Bioprinting on Organ-on-Chip: Development and Applications.

Organs-on-chips (OoCs) are microfluidic devices that contain bioengineered tissues or parts of natural tissues or organs and can mimic the crucial structures and functions of living organisms. They are designed to control and maintain the cell- and tissue-specific microenvironment while also providing detailed feedback about the activities that are taking place. Bioprinting is an emerging technology for constructing artificial tissues or organ constructs by combining state-of-the-art 3D printing methods with biomaterials. The utilization of 3D bioprinting and cells patterning in OoC technologies reinforces the creation of more complex structures that can imitate the functions of a living organism in a more precise way. Here, we summarize the current 3D bioprinting techniques and we focus on the advantages of 3D bioprinting compared to traditional cell seeding in addition to the methods, materials, and applications of 3D bioprinting in the development of OoC microsystems.

Laser Bioprinting of Cells Using UV and Visible Wavelengths: A Comparative DNA Damage Study.

Laser-based techniques for printing cells onto different substrates with high precision and resolution present unique opportunities for contributing to a wide range of biomedical applications, including tissue engineering. In this study, laser-induced forward transfer (LIFT) printing was employed to rapidly and accurately deposit patterns of cancer cells in a non-contact manner, using two different wavelengths, 532 and 355 nm. To evaluate the effect of LIFT on the printed cells, their growth and DNA damage profiles were assessed and evaluated quantitatively over several days. The damaging effect of LIFT-printing was thoroughly investigated, for the first time at a single cell level, by counting individual double strand breaks (DSB). Overall, we found that LIFT was able to safely print patterns of breast cancer cells with high viability with little or no heat or shear damage to the cells, as indicated by unperturbed growth and negligible gross DNA damage.

Indium Tin Oxide-Free Inverted Organic Photovoltaics Using Laser-Induced Forward Transfer Silver Nanoparticle Embedded Metal Grids.

Laser-induced forward transfer (LIFT) printing has emerged as a valid digital printing technique capable of transferring and printing a wide range of electronic materials. In this paper, we present for the first time LIFT printing as a method to fabricate silver (Ag) nanoparticle (np) grids for the development of indium tin oxide (ITO)-free inverted PM6:Y6 nonfullerene acceptor organic photovoltaics (OPVs). Limitations of the direct use of LIFT-printed Ag np grids in inverted ITO-free OPVs are addressed through a Ag grid embedding process. The embedded laser-printed Ag grid lines have high electrical conductivity, while the Ag metal grid transparency is varied by altering the number of Ag grid lines within the inverted OPVs' ITO-free bottom electrode. Following the presented Ag-grid embedding (EMP) process, metal-grid design optimizations, and device engineering methods incorporating an EMB-nine-line Ag np grid/PH500/AI4083/ZnO bottom electrode, we have demonstrated inverted ITO-free OPVs incorporating laser-printed Ag grids with 11.0% power conversion efficiency.

Laser-Induced Forward Transfer Printing on Microneedles for Transdermal Delivery of Gemcitabine.

Cancer treatment with chemotherapeutic drugs remains to be challenging to the physician due to limitations associated with lack of efficacy or high toxicities. Typically, chemotherapeutic drugs are administered intravenously, leading to high drug concentrations that drive efficacy but also lead to known side effects. Delivery of drugs through transdermal microneedles (MNs) has become an important alternative treatment approach. Such delivery options are well suited for chemotherapeutic drugs in which sustained levels would be desirable. In the context of developing a novel approach, laser-induced forward transfer (LIFT) was applied for bioprinting of gemcitabine (Gem) to coat polymethylmethacrylate MNs. Gem, a chemotherapeutic agent used to treat various types of cancer, is a good candidate for MN-assisted transdermal delivery to improve the pharmacokinetics of Gem while reducing efficiency limitations. LIFT bioprinting of Gem for coating of MNs with different drug amounts and successful transdermal delivery in mice is presented in this study. Our approach produced reproducible, accurate, and uniform coatings of the drug on MN arrays, and on in vivo transdermal application of the coated MNs in mice, dose-proportional concentrations of Gem in the plasma of mice was achieved. The developed approach may be extended to several chemotherapeutics and provide advantages for metronomic drug dosing.

