Application of inkjet printing technique for biological material delivery and antimicrobial assays

A modified commercial inkjet printer was developed to deliver biological samples. The active Escherichia coli cells were directly printed at precisely targeted positions on agar-coated substrates via this technique to generate complex bacterial colony patterns. Viable cell arrays with a high density of 400 dots/cm² were obtained without the addition of any surfactants or other chemicals. Moreover, an applicable example of multiple-layer inkjet printing technique was adapted to deposit bacteria and antibiotics for antimicrobial potential assays. After fluorescent E. coli cells were printed, gradient concentrations of water-soluble antibiotics were ejected onto them to determine its minimum inhibitory concentration (MIC) to test the antimicrobial activities. This approach simplifies the experimental manipulation by replacing laborious manual loading processes with automatically controlled printing procedures, which makes it a versatile tool for high-throughput applications.     

Cell damage evaluation of thermal inkjet printed Chinese hamster ovary cells

Thermal inkjet printing technology has been applied successfully to cell printing. However, there are concerns that printing process may cause cell damages or death. We conducted a comprehensive study of thermal inkjet printed Chinese hamster ovary (CHO) cells by evaluating cell viability and apoptosis, and possible cell membrane damages. Additionally, we studied the cell concentration of bio‐ink and found optimum printing of concentrations around 8 million cells per mL. Printed cell viability was 89% and only 3.5% apoptotic cells were observed after printing. Transient pores were developed in the cell membrane of printed cells. Cells were able to repair these pores within 2 h after printing. Green fluorescent protein (GFP) DNA plasmids were delivered to CHO‐S cells by co‐printing. The transfection efficiency is above 30%. We conclude that thermal inkjet printing technology can be used for precise cell seeding with minor effects and damages to the printed mammalian cells. The printing process causes transient pores in cell membranes, a process which has promising applications for gene and macroparticles delivery to induce the biocompatibility or growth of engineered tissues. Biotechnol. Bioeng. 2010;106: 963–969. © 2010 Wiley Periodicals, Inc.     

Continuous-flow microfluidic printing of proteins for array-based applications including surface plasmon resonance imaging

Arraying proteins is often more challenging than creating oligonucleotide arrays. Protein concentration and purity can severely limit the capacity of spots created by traditional pin and ink jet printing techniques. To improve protein printing methods, we have developed a three-dimensional microfluidic system to deposit protein samples within discrete spots (250-microm squares) on a target surface. Our current technology produces a 48-spot array within a 0.5 x 1 cm target area. A chief advantage of this method is that samples may be introduced in continuous flow, which makes it possible to expose each spot to a larger volume of sample than would be possible with standard printing methods. Using Biacore Flexchip (Biacore AB) surface plasmon resonance array-based biosensor as a chip reader, we demonstrate that the microfluidic printer is capable of spotting proteins that are dilute (<0.1 microg/ml) and contain high concentrations of contaminating protein (>10,000-fold molar excess). We also show that the spots created by the microfluidic printer are more uniform and have better-defined borders than what can be achieved with pin printing. The ability to readily print proteins using continuous flow will help expand the application of protein arrays.     

Printing multistrain bacterial patterns with a piezoelectric inkjet printer

Many studies involving interacting microorganisms would benefit from simple devices able to deposit cells in precisely defined patterns. We describe an inexpensive bacterial piezoelectric inkjet printer (adapted from the design of the POSaM oligonucleotide microarrayer) that can be used to "print out" different strains of bacteria or chemicals in small droplets onto a flat surface at high resolution. The capabilities of this device are demonstrated by printing ordered arrays comprising two bacterial strains labeled with different fluorescent proteins. We also characterized several properties of this piezoelectric printer, such as the droplet volume (of the order of tens of pl), the distribution of number of cells in each droplet, and the dependence of droplet volume on printing frequency. We established the limits of the printing resolution, and determined that the printed viability of Escherichia coli exceeded 98.5%.     

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices^1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.Direct-write assembly^4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 μm). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (< 1 μm) can be patterned and assembled into larger arrays and multidimensional architectures.In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.     

