Systematic and Rational Design of Protein Arrays in Noncontact Printers: Pipeline and Critical Aspects.

The systematic design and construction of customized protein microarrays are critical for the further successful screening of biological samples in biomedical research projects. In general protein microarrays are classified according to the content, detection method, and printing methodology, among others. Here, we are focused on the type of printing: contact and noncontact. Both approaches have advantages and disadvantages; however, in any of the approaches, a prior well design and systematic preparation of materials and/or instruments required for the customized antibody arrays is critical. In this chapter, the process for an antibody microarray by a noncontact printer is described in detail from the preparation of array content to the analysis, including quality control steps.

Curing of UV prints - Assessment of possible toxicological hazard for consumers.

In an experimental setting a laboratory analysis of substances migrating from UV prints under mechanical stress into sweat and saliva simulant was performed. The influence of paper type and curing degree on UV prints was investigated. Five substances were identified at concentrations above the limit of detection in the simulants PPG-3 glyceryl triacrylate, ethoxylated trimethylolpropane triacrylate, trimethylolpropane triacrylate, 2/4-isopropylthioxanthone (ITX), and 2,4-diethylthioxanthone (DETX). Migration of the acrylates and photoinitiators into saliva and sweat simulants were increased when the UV inks were printed on uncoated paper in comparison to coated paper. With an exposure scenario considering a person to leaf through 80 pages of UV-printed paper per day while touching each page with a licked fingertip, Risk Characterisation Ratios (RCR) for oral exposure well below 1 were obtained for all five substances indicating no risk for the general population. The three acrylates are classified for skin sensitisation. The migrated amounts per skin surface area of these three were compared with the EC3 value for a hypothetical substance that could be categorised as strong sensitiser (EC3 = 0.1%). The results show that the risk of skin sensitisation even under worst case conditions can be considered as negligible.

Release and cytotoxicity screening of the printer emissions of a CdTe quantum dots-based fluorescent ink.

The fluorescent properties of cadmium telluride (CdTe) containing quantum dots (QDs) have led to novel products and applications in the ink and pigment industry. The toxic effects of the emissions associated to the use of printing ink containing CdTe QDs might differ from those of conventional formulations which do not integrate nanoparticles, as CdTe QDs might be emitted. Within this work, the airborne emissions of a water-soluble fluorescent ink containing polyethylene glycol (PEG)-coated CdTe QDs of 3-5 nm diameter have been characterized and studied under controlled conditions during household inkjet printing in a scenario simulating the use phase. Subsequently, the cytotoxicological potential of atomized CdTe QDs ink in an acute exposure regimen simulating an accidental, worse-case scenario has been evaluated in vitro at the air-liquid interface (ALI) using the pulmonary cell line BEAS-2B. Endpoints screened included cell viability, oxidative stress and inflammatory effects. We have observed that CdTe QDs ink at 54.7 ng/mL decreased cell viability by 25.6 % when compared with clean air after 1h of exposure; a concentration about 65 times higher was needed to observe a similar effect in submerged conditions. However, we did not observe oxidative stress or inflammatory effects. The present study integrates the development of scenarios simulating the use phase of nano-additivated inks and the direct cell exposure for in vitro effects assessment, thus implementing a life-cycle oriented approach in the assessment of the toxicity of CdTe QDs.

Comparative investigations on key factors and print head designs for pharmaceutical inkjet printing.

The interest in using inkjet printing as manufacturing technology for personalized medicine has increased in recent years. The print head is the centrepiece of an inkjet printer. For pharmaceutical approaches, various types of printing equipment were tested in the past, but comparative investigations in relation to pharmaceutical use are still lacking. In the present study, two piezoelectric print heads of different designs (Spectra SE-128 AA and Konica Minolta KM512SHX) were systematically compared with the objective of deepening the process understanding and identifying the key factors on the resulted quantities. As substrates, oral thin films made from 15% (w/w) hypromellose (HPMC) casting solution were used. The Spectra print head with bigger nozzles was more efficient in one pass and resulted in less scattering (RSD ≤ 5%). Furthermore, it was found that liquid excipients like polyethylene glycol 400 characterized by low vapour pressure and limited penetration into the HPMC based films are not suitable. The choice of the printed geometry plays a subordinate role when printing the same surface area, whereas the composition of the inks, set process parameters as well as the size and functionality of the nozzles have a significant impact on the final printed quantity.