Parametric Study of Jet/Droplet Formation Process during LIFT Printing of Living Cell-Laden Bioink.

Bioprinting offers great potential for the fabrication of three-dimensional living tissues by the precise layer-by-layer printing of biological materials, including living cells and cell-laden hydrogels. The laser-induced forward transfer (LIFT) of cell-laden bioinks is one of the most promising laser-printing technologies enabling biofabrication. However, for it to be a viable bioprinting technology, bioink printability must be carefully examined. In this study, we used a time-resolved imaging system to study the cell-laden bioink droplet formation process in terms of the droplet size, velocity, and traveling distance. For this purpose, the bioinks were prepared using breast cancer cells with different cell concentrations to evaluate the effect of the cell concentration on the droplet formation process and the survival of the cells after printing. These bioinks were compared with cell-free bioinks under the same printing conditions to understand the effect of the particle physical properties on the droplet formation procedure. The morphology of the printed droplets indicated that it is possible to print uniform droplets for a wide range of cell concentrations. Overall, it is concluded that the laser fluence and the distance of the donor-receiver substrates play an important role in the printing impingement type; consequently, a careful adjustment of these parameters can lead to high-quality printing.

Eco-Friendly Lead-Free Solder Paste Printing via Laser-Induced Forward Transfer for the Assembly of Ultra-Fine Pitch Electronic Components.

Current challenges in printed circuit board (PCB) assembly require high-resolution deposition of ultra-fine pitch components (<0.3 mm and <60 µm respectively), high throughput and compatibility with flexible substrates, which are poorly met by the conventional deposition techniques (e.g., stencil printing). Laser-Induced Forward Transfer (LIFT) constitutes an excellent alternative for assembly of electronic components: it is fully compatible with lead-free soldering materials and offers high-resolution printing of solder paste bumps (<60 µm) and throughput (up to 10,000 pads/s). In this work, the laser-process conditions which allow control over the transfer of solder paste bumps and arrays, with form factors in line with the features of fine pitch PCBs, are investigated. The study of solder paste as a function of donor/receiver gap confirmed that controllable printing of bumps containing many microparticles is feasible for a gap < 100 µm from a donor layer thickness set at 100 and 150 µm. The transfer of solder bumps with resolution < 100 µm and solder micropatterns on different substrates, including PCB and silver pads, have been achieved. Finally, the successful operation of a LED interconnected to a pin connector bonded to a laser-printed solder micro-pattern was demonstrated.

A Miniature Bio-Photonics Companion Diagnostics Platform for Reliable Cancer Treatment Monitoring in Blood Fluids.

In this paper, we present the development of a photonic biosensor device for cancer treatment monitoring as a complementary diagnostics tool. The proposed device combines multidisciplinary concepts from the photonic, nano-biochemical, micro-fluidic and reader/packaging platforms aiming to overcome limitations related to detection reliability, sensitivity, specificity, compactness and cost issues. The photonic sensor is based on an array of six asymmetric Mach Zender Interferometer (aMZI) waveguides on silicon nitride substrates and the sensing is performed by measuring the phase shift of the output signal, caused by the binding of the analyte on the functionalized aMZI surface. According to the morphological design of the waveguides, an improved sensitivity is achieved in comparison to the current technologies (<5000 nm/RIU). This platform is combined with a novel biofunctionalization methodology that involves material-selective surface chemistries and the high-resolution laser printing of biomaterials resulting in the development of an integrated photonics biosensor device that employs disposable microfluidics cartridges. The device is tested with cancer patient blood serum samples. The detection of periostin (POSTN) and transforming growth factor beta-induced protein (TGFBI), two circulating biomarkers overexpressed by cancer stem cells, is achieved in cancer patient serum with the use of the device.

