AVS2018 Session BI+AS+IPF+NS-TuA: IoT Session: Biofabrication, Bioanalytics, Biosensors and Diagnostics

Continuous monitoring of biomarkers in biological environments is a key challenge for the development of biosensors capable of providing real-time feedback¹. These sensors promise to aid in the treatment of diseases with a highly dynamic nature however current technologies remain scarce¹. Nanoparticle based “optodes” have emerged as sensitive and tuneable biosensors, using chromo/ionophores to generate analyte-specific changes in fluorescence spectra in a dynamic and reversible manner. Currently this type of sensor suffers from limitations including leaching of reagents from the nanoparticles over time, combined with poor colloidal stability and resistance to fouling in biological fluids.

An organosilica core-shell pH sensitive nanoparticle containing a mixture of covalently incorporated pH-sensitive (shell) and pH-insensitive (core) fluorescent dyes has been developed. This platform demonstrates good long term stability (80 days), fast response time (<100 ms) and resistance to fouling in biological conditions². This presentation will describe the modification of these pH sensing particles towards the production of a lactate responsive particle for sensing through coupling with lactate dehydrogenase. Here we will present our latest results focussed on enzyme encapsulation in addition to modulation of shell parameters including thickness and degree of crosslinking in order to tune response kinetics for application in biological tissues.

¹ Corrie, S. R. et al., Analyst, 2015, 140, 4350-4364

² Robinson, K. J. et al., ACS Sensors, 2018

Silicon-based field-effect devices have been widely studied for label-free DNA detection in recent years. These devices rely on the detection of changes in the electrical surface potential during the DNA recognition event and thus require a reliable and selective immobilization of charged biomolecules on the device surface [1]. The preparation of self-assembled monolayers of phosphonic acids (SAMPs) on metal oxide surfaces is an efficient approach to generate well-defined organic interfaces with a high density of receptor binding sites close to the sensing surface [2,3]. In this work, we report the functionalization and characterization of silicon/silicon nitride surfaces with different types of peptide nucleic acid (PNA), a synthetic analogue to DNA [4].

Differently modified PNA molecules are covalently immobilized on the underlying SAMPs either in a multidentate or monodentate fashion to investigate the effect of different binding modes on receptor density and morphology important for PNA-DNA hybridization. Multidentate immobilization of the bioreceptors via C₆-SH attachment groups at the γ-points along the PNA backbone provides a rigid, lying configuration on the device surface (PNA 1), whereas a monodentate immobilization by Cys-capped PNA molecules (PNA 2) results in more flexible and more accessible receptor binding sites. Our results indicate that the presented functionalization scheme can be successfully applied to produce morphologically and electrochemically different PNA bioreceptor binding sites on silicon/silicon nitride surfaces. Consequently, a well-chosen modification of the PNA backbone is a valid approach to influence the sensing properties of surface-immobilized PNA bioreceptors, which might provide an additional parameter to further tune and tailor the sensing capabilities of PNA-based biosensing devices.

[1] Ingebrandt S. and Offenhausser A., Phys. Status Solidi A 203 (2006), 3399–3411.

[2] Chaki N. K. and Vijayamohanan K., Biosens. & Bioelectron. 17 (2002), 112.

[3] Stutzmann M., Garrido J. A., Eickhoff M. and Brandt M. S., Phys. Status Solidi A 203 (2006), 3424–3437.

[4] Nielsen P. E. and Egholm M. (ed.), Peptide Nucleic Acids, Horizon Scientific Press (1999).

The detection of endogenous gases including H₂S is of immense interest nowadays as it opens the way to predict some diseases as well as an early stage diagnosis. These three gasotransmitter (H₂S, NO and CO) gaseous molecules transfer the information and give the signal for mainly cardiovascular diseases Therefore, its detection has crucial importance in bio-medical science. Here, we demonstrate H₂S detection from living cells using silver nanorods arrays fabricated by glancing angle deposition method. Colorimetric and wettability properties of silver nanorods are being observed for the gaseous detection. We use the model organism E.coli to demonstrate the feasibility of the method for the determination of live and resistant strains of the bacteria. For the human cell, we have used Hela cell line for the same. For the simplicity and feasibility of the technique, Android based mobile app has been developed for the colorimetric detection. Data obtained in this study show the potency of the system to identify live/dead bacteria with or without antibiotic treatment and compared with the time-consuming standard plating method, it a simple and cost-effective method for the estimation of living and resistant microorganism. The performance of AgNRs as H₂S gas sensor is investigated by its sensing ability of 5 ppm of gas with an exposure time of only 30 s. It has potential application in the area of antimicrobial resistance and bio-medical healthcare.

Transplantation of whole pancreas/cadaveric islets is the most commonly acceptable treatment option for uncontrolled diabetes. However, the wider clinical applicability of these approaches is limited due to unavailability of donors and continuous need for the administration of immunosuppressant. Here we report a new strategy for efficient in-vitro trans-differentiation of human-hepatic progenitor cells (hHPCs) into insulin producing cells (iPCs) on biologically compatible micro-chips of 2D platforms. The physiological function of transdifferentiated hHPCs confirmed the activation of intracellular Ca⁺⁺ signaling and activation of pancreatic transcription factors (pTFs) which triggers the insulin exocytosis during hyperglycemic challenge. The iPCs on these micro-chips showed upregulated expression of master regulator Pdx-1, β-cell specific marker Nkx-6.1 and more importantly C-peptide similar to human pancreatic b-cells during hyperglycemic challenge. These platforms may provide long-term survival and function of iPCs which could be better technology for developing effective therapeutic options for the management of diabetic.