Plasticizer-Free Thin-Film Sodium-Selective Optodes Inkjet-Printed on Transparent Plastic for Sweat Analysis.

A novel strategy to functionalize transparent flexible plastic films with an optical ion-sensing layer using an inkjet-printing technology is described. The hydrophobic sensing chemicals that include a sodium ionophore, a lipophilic proton chromoionophore, and a lipophilic ion-exchanger are co-deposited onto substrates such as transparent polyester film sheets in the absence of any plasticizer and/or hydrophobic polymer matrix. The inkjet-printing process enables the formation of optode films with nanoscale thickness/roughness that readily facilitate interfacing with aqueous samples. Using a smartphone detector, the colorimetric response of the optodes is shown to reach 95% of equilibrium values within 100 s in response to different concentrations of sodium ions, which is more rapid than traditional ion-selective optodes based on plasticized PVC films as the sensing layer. The new optodes also exhibit high selectivity to Na+ over interfering ions including K+, Ca2+, and Mg2+. Chemical leaching experiments show that the highly hydrophobic optode components remain in place on the plastic substrate surface. Hence, excellent sensor stability and fully reversible optical responses are obtained, which is essential for potential continuous monitoring applications. Further testing of the sensors with undiluted human sweat samples is shown to yield accurate values for sodium concentrations. Therefore, the use of plasticizer-free ion-selective optode nanolayers that enable highly selective ion sensing on a clear plastic support is likely to expand the range of available chemical sensors suited for preparing wearable real-time sweat analysis devices.

Ejection of cell laden RPMI-1640 culture medium by Electrohydrodynamic method.

Sample deposition based on micro-droplet ejection has broad application prospects in the field of biomedicine. Ejection of RPMI-1640 medium (with and without cells) is investigated experimentally using a home-build electrohydrodynamic (EHD) ejection system, consisting of a liquid supplier and a nozzle, a high voltage source, a droplet collector, and a high speed photography module. High electric voltage is applied between the nozzle and the droplet collector. The liquid surface is electrically charged and the ejection takes place when electric force overcomes the surface tension. The ejection process is studied by using high speed photography and image processing. At low voltage, a stable ejection state is established with ejection frequency ranging from a few to a few tens of Hertz. At high voltage, another stable ejection state is reached with ejection frequency as high as 1300 Hz. At the transition voltage range, the ejection exhibits a periodic behaviour. During each cycle, the meniscus rapidly oscillates with gradually increased amplitude, and with several non-uniform droplets ejected at the final stage of the cycle. Human peripheral blood mononuclear cells, after ejection, shows survival rates higher than 79%, manifesting EHD ejection as a promising technique for cell printing.

Assessment of fine particles released during paper printing and shredding processes.

