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3D Printing Natural Organic Materials by Photochemistry
Abstract
In previous works, we have used two-photon induced photochemistry to fabricate 3D microstructures based on proteins, anti-bodies, and enzymes for different types of bio-applications. Among them, we can cite collagen lines to guide the movement of living cells, peptide modified GFP biosensing pads to detect Gram positive bacteria, anti-body pads to determine the type of red blood cells, and trypsin columns in a microfluidic channel to obtain a real time biochemical micro-reactor. In this paper, we report for the first time on two-photon 3D microfabrication of DNA material. We also present our preliminary results on using a commercial 3D printer based on a video projector to polymerize slicing layers of gelatine-objects.
Les technologies de photopolymérisation en cuve émergent rapidement dans le domaine de la fabrication additive. Pour suivre cette expansion rapide du marché, des résines photosensibles très efficaces et abordables sont nécessaires. Dans ce travail, nous introduisons un nouveau système photoamorceur à trois composants (3K PIS) basé sur le colorant Safranine O (SFH+) qui a été identifié comme un composé très efficace dans plusieurs 3K PIS pour les processus de photopolymérisation.Le colorant est combiné avec un sel de tétraphénylborate (TPB) comme donneur d'électrons et un dérivé de la triazine (TA) comme accepteur d'électrons pour former un système photocyclique. Le mécanisme réactionnel est exploré par photolyse laser éclair (LFP) et la photopolymérisation est étudiée par spectroscopie infrarouge à transformée de Fourier en temps réel (RT-FTIR). Des expériences infrarouges avec plusieurs irradiances permettent de créer un modèle empirique prédisant la conversion en fonction du temps et de l'intensité lumineuse.Ensuite, des expériences de profondeur de polymérisation sont menées suivant l'équation de Jacobs, permettant d’obtenir les paramètres d'impression3D de la résine ; à savoir l'énergie critique (Ec) et la profondeur de pénétration (Dp). Ces paramètres sont reliés aux analyses RT-FTIR, ce qui permet de déterminer le temps critique (tc) et la conversion au point de gel.Enfin, des pièces complexes de haute résolution sont imprimées avec la résine dont la composition a été sélectionnée en fonction de nos résultats, démontrant la viabilité de cette formulation pour l'impression 3D DLP.
This commentary discusses current capabilities of vat photopolymerization, an additive manufacturing (AM) technique also known as VP, with recent advances in the literature, current challenges/limitations, and future outlook in novel materials design. Current trends and recent research advances are broadly discussed covering a spectrum of material classes such as performance, medicine, energy, and active materials in parallel to their importance in diverse technologies. Current challenges and limitations of VP are also discussed in terms of material properties, photodegradation, and material toxicity with directions in future material design to overcome these challenges. This commentary paper is intended to be of broad interest to both chemists and engineers actively involved in the AM field, in terms of future material design and processing for further development of VP-based AM technology.
In this paper, we present a new fabrication of micro‐lens array (MLA) with pinhole array—pinhole/micro‐lens array (P/MLA) for integral imaging 3‐D display (II), which combine lithography and ink‐jet printing. A black circular groove array (BCGA) is used as pinhole array, and laser 3‐D microscope and a homemade setup have been used for the characterization of P/MLA. The results show that high‐precision P/MLA can be obtained using BCGA as templates. By controlling the driving voltage at different steps, the distance between nozzle and substrate, as well as the number of liquid droplets, P/MLA with smooth morphology, different sizes, good repeatability of geometry parameters, great uniformity of focusing, and good converging performance can be achieved. For demonstration, P/MLA with curvature, focal length, numerical aperture, and F‐number of 815.8 μm, 1.60 mm, 0.1311, and 3.8 are applied for the reconstruction in II, exhibiting good reconstruction performance with high resolution, and BCGA reduces the influence of stray light on II and improves the quality of the reconstructed image. We present a new structure of micro‐lens arrays with pinhole arrays—pinhole/micro‐lens arrays, for improving resolution of integral imaging 3‐D display, which can fabricate by combination of lithography and ink‐jet printing. Millet III is used as monitor to display elemental images, which are produced by matlab, and integral imaging prototype built with monitor and pinhole/micro‐lens arrays can produce reconstructed images with high quality.
