening, Engineering muscle, Human pharmacological response, Physiological similarity, Skeletal muscle

Tissue engineering (TE) aims to restore function or replace damaged tissue through biological principles and engineering. Nanofibers are attractive substrates for tissue regeneration applications because they structurally mimic the native extracellular matrix. Composite nanofibers, which are hybrid nanofibers blended from natural and synthetic polymers, represent a major advancement in TE and regenerative medicine, since they take advantage of the physical properties of the synthetic polymer and the bioactivity of the natural polymer while minimizing the disadvantages of both. Although various nanofibrous matrices have been applied to almost all the areas of TE, in this chapter we will focus on nanofiber composites scaffolds for vascular TE.

Keywords: Blood vessels, Nanofiber composite, Tissue engineering, Vascularized tissue