In this study, we investigated the airborne particles released during paper printing and paper shredding processes in an attempt to characterize and differentiate these particles. Particle characteristics were studied with real time instruments (RTIs) to measure concentrations and with samplers to collect particles for subsequent microscopy and cytotoxicity analysis. The particles released by paper shredding were evaluated for cytotoxicity by using in vitro human lung epithelial cell models. A substantial amount of particles were released during both the shredding and printing processes. We found that the printing process caused substantial release of particles with sizes of less than 300 nm in the form of metal granules and graphite. These released particles contained various elements including Al, Ca, Cu, Fe, Mg, N, K, P, S and Si. The particles released by the paper shredding processes were primarily nanoparticles and had a peak size between 27.4 nm and 36.5 nm. These paper particles contained elements including Al, Br Ca, Cl, Cr, Cu, Fe, Mg, N, Na, Ni P, S and Si, as determined by scanning electron microscope-energy dispersive X-ray spectroscopy (SEM-EDS) and single-particle inductively coupled plasma-mass spectroscopy (SP-ICP-MS) analysis. Although various metals were identified in the paper particles, these particles did not elicit cytotoxicity to simian virus-transformed bronchial epithelial cells (BEAS2B) and immortalized normal human bronchial epithelial cells (HBE1). However, future studies should investigate other cytotoxicity effects of these paper particles in various types of lung cells to identify potential health effects of the particles.

Laser printing-enabled direct creation of cellular heterogeneity in lab-on-a-chip devices.

Lab-on-a-chip devices, capable of culturing living cells in continuously perfused, micrometer-sized channels, have been intensively investigated to model physiological microenvironments for cell-related testing and evaluation applications. Various chemical, physical, and/or biological culture cues are usually expected in a designed chip to mimic the in vivo environment with defined spatial heterogeneity of cells and biomaterials. To create such heterogeneity within a given chip, typical methods rely heavily on sophisticated fabrication and cell seeding processes, and chips fabricated with these methods are difficult to readily adapt for other applications. In this study, laser-induced forward transfer (LIFT)-based printing has been implemented to create heterogeneous cellular patterns in a lab-on-a-chip device to achieve the efficiency in creating heterogeneous cellular patterns as well as the flexibility in adapting different evaluation configurations in lab-on-a-chip devices. Two applications, parallel evaluation of cellular behavior and targeted drug delivery to cancer cells, have been implemented as proof-of-concept demonstrations of the proposed fabrication method. For the first application, the morphology of cells in different extracellular matrix (ECM) materials cultured under varying conditions has been investigated. It is found that less stiff ECM and dynamic culturing are preferred for spreading of fibroblasts. For the second application, different drug carriers have been utilized for targeted delivery of anticancer drugs to breast cancer cells. It is found that targeted drug delivery is important to realize effective chemotherapy and drug release rate from drug carriers affects the chemotherapy effect. Consequently, the proposed laser printing-based method enables direct creation of heterogeneous cellular patterns within lab-on-a-chip devices which improves the efficiency and versatility of cell-related sensing and evaluation using lab-on-a-chip devices.

Engineering the Surface Properties of a Zwitterionic Polymer Brush to Enable the Simple Fabrication of Inkjet-Printed Point-of-Care Immunoassays.

Motivated by the lack of adventitious protein adsorption on zwitterionic polymer brushes that promise low noise and hence high analytical sensitivity for surface-based immunoassays, we explored their use as a substrate for immunoassay fabrication by the inkjet printing of antibodies. We observed that a poly(sulfobetaine)methacrylate brush on glass is far too hydrophilic to enable the noncovalent immobilization of antibodies by inkjet printing. To circumvent this limitation, we developed a series of hybrid zwitterionic-cationic surface coatings with tunable surface wettability that are suitable for the inkjet printing of antibodies but also have low protein adsorption. We show that in a microarray format in which both the capture and detection antibodies are discretely printed as spots on these hybrid brushes, a point-of-care sandwich immunoassay can be carried out with an analytical sensitivity and dynamic range that is similar to or better than those of the same assay fabricated on a PEG-like brush. We also show that the hybrid polymer brushes do not bind anti-PEG antibodies that are ubiquitous in human blood, which can be a problem with immunoassays fabricated on PEG-like coatings.

Reactive Inkjet Printing of Functional Silk Stirrers for Enhanced Mixing and Sensing.