Antibody separation is a key biopharmaceutical process, which requires high specificity and efficiency in isolating the biomacromolecule from a complex biological fluid. Development of the separation adsorbent benefits diagnostics and therapeutics, such as point-of-care testing, treatment of cancer and autoimmune disease. In the process of antibody separation, Protein A chromatography is a commonly employed adsorbent, which could obtain antibody in high purity from serum or ascites. In the process-scale purification and therapeutic plasma exchange, safety issues, e.g. leakage and instability of the immobilized Protein A, and cross-contamination during regeneration, are overwhelmed in biopharmaceutics. An alternative approach to Protein A chromatography is using synthetic ligand, molecular weight of which is commonly less than 200 Da. The main advantages of synthetic ligand are well-controlled chemical structure, low cost, ease in clean-in-place, and repeatable regeneration capability in harsh conditions. However, it is a challenge to adsorb antibody in a highly selective manner from a complex biological fluid, which consists a variety of proteins with a broad range of concentrations.

The functionalization of material surfaces with proteins is of great importance to a number of technologies, from industrial processes to biomedical diagnostics. However, while it has been proposed that orientation may be important to the function of such biomaterials, efforts to study such roles are hampered by a lack of rapid, quantitative, and orientation-specific immobilization techniques which will reduce non-specific fouling, and allow substoichiometric attachment of proteins onto surfaces in an orientation-controlled manner. Here, Carbonic Anydrase II (HCA)—a 30 kDA, monomeric metalloenzyme which catalyzes the interconversion of carbon dioxide to bicarbonate—is immobilized onto strained trans-cyclooctene (sTCO)-functionalized magnetic resin in an orientation-specific manner via bioorthogonal ligation with a site-specifically installed tetrazine-containing amino acid (Tet2.0).

Many of the exceptional properties of natural materials (e.g. fracture toughness of bones, strength to weight ratio of bamboo) can be attributed to their structural hierarchy, which originates, in part, from the nanoscale organization of the enzymes that synthesize these materials. In order to best utilize such enzymes ex vivo to grow engineered biomaterials, the role of this multiscale organization must be understood. Here, we report a novel strategy for studying the activity of arrangements of enzymes within a multifunctional material in a high throughput manner. In particular, we use top-down patterning techniques in conjunction with small molecule self-assembly to designate enzyme-binding regions amidst a non-binding, hydrophobic background. Key to this experimental scheme is the parallel nature of both the fabrication and the characterization processes that enable the efficient study of many geometric parameters of the enzyme-binding features. These parameters include, (1) feature size, (2) density of enzyme within each feature, and (3) distance between features. This level of control can in principle allow us to separate effects of reaction kinetics and substrate diffusion. Two strategies have been explored for the immobilization of enzymes including click chemistry to non-natural amino acids and binding to poly-histidine affinity tags. Top-down lithography and enzyme assembly were verified using a variety of surface characterization techniques including atomic force microscopy, X-ray photoelectron spectroscopy, infrared spectroscopy, spectroscopic ellipsometry, and contact angle goniometry. Initially, this high throughput paradigm is used to develop a fluorimetric assay to quantify the activity of surface-bound enzymes as a function of their spatial organization. Together with the widespread utilization of high throughput techniques in synthetic biology, the ability to study spatial organization in a rapid fashion is expected to dramatically improve ex vivo applications of enzymes.

Polylactic acid (PLA, [C₃H₄O₂]_n, CAS 26161-42-2) is a biodegradable and thermoplastic polymer. PLA is naturally produced and can be extracted from many plants such as sugarcane, cornstarch or cassava roots. Typical industrial production processes for PLA are direct condensation of lactic acid monomers (~100°C - 160°C) and ring-opening polymerization of lactide with metal catalysts. For applications in bulk forms, PLA can be produced by extrusion, casting, injection molding and spin coating or even 3d printing.

In cell and tissue engineering applications, amino acids are essential ingredients for cell-tissue culture, implants/replacements, drugs and treatment tests. There are twenty amino acids appearing in human genetic codes by triplet codons and usually categorized according to their polarity, acidity/basicity.

In this study, we fabricate nanofibers by electrospinning on a spin-coated PLA film. This specially designed combination of PLA films and nanofibers is meant to have enduring interfacial adhesion between the two for biomedical applications such as implants. Both PLA nanofibers and films are mixed with selected amino acids. Five amino acids were chosen: tryptophan (Trp, ), methionine (Met, ), serine (Ser, ), glutamate (Glu, ) and arginine (Arg, ). The selection is based on the different electrical polarity of each amino acid. The electrical polarity has profound effects on the solubility, pH acidity of amino acids in water and many other associated biochemical functions. These amino acids are representatives of certain biochemical features for potentially different influences in our applications for cell culture.

The electrospinning process is controlled by several parameters such as the voltage of power supply, feeding velocity of polymer solution through the syringe pump, electrical field strength and distance to the collection plate of nanofibers. Different combinations of these parameters are studied to determine an optimal control for fiber formation. Properties of and microstructures of deposited films and nanofibers are investigated as following: thickness and deposition rate by surface profilometer; microstructures by Fourier transform infrared spectrometer (FTIR); surface morphology by scanning electron microscope (SEM); optical properties by UV-Visible-IR spectrometer and wettability by the contact angle.