Supramolecular chemistry continues to experience widespread growth, as fine-tuned chemical structures lead to well-defined bulk materials. Previous literature described the roles of hydrogen bonding, ionic aggregation, guest/host interactions and π-π stacking to tune mechanical, viscoelastic, and processing performance. The versatility of reversible interactions enables the more facile manufacturing of molded parts with tailored hierarchical structures such as tissue engineered scaffolds for biological applications. Recently, supramolecular polymers and additive manufacturing processes merged to provide parts with control of the molecular, macromolecular, and feature length scales. Additive manufacturing, or 3D printing, generates customizable constructs desirable for many applications, and the introduction of supramolecular interactions will potentially increase production speed, offer a tunable surface structure for controlling cell-scaffold interactions, and impart desired mechanical properties through reinforcing inter-layer adhesion and introducing gradients or self-assembled structures. This review details the synthesis and characterization of supramolecular polymers suitable for additive manufacture and biomedical applications as well as the use of supramolecular polymers in additive manufacturing for drug delivery and complex tissue scaffold formation. The effect of supramolecular assembly and its dynamic behavior offers potential for controlling anisotropy of the printed objects with exquisite geometrical control. The potential for supramolecular polymers to generate well-defined parts, hierarchical structures, and scaffolds with gradient properties/tuned surfaces provides an avenue for developing next-generation biomedical devices and tissue scaffolds.
In this study, bio-sensing pads are proposed to capture living cells, which are fabricated on cover glasses by cross-linking proteins/antibodies using laser induced photochemistry. The biological functions of the cross-linked protein/antibody were verified by capturing Staphylococcus aureus (S. aureus), Leptospira, and red blood cells (RBCs), separately, with associated protein/antibody sensing pads. The experimental results show that S. aureus were bound on GFP-AcmA’ pad after minutes of incubation and phosphate buffered saline (PBS) rinsing. No binding was observed with reference pad
made of neutral bovine serum albumin (BSA). Second, A-type RBCs were chosen as the model cell to demonstrate the blood typing feasibility of the anti-A pad in microchannel. The A-type RBCs were captured only by the anti-A pad, but not the reference pad made of BSA. The same experimental model was carried out on the Leptospira, which stuck on the blood serum pad after PBS rinsing, but not BSA pad. This study provides a potential platform for simple and direct detection of living full cells without culture that could be used in point-of-care settings.
A serviceable bioprobe is one of the important components for the developments of microfluidic and lab-on-a-chip systems. In this paper, we report a novel bioprobe fabricated by laser-induced cross-linking technology for simple and direct screen of Gram-positive bacteria on a biochip. The AcmA' protein is known to bind specifically to peptidoglycan (PG) that forms the thick outside layer of Gram-positive bacteria. Moreover, the AcmA' protein has a much broad spectrum of bacterium types than do antibodies that are more specific to only one bacterium type because the AcmA' protein is a generic characteristic of Gram-positive bacteria. Green fluorescent protein (GFP) is generally used as a mecular marker. In this study, the GFP was fused with the AcmA' protein to act as an indicator for tracing the AcmA' binding activity on PG by green fluorescence. The GFP-AcmA’ protein was three-dimensionally structured by laser induced cross-linking photochemistry technology to fabricate a bioprobe for capturing Gram-positive bacteria. The positive and negative tests on Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus and Streptococcus agalactiae were demonstrated respectively. Screening is readily performed using optical microscopy observation. The experiments show that only Gram-positive bacteria were bound on GFP-AcmA’ probes after minutes of incubation and phosphate buffered saline (PBS) rinsing. No binding was observed in the Gram-negative bacteria or in reference probes composed of neutral bovine serum albumin (BSA). The repeated experiments indicate that our bioprobes are reusable. Finally, a 3D wedge-shaped GFP-AcmA' probe was demonstrated in a microfluidic channel. This study provides a novel platform for convenient Gram-positive bacteria screen that could potentially be used in lab-on-a-chip applications.