Stirring small volumes of solution can reduce immunoassay readout time, homogenize cell cultures, and increase enzyme reactivity in bioreactors. However, at present many small scale stirring methods require external actuation, which can be cumbersome. To address this, here, reactive inkjet printing is shown to be able to produce autonomously rotating biocompatible silk-based microstirrers that can enhance fluid mixing. Rotary motion is generated either by release of a surface active agent (small molecular polyethylene glycol) resulting in Marangoni effect, or by catalytically powered bubble propulsion. The Marangoni driven devices do not require any chemicals to be added to the fluid as the "fuel," while the catalytically powered devices are powered by decomposing substrate molecules in solution. A comparison of Marangoni effect and enzyme powered stirrers is made. Marangoni effect driven stirrers rotate up to 600 rpm, 75-100-fold faster than enzyme driven microstirrers, however enzyme powered stirrers show increased longevity. Further to stirring applications, the sensitivity of the motion generation mechanisms to fluid properties allows the rotating devices to also be exploited for sensing applications, for example, acting as motion sensors for water pollution.

Multifunctional self-assembled monolayers via microcontact printing and degas-driven flow guided patterning.

Soft lithography-based patterning techniques have been developed to investigate biological and chemical phenomena. Until now, micropatterning with various materials required multiple procedural steps such as repeating layer-by-layer patterning, aligning of stamps, and incubating printed inks. Herein, we describe a facile micropatterning method for producing chemically well-defined surface architectures by combining microcontact (µCP) and microfluidic vacuum-assisted degas-driven flow guided patterning (DFGP) with a poly(dimethylsiloxane) (PDMS) stamp. To demonstrate our concept, we fabricated a bi-composite micropatterned surface with different functional molecular inks such as fluorescein isothiocyanate labelled bovine serum albumin (FITC-BSA) and polyethylene glycol (PEG)-silane for a biomolecule array, and 3-aminopropyltriethoxysilane (APTES) and PEG-silane pattern for a self-assembled colloid gold nanoparticle monolayer. With a certain composition of molecular inks for the patterning, bi-composite surface patterns could be produced by this µCP-DFGP approach without any supplementary process. This patterning approach can be used in microfabrication and highly applicable to biomolecules and nanoparticles that spread as a monolayer.

Lift-off cell lithography for cell patterning with clean background.

We developed a highly efficient method for patterning cells by a novel and simple technique called lift-off cell lithography (LCL). Our approach borrows the key concept of lift-off lithography from microfabrication and utilizes a fully biocompatible process to achieve high-throughput, high-efficiency cell patterning with nearly zero background defects across a large surface area. Using LCL, we reproducibly achieved >70% patterning efficiency for both adherent and non-adherent cells with <1% defects in undesired areas.

Dynamic inking of large-scale stamps for multiplexed microcontact printing and fabrication of cell microarrays.

Microcontact printing has become a versatile soft lithography technique used to produce molecular micro- and nano-patterns consisting of a large range of different biomolecules. Despite intensive research over the last decade and numerous applications in the fields of biosensors, microarrays and biomedical applications, the large-scale implementation of microcontact printing is still an issue. It is hindered by the stamp-inking step that is critical to ensure a reproducible and uniform transfer of inked molecules over large areas. This is particularly important when addressing application such as cell microarray manufacturing, which are currently used for a wide range of analytical and pharmaceutical applications. In this paper, we present a large-scale and multiplexed microcontact printing process of extracellular matrix proteins for the fabrication of cell microarrays. We have developed a microfluidic inking approach combined with a magnetic clamping technology that can be adapted to most standard substrates used in biology. We have demonstrated a significant improvement of homogeneity of printed protein patterns on surfaces larger than 1 cm2 through the control of both the flow rate and the wetting mechanism of the stamp surface during microfluidic inking. Thanks to the reproducibility and integration capabilities provided by microfluidics, we have achieved the printing of three different adhesion proteins in one-step transfer. Selective cell adhesion and cell shape adaptation on the produced patterns were observed, showing the suitability of this approach for producing on-demand large-scale cell microarrays.