Facile and Low-Cost SPE Modification Towards Ultra-Sensitive Organophosphorus and Carbamate Pesticide Detection in Olive Oil.

Despite the fact that a considerable amount of effort has been invested in the development of biosensors for the detection of pesticides, there is still a lack of a simple and low-cost platform that can reliably and sensitively detect their presence in real samples. Herein, an enzyme-based biosensor for the determination of both carbamate and organophosphorus pesticides is presented that is based on acetylcholinesterase (AChE) immobilized on commercially available screen-printed carbon electrodes (SPEs) modified with carbon black (CB), as a means to enhance their conductivity. Most interestingly, two different methodologies to deposit the enzyme onto the sensor surfaces were followed; strikingly different results were obtained depending on the family of pesticides under investigation. Furthermore, and towards the uniform application of the functionalization layer onto the SPEs' surfaces, the laser induced forward transfer (LIFT) technique was employed in conjunction with CB functionalization, which allowed a considerable improvement of the sensor's performance. Under the optimized conditions, the fabricated sensors can effectively detect carbofuran in a linear range from 1.1 × 10-9 to 2.3 × 10-8 mol/L, with a limit of detection equal to 0.6 × 10-9 mol/L and chlorpyrifos in a linear range from 0.7 × 10-9 up to 1.4 × 10-8 mol/L and a limit of detection 0.4 × 10-9 mol/L in buffer. The developed biosensor was also interrogated with olive oil samples, and was able to detect both pesticides at concentrations below 10 ppb, which is the maximum residue limit permitted by the European Food Safety Authority.

Bioprinting for Liver Transplantation.

Bioprinting techniques can be used for the in vitro fabrication of functional complex bio-structures. Thus, extensive research is being carried on the use of various techniques for the development of 3D cellular structures. This article focuses on direct writing techniques commonly used for the fabrication of cell structures. Three different types of bioprinting techniques are depicted: Laser-based bioprinting, ink-jet bioprinting and extrusion bioprinting. Further on, a special reference is made to the use of the bioprinting techniques for the fabrication of 2D and 3D liver model structures and liver on chip platforms. The field of liver tissue engineering has been rapidly developed, and a wide range of materials can be used for building novel functional liver structures. The focus on liver is due to its importance as one of the most critical organs on which to test new pharmaceuticals, as it is involved in many metabolic and detoxification processes, and the toxicity of the liver is often the cause of drug rejection.

Selective Laser Sintering of Laser Printed Ag Nanoparticle Micropatterns at High Repetition Rates.

The increasing development of flexible and printed electronics has fueled substantial advancements in selective laser sintering, which has been attracting interest over the past decade. Laser sintering of metal nanoparticle dispersions in particular (from low viscous inks to high viscous pastes) offers significant advantages with respect to more conventional thermal sintering or curing techniques. Apart from the obvious lateral selectivity, the use of short-pulsed and high repetition rate lasers minimizes the heat affected zone and offers unparalleled control over a digital process, enabling the processing of stacked and pre-structured layers on very sensitive polymeric substrates. In this work, the authors have conducted a systematic investigation of the laser sintering of micro-patterns comprising Ag nanoparticle high viscous inks: The effect of laser pulse width within the range of 20⁻200 nanoseconds (ns), a regime which many commercially available, high repetition rate lasers operate in, has been thoroughly investigated experimentally in order to define the optimal processing parameters for the fabrication of highly conductive Ag patterns on polymeric substrates. The in-depth temperature profiles resulting from the effect of laser pulses of varying pulse widths have been calculated using a numerical model relying on the finite element method, which has been fed with physical parameters extracted from optical and structural characterization. Electrical characterization of the resulting sintered micro-patterns has been benchmarked against the calculated temperature profiles, so that the resistivity can be associated with the maximal temperature value. This quantitative correlation offers the possibility to predict the optimal process window in future laser sintering experiments. The reported computational and experimental findings will foster the wider adoption of laser micro-sintering technology for laboratory and industrial use.