This study highlights some of the effects of UV crosslinking of DNA-CTMA
on its electrical and optical characteristics. The crosslinking of
DNA-CTMA occurs via the photodimerization of attached coumarin moieties
under UV irradiation. An exposure time of 30 min to UV light with an
output power of 166 mW/cm2 is needed to complete the
crosslinking process. The UV-crosslinked films show a significant
increase in the electrical resistivity (decrease in leakage current) and
a markedly lower dielectric constant. This paper,
originally published on 1 October 2013, was replaced with a
corrected/revised version on 25 October 2013. If you downloaded the
original PDF but are unable to access the revision, please contact SPIE
Digital Library Customer Service for assistance.
Measurements of optical tweezers forces on biological micro-objects can be used to develop innovative We review our recent results towards the development of a turnkey 3D laser printer, based on self-Q-switched microchip Nd:YAG lasers, with reproducible sub-100nm resolution, and with large-scale (cm) and fast-speed (cm/sec) capability at micron resolution. First of all, we report on line fabrication with 70nm lateral, and 150nm longitudinal resolutions without significant shrinking. This is due to the tight focusing with green visible wavelength, large numerical aperture, and excellent resin properties. Secondly, we report on two-photon sensitive photoacid generators that lead to efficient 3D microfabrication with epoxy SU-8 resin. Thirdly, we demonstrate high-speed microfabrication of large scale, millimeter size, scaffolds and cemtimeter height needle with high repetition rate (130 Khz), and high average power (1W) amplified microchip laser. Finally we demonstrate the two-photon induced cross-linking of antibodies to determine the type of red blood cells in microfluidic channels.
We review our recent advances in two-photon induced photochemistry to fabricate three-dimensional micro-objects made in polymers, proteins and noble metals using Q-switched Nd:YAG microchip lasers. We have synthesized a new photoinitiator that is about 4 times more sensitive for two-photon polymerisation with sub-nanosecond pulses at 532 nm. We describe the improvement of our fabrication process and strategies to obtain solid microstructures that correspond to their models. We report on our progress to make silver microstructures by the photoreduction of silver nitrate with microchip lasers at 1064nm.
A simple polymerisation strategy allows water solubilisation of chromophores for biophotonics, with good conservation of their fluorescence quantum-yield. Preliminary investigations show that the resulting objects are valuable candidates for photodynamic therapy and two-photon fluorescence imaging.
In recent years, two-photon laser scanning microscopy (TPLSM), especially intravital, has emerged as an essential tool
in biomedical research and has become a routine technique to track biological processes down to the cellular level in living animals. In this context, fuorophores presenting electronic transitions of the “intramolecular charge transfer” (ICT) type are considered as a particularly appealing class of molecules. The peculiar linear (environmental sensitivity, olvatochromism) and nonlinear (two-photon absorption, second harmonic generation) spectroscopic properties ssociated with ICT transitions have been taken advantage of for the development of functional materials, including H or
polarity probesand biosensors with one or two-photon induced fuorescence. These fuorophores are inherently lipophilic, because of their extended conjugated carbon backbone. Thus, major efforts have been put into the improvement of their solubility in biological media and their biocompatibility. In this context, it has been shown by Müllen et al. that controlled growth of a star-shaped polymer shell around a central fuorophore is a particularly effective strategy. Moreover, we have recently provided proof of the principle that such structures are valuable candidates for the ectorization of twophoton fuorophores into cells. We showed that the resulting structures display good-to-very-good conservation of their twophoton brightness in aqueous media, with emission wavelengths up to 570 nm. This result was however not fully optimal for in vivo two-photon imaging applications, since it is desirable that the probe features maximal Fuorescence in the biological window, i.e. at a wavelength between 700–1000 nm.