Effect of polymethyl methacrylate on in situ patterning of perovskite quantum dots by inkjet printing

The preparation of perovskite quantum dots (PQDs) using an in situ inkjet printing method is beneficial for improving the problems of aggregation and photoluminescence (PL) quenching during long-term storage. However, the stability of PQDs prepared using this method is still not ideal, and the morphology of in situ-printed patterns needs to be optimized. To address these problems, this study introduced polymethyl methacrylate (PMMA) into the process of in situ inkjet printing of PQDs and explored the effect of PMMA on the in situ patterning effect of PQDs. The results showed that using a mixed precursor solution containing a small amount of PMMA as the printing ink can slow down the shrinkage process of ink droplets and improve the uniformity of film formation. As the printing substrate, PMMA provided a suitable high-viscosity environment for the in situ growth of PQDs. This could effectively suppress the coffee ring effect. In addition, the interaction between the C=O=C group in PMMA and metal ion Pb²⁺ in the CsPbBr₃ precursor molecules was favourable to enhancing the density of PQDs. The prepared PMMA-coated CsPbBr₃ quantum dots (QDs) pattern had high stability and could maintain at 90.08% PL intensity after 1 week of exposure to air.     

Improving piezoelectric cell printing accuracy and reliability through neutral buoyancy of suspensions

The sedimentation and aggregation of cells within inkjet printing systems has been hypothesized to negatively impact printer performance. The purpose of this study was to investigate this influence through the use of neutral buoyancy. Ficoll PM400 was used to create neutrally buoyant MCF‐7 breast cancer cell suspensions, which were ejected using a piezoelectric drop‐on‐demand inkjet printing system. It was found that using a neutrally buoyant suspension greatly increased the reproducibility of consistent cell counts, and eliminated nozzle clogging. Moreover, the use of Ficoll PM400 was shown to not affect cellular viability. This is the first demonstration of such scale and accuracy achieved using a piezoelectric inkjet printing system for cellular dispensing. Biotechnol. Bioeng. 2012; 109: 2932–2940. © 2012 Wiley Periodicals, Inc.     

The impact of fabrication parameters and substrate stiffness in direct writing of living constructs

Biomolecules and living cells can be printed in high‐resolution patterns to fabricate living constructs for tissue engineering. To evaluate the impact of processing cells with rapid prototyping (RP) methods, we modeled the printing phase of two RP systems that use biomaterial inks containing living cells: a high‐resolution inkjet system (BioJet) and a lower‐resolution nozzle‐based contact printing system (PAM²). In the first fabrication method, we reasoned that cell damage occurs principally during drop collision on the printing surface, in the second we hypothesize that shear stresses act on cells during extrusion (within the printing nozzle). The two cases were modeled changing the printing conditions: biomaterial substrate stiffness and volumetric flow rate, respectively, in BioJet and PAM². Results show that during inkjet printing impact energies of about 10^?8 J are transmitted to cells, whereas extrusion energies of the order of 10^?11 J are exerted in direct printing. Viability tests of printed cells can be related to those numerical simulations, suggesting a threshold energy of 10^?9 J to avoid permanent cell damage. To obtain well‐defined living constructs, a combination of these methods is proposed for the fabrication of scaffolds with controlled 3D architecture and spatial distribution of biomolecules and cells. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Piezoelectric inkjet printing of a cross-hatch immunoassay on a disposable nylon membrane

The development of a cost-effective method for manufacturing immunoassays is a key step towards their commercial use. In this study, a piezoelectric inkjet printer and a nylon membrane were used to fabricate a disposable immunoassay. Using a piezoelectric inkjet printer, a cross-hatch pattern of goat anti-mouse antibody (GαM) and rabbit anti-horseradish peroxidase (RαHRP) antibody were deposited on the nylon membrane. These patterns were subsequently treated with a solution containing rabbit anti-goat antibody labeled with horseradish peroxidase (RαG-HRP). The effectiveness of the immobilization process was examined using tetramethylbenzidine (TMB), which oxidizes in the presence of HRP to form a visible precipitate. Optical evaluation of the TMB precipitate was used to assess the precision of the features in the inkjet-printed pattern as well as antibody functionality following inkjet printing. Uniform patterns that contained functional antibodies were fabricated using the piezoelectric inkjet printer. These results suggest that piezoelectric inkjet printing may be used to fabricate low-cost disposable immunoassays for biotechnology and healthcare applications.     