Phosphate Modified Screen Printed Electrodes by LIFT Treatment for Glucose Detection.

The design of new materials as active layers is important for electrochemical sensor and biosensor development. Among the techniques for the modification and functionalization of electrodes, the laser induced forward transfer (LIFT) has emerged as a powerful physisorption method for the deposition of various materials (even labile materials like enzymes) that results in intimate and stable contact with target surface. In this work, Pt, Au, and glassy carbon screen printed electrodes (SPEs) treated by LIFT with phosphate buffer have been characterized by scanning electron microscopy and atomic force microscopy to reveal a flattening effect of all surfaces. The electrochemical characterization by cyclic voltammetry shows significant differences depending on the electrode material. The electroactivity of Au is reduced while that of glassy carbon and Pt is greatly enhanced. In particular, the electrochemical behavior of a phosphate LIFT treated Pt showed a marked enrichment of hydrogen adsorbed layer, suggesting an elevated electrocatalytic activity towards glucose oxidation. When Pt electrodes modified in this way were used as an effective glucose sensor, a 1⁻10 mM linear response and a 10 µM detection limit were obtained. A possible role of phosphate that was securely immobilized on a Pt surface, as evidenced by XPS analysis, enhancing the glucose electrooxidation is discussed.

Single Step Laser Transfer and Laser Curing of Ag NanoWires: A Digital Process for the Fabrication of Flexible and Transparent Microelectrodes.

Ag nanowire (NW) networks have exquisite optical and electrical properties which make them ideal candidate materials for flexible transparent conductive electrodes. Despite the compatibility of Ag NW networks with laser processing, few demonstrations of laser fabricated Ag NW based components currently exist. In this work, we report on a novel single step laser transferring and laser curing process of micrometer sized pixels of Ag NW networks on flexible substrates. This process relies on the selective laser heating of the Ag NWs induced by the laser pulse energy and the subsequent localized melting of the polymeric substrate. We demonstrate that a single laser pulse can induce both transfer and curing of the Ag NW network. The feasibility of the process is confirmed experimentally and validated by Finite Element Analysis simulations, which indicate that selective heating is carried out within a submicron-sized heat affected zone. The resulting structures can be utilized as fully functional flexible transparent electrodes with figures of merit even higher than 100. Low sheet resistance (<50 Ohm/sq) and high visible light transparency (>90%) make the reported process highly desirable for a variety of applications, including selective heating or annealing of nanocomposite materials and laser processing of nanostructured materials on a large variety of optically transparent substrates, such as Polydimethylsiloxane (PDMS).

Direct Creation of Biopatterns via a Combination of Laser-Based Techniques and Click Chemistry.

In this paper, we present the immobilization of thiol-modified aptamers on alkyne- or alkene-terminated silicon nitride surfaces by laser-induced click chemistry reactions. The aptamers are printed onto the surface by laser-induced forward transfer (LIFT), which also induces the covalent bonding of the aptamers by thiol-ene or thiol-yne reactions that occur upon UV irradiation of the thiol-modified aptamers with ns laser pulses. This combination of LIFT and laser-induced click chemistry allows the creation of high-resolution patterns without the need for masks. Whereas the click chemistry already takes place during the printing process (single-step process) by the laser pulse used for the printing process, further irradiation of the LIFT-printed aptamers by laser pulses (two-step process) leads to a further increase in the immobilization efficiency.

Laser Induced Forward Transfer: a study on the enhancement of sensitivity for multianalyte sensing.

In this article novel approaches for the improvement of the recorded signal coupled with the feasibility of multiple analyte detection, irrespective of the biosensor platform are being presented. The techniques that have been developed address commonly encountered issues that have traditionally hindered the commercialization of biosensors, such as cost, reproducibility and sensitivity and most importantly multianalyte detection. The fluorescence-based detection of copper is being described as an example of the use of Laser Induced Forward Transfer technique (LIFT) for the immobilization of biomolecules with high spatial resolution, in addition to a technique that involves the displacement of a short complementary strand to the immobilized probe molecule for the quantification of analyte binding and the enhancement of the recorded signal.