Printable nanocomposites of polymers and silver nanoparticles for antibacterial devices produced by DoD technology.

Silver nanoparticles (Ag-NPs) are known for their efficient bactericidal activity and are widely used in industry. This study aims to produce printable antibacterial devices by drop-on-demand (DoD) inkjet technology, using Ag-NPs as the active part in complex printable fluids. The synthesis of this active part is described using two methods to obtain monodisperse NPs: chemical and microwave irradiation. The synthesized NPs were characterized by UV-VIS, STEM, TEM, DLS and XRD. Two printable fluids were produced based: one with Ag-NPs and a second one, a polymeric nanocomposite, using silver nanoparticles and polyvinyl butyral (Ag-NPs/PVB). Cellulose acetate was used as a flexible substrate. The ecotoxicity analysis of fluids and substrate was performed with Artemia franciscana nauplii. Optimized electric pulse waveforms for drop formation of the functional fluids were obtained for the piezoelectric-based DoD printing. Activity of printed antibacterial devices was evaluated using the Kirby-Bauer method with Staphylococcus aureus and Escherichia coli. The results show that the printed device with Ag-NP fluids evidenced a bacterial inhibition. An important advantage in using the DoD process is the possibility of printing, layer by layer or side by side, more than one active principle, allowing an interleaved or simultaneous release of silver NP and other molecules of interest as for example with a second functional fluid to ensure effectiveness of Ag activity.

High-resolution Patterning Using Two Modes of Electrohydrodynamic Jet: Drop on Demand and Near-field Electrospinning.

Electrohydrodynamic (EHD) jet printing has drawn attention in various fields because it can be used as a high-resolution and low-cost direct patterning tool. EHD printing uses a fluidic supplier to maintain the extruded meniscus by pushing the ink out of the nozzle tip. The electric field is then used to pull the meniscus down to the substrate to produce high-resolution patterns. Two modes of EHD printing have been used for fine patterning: continuous near-field electrospinning (NFES) and dot-based drop-on-demand (DOD) EHD printing. According to the printing modes, the requirements for the printing equipment and ink viscosity will differ. Even though two different modes can be implemented with a single EHD printer, the realization methods significantly differ in terms of ink, fluidic system, and driving voltage. Consequently, without a proper understanding of the jetting requirements and limitations, it is difficult to obtain the desired results. The purpose of this paper is to present a guideline so that inexperienced researchers can reduce the trial and error efforts to use the EHD jet for their specific research and development purposes. To demonstrate the fine-patterning implementation, we use Ag nanoparticle ink for the conductive patterning in the protocol. In addition, we also present the generalized printing guidelines that can be used for other types of ink for various fine-patterning applications.

Valvejet Technology for the Production of a Personalised Fixed Dose Combination of Ramipril and Glimepiride: an Investigative Study on the Stability of Ramipril.

PURPOSE: To use valvejet technology for printing a fixed dose combination of ramipril and glimepiride, and to investigate the stability profile of ramipril, which is susceptible to a range of processing and storage conditions. METHODS: Inks of ramipril and glimepiride were formulated and printed on to HPMC film and the films were evaluated for the chemical and solid-state integrity of the APIs using HPLC and XRPD. The stability of the APIs in the inks and in the printed samples was investigated using Raman and NMR techniques. RESULTS: The printed samples demonstrated excellent precision and accuracy in the doses of APIs deposited. Both drugs were chemically intact in the freshly printed samples and ramipril was found to be in its amorphous form. Ramipril in the printed samples has transformed into ramipril diketopiperazine when stored at 40°C with 75% RH, but remained stable when stored in a desiccator. Results from the stability study of ramipril ink show that the API has undergone degradation when stored both at room temperature and at 40°C but remained stable when stored in a refrigerator. CONCLUSION: An FDC of ramipril and glimepiride was successfully printed using valvejet technology. The significance of inkjet printing in producing amorphous dosage forms from solution based inks and personalised dosage forms of drugs susceptible to processing conditions was demonstrated using ramipril. This study illustrates the significance of examining the stability of the APIs in the inks and the importance of appropriate storing of both the inks and printed samples.