We report on the laser fabrication of BSA and type I collagen microstructures using Eosin Y or Rose Bengal photo sensitizers. Similar cross-linking results have been obtained by one-photon absorption or two-photon absorption using a green Q-switched microlaser or a near-infrared mode-locked Ti:sapphire laser, respectively. The microstructure resolution depends on the laser power, but is typically limited by the size of the laser beam within the sample. The cross-linking of type I collagen with Rose Bengal is obtained in acidic conditions with a large photosensitizer concentration in the millimole range. The biocompatibility of microstructures was demonstrated by observing the specific adhesion of cells on collagen lines.
Air pollution is a potential driver for the increasing prevalence of allergic disease, and post-translational modification by air pollutants can enhance the allergenic potential of proteins. Here, the kinetics and mechanism of protein oligomerization upon ozone (O<sub>3</sub>) exposure were studied in coated-wall flow tube experiments at environmentally relevant O<sub>3</sub> concentrations, relative humidities and protein phase states (amorphous solid, semi-solid, and liquid). We observed the formation of protein dimers, trimers, and higher oligomers, and attribute the cross-linking to the formation of covalent intermolecular dityrosine species. The oligomerization proceeds fast on the surface of protein films. In the bulk material, reaction rates are limited by diffusion depending on phase state and humidity. From the experimental data we derive a chemical mechanism and rate equations for a kinetic multi-layer model of surface and bulk reaction enabling the prediction of oligomer formation. Increasing levels of tropospheric O<sub>3</sub> in the Anthropocene may promote the formation of protein oligomers with enhanced allergenicity and may thus contribute to the increasing prevalence of allergies.<sub>.
The exposure to UV-A radiation of bovine serum albumin (BSA) in aerated aqueous solution in the presence of pterin (Ptr), results in chemical and conformational modifications of the protein. Ptr belongs to a family of heterocyclic compounds that are well-known type I (electron-transfer) and type II (singlet oxygen) photosensitizers. The evolution of the photosensitized processes was followed by UV/vis spectrophotometry and fluorescence spectroscopy indicating that tryptophan (Trp) and tyrosine (Tyr) residues were affected. Additionally, conformational changes were evaluated by electrophoresis (SDS-PAGE) and size exclusion chromatography coupled with dynamic light scattering detection, showing that BSA undergoes dimerization, via the formation of Tyr radicals. The degradation of Trp residues takes place faster than the oligomerization of the protein. The photosensitized process is initiated by an electron transfer from BSA to the triplet excited stated of Ptr, being a purely dynamic mechanism.
Biopolymers such as DNA can be used as a host material for nonlinear
optical dyes for photonic applications. In previous work by Heckman et
al. (Proc. SPIE 6401, 640108-2), the chromophore Disperse Red 1 (DR1)
was combined with CTMA-DNA (a water-insoluble DNA/surfactant complex) to
produce an electro-optic waveguide modulator. However, DR1 does not bind
strongly to DNA and has a low first hyperpolarizability (β). We
have used theory-aided design to develop and synthesize a novel
chromophore with strong affinity for DNA and higher β than DR1. We
have also developed a surfactant containing a photocrosslinkable moiety
that can be used to harden thin films of the DNA/surfactant/dye
composite under ultraviolet light. The optical and thermal properties of
these materials and outlook for device applications will be discussed.