Label-free DNA biosensor based on resistance change of platinum nanoparticles assemblies.

A novel nanoparticle based biosensor for the fast and simple detection of DNA hybridization events is presented. The sensor utilizes hybridized DNA's charge transport properties, combining them with metallic nanoparticle networks that act as nano-gapped electrodes. The DNA hybridization events can be detected by a significant reduction in the sensor's resistance due to the conductive bridging offered by hybridized DNA. By modifying the nanoparticle surface coverage, which can be controlled experimentally being a function of deposition time, and the structural properties of the electrodes, an optimized biosensor for the in situ detection of DNA hybridization events is ultimately fabricated. The fabricated biosensor exhibits a wide response range, covering four orders of magnitude, a limit of detection of 1nM and can detect a single base pair mismatch between probe and complementary DNA.

Superamphiphobic polymeric surfaces sustaining ultrahigh impact pressures of aqueous high- and low-surface-tension mixtures, tested with laser-induced forward transfer of drops.

Superamphiphobic, (quasi-)ordered plasma-textured surfaces, coated with a perfluorinated monolayer, exhibit extreme resistance against drop-pinning for both water-like and low-surface-tension mixtures (36 mN m(-1)). The highest values reported here are 36 atm for a water-like mixture, 5 times higher than previously reported in the literature, and 7 atm for a low-surface-tension mixture, the highest ever reported value for lotus-leaf-inspired surfaces.

A photosynthetic biosensor with enhanced electron transfer generation realized by laser printing technology.

One of the limits of current electrochemical biosensors is a lack of methods providing stable and highly efficient junctions between biomaterial and solid-state devices. This paper shows how laser-induced forward transfer (LIFT) can enable efficient electron transfer from photosynthetic biomaterial immobilized on screen-printed electrodes (SPE). The ideal pattern, in terms of photocurrent signal of thylakoid droplets giving a stable response signal with a current intensity of approximately 335 ± 13 nA for a thylakoid mass of 28 ± 4 ng, was selected. It is shown that the efficiency of energy production of a photosynthetic system can be strongly enhanced by the LIFT process, as demonstrated by use of the technique to construct an efficient and sensitive photosynthesis-based biosensor for detecting herbicides at nanomolar concentrations.

Detection of DNA mutations using a capacitive micro-membrane array.

The detection of DNA hybridization using capacitive readout and a biosensor array of ultrathin Si membranes is presented. The biosensor exploits the ability of the ultrathin membranes to deflect upon surface stress variations caused by biological interactions. Probe DNA molecules are immobilized on the membrane surface and the surface stress variations during hybridization with their complementary strands force the membrane to deflect and effectively change the capacitance between the flexible membrane and the fixed substrate. The sensor array comprises 256 such sensing sites thus allowing the concurrent sensing of multiple DNA mutations. The biosensor and its performance for the detection of complementary DNA strands are demonstrated using beta-thalassemia oligonucleotides. The experimental results show that the presented sensors are able to detect DNA hybridization and to discriminate single nucleotide mismatches.

Ballistic laser-assisted solid transfer (BLAST) from a thin film precursor.

A novel technique for the laser-induced forward transfer (LIFT) of material in solid phase from a thin film precursor is presented. Multiple, sub-threshold energy femtosecond pulses are used to lessen the adhesion of a donor film to a support substrate to facilitate forward transfer of solid 'pellets' of donor material to a receiver. A relatively higher intensity outer ring is added to the transfer laser pulses, by means of the near-field diffraction pattern of a circular aperture, to define the area for transfer in the donor film and allow for more reproducible pellet shapes. This technique has been termed Ballistic Laser-Assisted Solid Transfer (BLAST).