Finger-Based Printed Sensors Integrated on a Glove for On-Site Screening Of Pseudomonas aeruginosa Virulence Factors.

Early screening of clinically relevant pathogens in the environment is a highly desirable goal in clinical care, providing precious information that will improve patient-care outcomes. In this work, a glove-based electrochemical sensor has been designed for point-of-use screening of Pseudomonas aeruginosa's virulence factors. The methodology used for the elaboration of the fabric platform relied on printing the conductive inks on the index and middle fingers of the glove, with the goal of screening pyocyanin and pyoverdine targets. The analytical signatures of the analytes were recorded in about 4 min, via the rapid and selective square-wave-voltammetry technique. Finger-based sensors display good performance and discrimination between the targets and potential interferences, along with good reproducibility. The sensors featured linearity over the 0.01-0.1 µM range for pyocyanin and 5-50 µM range for pyoverdine, with sensitivities of 2.51 µA/µM for pyocyanin and 1.09 nA/µM for pyoverdine ( R2 = 0.990 and 0.995, respectively) and detection limits of 3.33 nM for pyocyanin and 1.66 µM for pyoverdine. Moreover, the sensors were tested in binary mixtures of analytes, with successful outcomes. In order to gain information from the surrounding environment, the active electronic areas of the printed fingers were coated with a conductive hydrogel matrix, and relevant target surfaces were "swiped for notification" of contaminants. The simple fabrication, low-cost, and reusability of the proposed glove are likely to underpin the progressive drive of wearable sensors toward decentralized environmental and healthcare applications.

Silk protein nanowires patterned using electron beam lithography.

Nanofabrication approaches to pattern proteins at the nanoscale are useful in applications ranging from organic bioelectronics to cellular engineering. Specifically, functional materials based on natural polymers offer sustainable and environment-friendly substitutes to synthetic polymers. Silk proteins (fibroin and sericin) have emerged as an important class of biomaterials for next generation applications owing to excellent optical and mechanical properties, inherent biocompatibility, and biodegradability. However, the ability to precisely control their spatial positioning at the nanoscale via high throughput tools continues to remain a challenge. In this study electron beam lithography (EBL) is used to provide nanoscale patterning using methacrylate conjugated silk proteins that are photoreactive 'photoresists' materials. Very low energy electron beam radiation can be used to pattern silk proteins at the nanoscale and over large areas, whereby such nanostructure fabrication can be performed without specialized EBL tools. Significantly, using conducting polymers in conjunction with these silk proteins, the formation of protein nanowires down to 100 nm is shown. These wires can be easily degraded using enzymatic degradation. Thus, proteins can be precisely and scalably patterned and doped with conducting polymers and enzymes to form degradable, organic bioelectronic devices.

A Low-Cost Inkjet-Printed Aptamer-Based Electrochemical Biosensor for the Selective Detection of Lysozyme.

Recently, inkjet-printing has gained increased popularity in applications such as flexible electronics and disposable sensors, as well as in wearable sensors because of its multifarious advantages. This work presents a novel, low-cost immobilization technique using inkjet-printing for the development of an aptamer-based biosensor for the detection of lysozyme, an important biomarker in various disease diagnosis. The strong affinity between the carbon nanotube (CNT) and the single-stranded DNA is exploited to immobilize the aptamers onto the working electrode by printing the ink containing the dispersion of CNT-aptamer complex. The inkjet-printing method enables aptamer density control, as well as high resolution patternability. Our developed sensor shows a detection limit of 90 ng/mL with high target selectivity against other proteins. The sensor also demonstrates a shelf-life for a reasonable period. This technology has potential for applications in developing low-cost point-of-care diagnostic testing kits for home healthcare.