Emphasis has been placed in this article dedicated to DNA damage on recent aspects of the formation and measurement of oxidatively generated damage in cellular DNA in order to provide a comprehensive and updated survey. This includes single pyrimidine and purine base lesions, intrastrand cross-links, purine 5',8-cyclonucleosides, DNA-protein adducts and interstrand cross-links formed by the reactions of either the nucleobases or the 2-deoxyribose moiety with the hydroxyl radical, one-electron oxidants, singlet oxygen, and hypochlorous acid. In addition, recent information concerning the mechanisms of formation, individual measurement, and repair-rate assessment of bipyrimidine photoproducts in isolated cells and human skin upon exposure to UVB radiation, UVA photons, or solar simulated light is critically reviewed.
Nonlinear multiphoton photo-cross-linking and photopolymerization of proteins and polymers in solution have been used to direct the three-dimensional assembly of micron scale objects. Two aspects of fabricated proteinatious matrixes are examined in this paper: the efficiency of protein photopolymerization and the application of fabricated matrixes as sustained release devices. The efficiency of photoactivated cross-linking of the proteins bovine serum albumin and fibrinogen, using rose bengal, have been determined and found to vary with photosensitizer concentration. This concentration dependence suggests that the mechanism for protein cross-linking is a direct hydrogen transfer between an amino acid residue of the protein and the dye molecule itself. A comparison of the surface structure of single and multiple protein oligomers is undertaken and shown to vary significantly depending on fabrication materials. Alkaline phosphatase bioactivity, upon entrapment in a protein structure, is maintained. The properties of fabricated protein matrixes as sustained release devices is also examined. The rates of diffusion of fluorescently labeled dextrans (10 and 40 kDa) from an optically fabricated BSA matrix vary with molecular weight and are linear with cross-link density. The half-life of release of 10 kDa dextran-TMR from a BSA micron scale structure is less than or equal to 6 min while 40 kDa dextran-TMR half-life of release is 25 min. Finally, rhodamine 610, a typical drug size molecule, was entrapped in an acrylamide structure, and its release is found to be diffusion-limited with half-lives of 10−31 min, depending on cross-link density.
Our research in nonlinear optic (NLO) polymer-based electro-optic (EO) modulators has centered on optimizing device performance through the using of polymer cladding layers with higher relative conductivities than the NLO core material. We have demonstrated as much as a 10 times increase in the effective EO coefficient of electrode poled, guest/host NLO polymers, compared to using passive polymer claddings. We have achieved the lowest poling voltage to date for maximum EO coefficient, 300 V, for a two-layer waveguide structure consisting of a 2 µm thick NLO polymer layer and 2 µm thick conductive cladding layer. Optimized polymer cladding materials posessing the desired optical and electromagnetic properties we find need to be balanced with materials processability. In addition to the conventional polymer materials under investigation, a novel material, deoxyribonucleic acid (DNA), derived from salmon sperm, has shown promise in providing both the desired optical and electromagnetic properties, as well as the desired resistance to various solvents used for NLO polymer device fabrication. Our investigation also includes intercalation of fluorescent dyes, photochromic dyes, nonlinear optic chromophores, two-photon dyes, and rare earth compounds into a DNA-based host material and comparing results with poly(methyl methacrylate) (PMMA)-based host materials.
A molecular engineering strategy based on rational variations of the bromine substitution pattern in two-photon absorbing singlet oxygen sensitizers allows studying the relations that exist between the positioning of an inter-system crossing promoter on the charge-transfer chromophore and its ability to generate singlet oxygen.
In this paper, we report on the fabrication of three-dimensional (3D) enzymatic microreactors within polydimethylsiloxane
microfluidic channels through a photocrosslinking mechanism mediated by the two-photon absorption process at the focal point
of pulse lasers, i.e., a sub-nanosecond Nd:YAG microlaser or a femtosecond Ti:Sapphire laser. This approach allows the building
of localized 3D trypsin structures with submicron resolution. The fabrication of two different trypsin structures was successfully
demonstrated using Eosin Y and Flavin Adenine Dinucleotide as biological photosensitizers: (i) arrays of 3D cylindrical rows
and (ii) 3D woodpile structure. The enzymatic activity of the fabricated structures was evaluated by fluorescence spectroscopy
using BODIPY FL casein as fluorogenic substrate. The real time investigation of the peptide cleavage into the microfluidic
channel demonstrated that the fabricated trypsin microstructures maintain their catalytic activity. This approach opens up
the way to complex multistep enzymatic reactions in well-localized regions of microfluidic devices, with great importance
in health screening and biomedical diagnostics.