A Modified Consumer Inkjet for Spatiotemporal Control of Gene Expression

This paper presents a low-cost inkjet dosing system capable of continuous, two-dimensional spatiotemporal regulation of gene expression via delivery of diffusible regulators to a custom-mounted gel culture of E. coli. A consumer-grade, inkjet printer was adapted for chemical printing; E. coli cultures were grown on 750 µm thick agar embedded in micro-wells machined into commercial compact discs. Spatio-temporal regulation of the lac operon was demonstrated via the printing of patterns of lactose and glucose directly into the cultures; X-Gal blue patterns were used for visual feedback. We demonstrate how the bistable nature of the lac operon''s feedback, when perturbed by patterning lactose (inducer) and glucose (inhibitor), can lead to coordination of cell expression patterns across a field in ways that mimic motifs seen in developmental biology. Examples of this include sharp boundaries and the generation of traveling waves of mRNA expression. To our knowledge, this is the first demonstration of reaction-diffusion effects in the well-studied lac operon. A finite element reaction-diffusion model of the lac operon is also presented which predicts pattern formation with good fidelity.     

Inkjet Printing of Back Electrodes for Inverted Polymer Solar Cells

Evaporation is the most commonly used deposition method in the processing of back electrodes in polymer solar cells used in scientific studies. However, vacuum‐based methods such as evaporation are uneconomical in the upscaling of polymer solar cells as they are throughput limiting steps in an otherwise fast roll‐to‐roll production line. In this paper, the applicability of inkjet printing in the ambient processing of back electrodes in inverted polymer solar cells with the structure ITO/ZnO/P3HT:PCBM/PEDOT:PSS/Ag is investigated. Furthermore, the limitation of screen printing, the commonly employed method in the ambient processing of back electrode, is demonstrated and discussed. Both inkjet printing and screen printing of back electrodes are studied for their impact on the photovoltaic properties of the polymer solar cells measured under 1000 Wm^?2 AM1.5. Each ambient processing technique is compared with evaporation in the processing of back electrode. Laser beam induced current (LBIC) imaging is used to investigate the impact of the processing techniques on the current collection in the devices. We report that inkjet printing of back electrode delivers devices having photovoltaic performance comparable to devices with evaporated back electrodes. We further confirm that inkjet printing represent an efficient alternative to screen printing.     

Scalable,Self‐Aligned Printing of Flexible Graphene Micro‐Supercapacitors

Graphene micro‐supercapacitors (MSCs) are an attractive energy storage technology for powering miniaturized portable electronics. Despite considerable advances in recent years, device fabrication typically requires conventional microfabrication techniques, limiting the translation to cost‐effective and high‐throughput production. To address this issue, we report here a self‐aligned printing process utilizing capillary action of liquid inks in microfluidic channels to realize scalable, high‐fidelity manufacturing of graphene MSCs. Microstructured ink receivers and capillary channels are imprinted on plastic substrates and filled by inkjet printing of functional materials into the receivers. The liquid inks move under capillary flow into the adjoining channels, allowing reliable patterning of electronic materials in complex structures with greatly relaxed printing tolerance. Leveraging this process with pristine graphene and ion gel inks, miniaturized all‐solid‐state graphene MSCs are demonstrated to concurrently achieve outstanding resolution (active footprint: <1 mm², minimum feature size: 20 µm) and yield (44/44 devices), while maintaining a high specific capacitance (268 µF cm^–2) and robust stability to extended cycling and bending, establishing an effective route to scale down device size while scaling up production throughput.     

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.     

Colorimetry as Quality Control Tool for Individual Inkjet-Printed Pediatric Formulations