KeywordsLaser microfabrication–Two-photon absorption–Microreactors–Enzymatic activity–Microfluidic channels
The objective of this study is to examine the feasibility of using visible light to form gels from polysaccharide precursors.
Hydrogel formation by visible light irradiation would be very beneficial because visible light is a benign light source and
ready available when compared with other light sources such as UV. Dextran-methacylate was synthesized and photocrosslinked
using (−)-riboflavin as a photoinitiator and L-arginine as a co-initiator under the visible light. The effect of various concentrations
of (−)-riboflavin and L-arginine on the photo-crosslinking of dextran-methacrylate hydrogel was investigated. The fabricated
hydrogel was characterized by FT-IR and SEM. The photoinitiator [(−)-riboflavin] and co-initiator (L-arginine) as well as
dextran precursor are completely biocompatible. The optimum condition for the biocompatible dextran-based hydrogel formation
under the harmless light source (visible light) was elucidated in this study. In general, the (−)-riboflavin, 0.01–0.5 %,
and L-arginine, 5–20 % of the weight of dextran-methacrylate were the best condition in forming dextran-based hydrogels under
the visible light. The three-dimensional hydrogel structure was verified by SEM morphology of swollen hydrogels. Photocrosslinking
under the visible light source would enlarge the applications of this type of photocrosslinking in the biomedical area (e.g.,
eyes or other light-sensitive organs).
A chromophore featuring a diyne bridge that connects two dibromobenzene moieties shows a much improved two-photon singlet oxygen generation ability in the biological transparency window compared to a related and relevant literature benchmark, as a result of a distorted ground state.
We report the ability to modify microscopic 3D topographies within dissociated cultures, providing a means to alter the development of neurons as they extend neurites and establish interconnections. In this approach, multiphoton excitation is used to focally excite noncytotoxic photosensitizers that promote protein crosslinking, such as BSA, into matrices having feature sizes ≥250 nm. Barriers, growth lanes, and pinning structures comprised of crosslinked proteins are fabricated under conditions that do not compromise the viability of neurons both on short time scales and over periods of days. In addition, the ability to fabricate functional microstructures from crosslinked avidin enables submicrometer localization of controllable quantities of biotinylated ligands, such as indicators and biological effectors. Feasibility is demonstrated for using in situ microfabrication to guide the contact position of cortical neurons with micrometer accuracy, opening the possibility for engineering well defined sets of synaptic interactions.
• biofabrication
• multiphoton cell patterning
• growth cone
Novel cationic dye and crosslikable surfactant for DNA biophotonics
- Carrie M Robinson
- Emily M Bartsch
- James Heckman
- Grote
Robinson, Carrie M. Bartsch, Emily M. Heckman, James Grote, "Novel cationic dye and crosslikable surfactant for
DNA biophotonics." Proc. SPIE, 8464, 84640D, 2012.
Recent advances in two-photon 3D laser lithography with self-Q-switched Nd: YAG microchip lasers
- P L Baldeck
- P Prabhakaran
- C Y Liu
- M Bouriau
- L Gredy
- O Stephan
- . . Lin
Baldeck, P. L., Prabhakaran, P., Liu, C. Y., Bouriau, M., Gredy, L., Stephan, O.,... & Lin, C. L. (2013, September). Recent
advances in two-photon 3D laser lithography with self-Q-switched Nd: YAG microchip lasers. In SPIE Organic Photonics+
Electronics (pp. 88270E-88270E). (2013)