Printing technologies were recently introduced to the pharmaceutical field for manufacturing of drug delivery systems. Printing allows on demand manufacturing of flexible pharmaceutical doses in a personalized manner, which is critical for a successful and safe treatment of patient populations with specific needs, such as children and the elderly, and patients facing multimorbidity. Printing of pharmaceuticals as technique generates new demands on the quality control procedures. For example, rapid quality control is needed as the printing can be done on demand and at the point of care. This study evaluated the potential use of a handheld colorimetry device for quality control of printed doses of vitamin Bs on edible rice and sugar substrates. The structural features of the substrates with and without ink were also compared. A multicomponent ink formulation with vitamin B₁, B₂, B₃, and B₆ was developed. Doses (4 cm²) were prepared by applying 1–10 layers of yellow ink onto the white substrates using thermal inkjet technology. The colorimetric method was seen to be viable in detecting doses up to the 5th and 6th printed layers until color saturation of the yellow color parameter (b*) was observed on the substrates. Liquid chromatography mass spectrometry was used as a reference method for the colorimetry measurements plotted against the number of printed layers. It was concluded that colorimetry could be used as a quality control tool for detection of different doses. However, optimization of the color addition needs to be done to avoid color saturation within the planned dose interval.     

Patterned Immobilization of Antibodies within Roll-to-Roll Hot Embossed Polymeric Microfluidic Channels

This paper describes a method for the patterned immobilization of capture antibodies into a microfluidic platform fabricated by roll-to-roll (R2R) hot embossing on poly (methyl methacrylate) (PMMA). Covalent attachment of antibodies was achieved by two sequential inkjet printing steps. First, a polyethyleneimine (PEI) layer was deposited onto oxygen plasma activated PMMA foil and further cross-linked with glutaraldehyde (GA) to provide an amine-reactive aldehyde surface (PEI-GA). This step was followed by a second deposition of antibody by overprinting on the PEI-GA patterned PMMA foil. The PEI polymer ink was first formulated to ensure stable drop formation in inkjet printing and the printed films were characterized using atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). Anti-CRP antibody was patterned on PMMA foil by the developed method and bonded permanently with R2R hot embossed PMMA microchannels by solvent bonding lamination. The functionality of the immobilized antibody inside the microfluidic channel was evaluated by fluorescence-based sandwich immunoassay for detection of C-reactive protein (CRP). The antibody-antigen assay exhibited a good level of linearity over the range of 10 ng/ml to 500 ng/ml (R² = 0.991) with a calculated detection limit of 5.2 ng/ml. The developed patterning method is straightforward, rapid and provides a versatile approach for creating multiple protein patterns in a single microfluidic channel for multiplexed immunoassays.     

Biological deinking of inkjet-printed paper using Vibrio alginolyticus and its enzymes

Recycling of office waste paper (photocopy, inkjet, and laser prints) is a major problem due to difficulty in removal of nonimpact ink. Biological deinking of office waste paper is reported using several microorganisms and their enzymes. We report here deinking and decolorization of the dislodged ink particles from inkjet printed paper pulp by a marine bacterium, Vibrio alginolyticus isolate no. NIO/DI/32, obtained from marine sediments. Decolorization of this pulp was achieved within 72 h by growing the bacterium in the pulp of 3–6% consistency suspended in seawater. Immobilized bacterial cells in sodium alginate beads were also able to decolorize this pulp within 72 h. The cell-free culture supernatant of the bacterium grown in nutrient broth was not effective in deinking. However, when the culture was grown in nutrient broth supplemented with starch or Tween 80, the cell-free culture supernatant could effectively deink and decolorize inkjet-printed paper pulp within 72 h at 30°C. The culture supernatant of V. alginolyticus grown in the presence of starch or Tween 80 showed 49 U ml⁻¹ and 33 U ml⁻¹ amylase and lipase activities, respectively. Dialysis of these culture supernatants through 10 kDa cut-off membrane resulted in a 35–40% reduction in their efficiency in decolorizing the pulp. It appears that amylase and lipase effectively help in dislodging the ink particles from the inkjet printed-paper pulp. We hypothesize that the bacterium might be inducing the formation of low molecular weight free radicals in the culture medium, which might be responsible for decolorization of the pulp.     

The Effect of Inkjet Printing over Polymeric Films as Potential Buccal Biologics Delivery Systems

The buccal mucosa appears as a promissory route for biologic drug administration, and pharmaceutical films are flexible dosage forms that can be used in the buccal mucosa as drug delivery systems for either a local or systemic effect. Recently, thin films have been used as printing substrates to manufacture these dosage forms by inkjet printing. As such, it is necessary to investigate the effects of printing biologics on films as substrates in terms of their physical and mucoadhesive properties. Here, we explored solvent casting as a conventional method with two biocompatible polymers, hydroxypropyl methylcellulose, and chitosan, and we used electrospinning process as an electrospun film fabrication of polycaprolactone fibers due to its potential to elicit mucoadhesion. Lysozyme was used as biologic drug model and was formulated as a solution for printing by thermal inkjet printing. Films were characterized before and after printing by mechanical and mucoadhesive properties, surface, and ultrastructure morphology through scanning electron microscopy and solid state properties by thermal analysis. Although minor differences were detected in micrographs and thermograms in all polymeric films tested, neither mechanical nor mucoadhesive properties were affected by these differences. Thus, biologic drug printing on films was successful without affecting their mechanical or mucoadhesive properties. These results open way to explore biologics loading on buccal films by inkjet printing, and future efforts will include further in vitro and in vivo evaluations.     

Organ printing: computer-aided jet-based 3D tissue engineering

Tissue engineering technology promises to solve the organ transplantation crisis. However, assembly of vascularized 3D soft organs remains a big challenge. Organ printing, which we define as computer-aided, jet-based 3D tissue-engineering of living human organs, offers a possible solution. Organ printing involves three sequential steps: pre-processing or development of "blueprints" for organs; processing or actual organ printing; and postprocessing or organ conditioning and accelerated organ maturation. A cell printer that can print gels, single cells and cell aggregates has been developed. Layer-by-layer sequentially placed and solidified thin layers of a thermo-reversible gel could serve as "printing paper". Combination of an engineering approach with the developmental biology concept of embryonic tissue fluidity enables the creation of a new rapid prototyping 3D organ printing technology, which will dramatically accelerate and optimize tissue and organ assembly.     

3D Printing Sulfur Copolymer‐Graphene Architectures for Li‐S Batteries

3D printing is becoming an efficient approach to facilely and accurately fabricate diverse complex architectures with broad applications. However, suitable inks and 3D print favorable architectures with high electrochemical performances for energy storage are still being explored. Here, sulfur copolymer‐graphene architectures with well‐designed periodic microlattices are 3D printed as a cathode for Li‐S batteries using a suitable ink composed of sulfur particles, 1,3‐diisopropenylbenzene (DIB), and condensed graphene oxide dispersion. Using thermal treatment, elemental sulfur can be reacted with DIB to produce sulfur copolymer, which can partially suppress the dissolution of polysulfides. Moreover, graphene in the architecture can provide high electrical conductivity for whole electrode. Hence, 3D printed sulfur copolymer‐graphene architecture exhibits a high reversible capacity of 812.8 mA h g^?1 and good cycle performance. Such a simple 3D printing approach can be further extended to construct many complex architectures for various energy storage devices.     

An amperometric glucose biosensor prototype fabricated by thermal inkjet printing

The prototype of an amperometric glucose biosensor was realized by thermal inkjet printing using biological and electronic water-based inks, containing a glucose oxidase (GOD) from Aspergillus niger and the conducting polymer blend poly(3,4-ethylenedioxythiophene/polystyrene sulfonic acid) (PEDOT/PSS), respectively. The biosensor was fabricated microdepositing PEDOT/PSS and GOD, in sequence, on ITO-glass, by a commercial inkjet printer, with the help of a commercial software. High density microdots matrices were so-realized, with a calculated resolution of about 221 x 221 dpi (dot per inch). By means of a rapid and easy assay it was demonstrated that no activity loss occurred upon the printing of GOD, despite of the use of a thermal printhead. The device was encapsulated in a semipermeable membrane of cellulose acetate, applied by dip-coating, in order to prevent dissolution of the enzyme and/or PEDOT/PSS in water. The preliminary response of the electrode was measured in an aqueous glucose solution in the presence of ferrocenemethanol (FeMeOH) as a mediator, and resulted linear up to 60 mM in glucose. The best sensitivity value achieved was 6.43 microAM(-1) cm(-2) (447 nAM(-1) U(-1) cm(-2)). The characteristics of the device, and the possible performance improvements have been analyzed and discussed. The reported findings indicate that inkjet printing could be a viable instrument for the easy construction of a working biosensor via direct digital design using biological and conductive polymer based inks. Such an approach may be seen as an example of "biopolytronics".