3D-Printable Bioactivated Nanocellulose-Alginate Hydrogels.

We describe herein a nanocellulose-alginate hydrogel suitable for 3D printing. The composition of the hydrogel was optimized based on material characterization methods and 3D printing experiments, and its behavior during the printing process was studied using computational fluid dynamics simulations. The hydrogel was biofunctionalized by the covalent coupling of an enhanced avidin protein to the cellulose nanofibrils. Ionic cross-linking of the hydrogel using calcium ions improved the performance of the material. The resulting hydrogel is suitable for 3D printing, its mechanical properties indicate good tissue compatibility, and the hydrogel absorbs water in moist conditions, suggesting potential in applications such as wound dressings. The biofunctionalization potential was shown by attaching a biotinylated fluorescent protein and a biotinylated fluorescent small molecule via avidin and monitoring the material using confocal microscopy. The 3D-printable bioactivated nanocellulose-alginate hydrogel offers a platform for the development of biomedical devices, wearable sensors, and drug-releasing materials.

Label-free C-reactive protein electronic detection with an electrolyte-gated organic field-effect transistor-based immunosensor.

In this contribution, we propose a label-free immunosensor, based on a novel type of electrolyte-gated field-effect transistor (EGOFET), for ultrasensitive detection of the C-reactive protein (CRP). The recognition layer of the biosensor is fabricated by physical adsorption of the anti-CRP monoclonal antibody onto a poly-3-hexyl thiophene (P3HT) organic semiconductor surface. A supplementary nonionic hydrophilic polymer is used as a blocking agent preventing nonspecific interactions and allowing a better orientation of the antibodies immobilized onto the P3HT surface. The whole biomolecule immobilization procedure does not require any pretreatment of the organic semiconductor surface, and the whole functionalization process is completed in less than 30 min. Surface plasmon resonance (SPR) measurements were performed to assess the amount of biomolecules physisorbed onto the P3HT and to evaluate the CRP binding proprieties of the deposited anti-CRP layer. A partial surface coverage of about 23 % of adsorbed antibody molecules was found to most efficiently sense the CRP. The electrical performance of the EGOFET immunosensor was comparable to that of a bare P3HT EGOFET device, and the obtained CRP calibration curve was linear over six orders of magnitude (from 4 pM to 2 µM). The relative standard deviation of the individual calibration points, measured on immunosensors fabricated on different chips, ranged between 1 and 14 %, and a detection limit of 2 pM (220 ng/L) was established. The novel electronic immunosensor is compatible with low-cost fabrication procedures and was successfully employed for the detection of the CRP biomarker in the clinically relevant matrix serum. Graphical abstract Schematic of the EGOFET immunosensor for CRP detection. The anti-CRP monoclonal antibody layer is physisorbed on the P3HT organic semiconductor and the CRP is directly measured by a label-free electronic EGOFET transducer.

Detection beyond Debye's length with an electrolyte-gated organic field-effect transistor.

Electrolyte-gated organic field-effect transistors are successfully used as biosensors to detect binding events occurring at distances from the transistor electronic channel that are much larger than the Debye length in highly concentrated solutions. The sensing mechanism is mainly capacitive and is due to the formation of Donnan's equilibria within the protein layer, leading to an extra capacitance (CDON) in series to the gating system.

Electrolyte-gated organic field-effect transistor sensors based on supported biotinylated phospholipid bilayer.

Anchored, biotinylated phospholipids forming the capturing layers in an electrolyte-gated organic field-effect transistor (EGOFET) allow label-free electronic specific detection at a concentration level of 10 nM in a high ionic strength solution. The sensing mechanism is based on a clear capacitive effect across the PL layers involving the charges of the target molecules.

Structural and functional characteristics of chimeric avidins physically adsorbed onto functionalized polythiophene thin films.

Stabilized bioreceptor layers are of great importance in the design of novel biosensors. In earlier work, chimeric avidins enabled immobilization of biotinylated antibodies onto gold surfaces with greater stability compared to more conventional avidins (wild-type avidin and streptavidin). In the present study, the applicability of chimeric avidins as a general binding scaffold for biotinylated antibodies on spin-coated functionalized polythiophene thin films has been studied by surface plasmon resonance and atomic force microscopy. Novel chimeric avidins showed remarkably increased binding characteristics compared with other avidins, such as wild-type avidin, streptavidin, and bacterial avidin when merely physically adsorbed onto the polythiophene surface. They gave the highest binding capacities, the highest affinity constant, and the highest stability for biotinylated probe immobilization. Introduction of carboxylic acid groups to polythiophene layer further enhanced the binding level of the avidins. Polythiophene layers functionalized with chimeric avidins thus offered a promising generic platform for biosensor applications.

Detection of DNA hybridisation in a diluted serum matrix by surface plasmon resonance and film bulk acoustic resonators.

Nanomolar quantities of single-stranded DNA products ~100 nucleotides long can be detected in diluted 1% serum by surface plasmon resonance (SPR) and film bulk acoustic resonators (FBARs). We have used a novel FBAR sensor in parallel with SPR and obtained promising results with both the acoustic and the optical device. Oligonucleotides and a repellent lipoamide, Lipa-DEA, were allowed to assemble on the sensor chip surfaces for only 15 min by dispensing. Lipa-DEA surrounds the analyte-binding probes on the surface and effectively reduces the non-specific binding of bovine serum albumin and non-complementary strands. In a highly diluted serum matrix, the non-specific binding is, however, a hindrance, and the background response must be reduced. Nanomolar concentrations of short complementary oligos could be detected in buffer, whereas the response was too low to be measured in serum. DNA strands that are approximately 100 base pairs long at concentrations as low as 1-nM could be detected both in buffer and in 1% serum by both SPR and the FBAR resonator.

Improved functionality of antibody-colloidal gold conjugates with the aid of lipoamide-grafted N-[tris(hydroxymethyl)methyl]acrylamide polymers.

Colloidal gold has been used as a label in sandwich assays for human IgG, in which intercalating N-[tris(hydroxymethyl)methyl]acrylamide (pTHMMAA) polymers have been employed to stabilise the particles coated with antibody fragments. A direct absorbance reading of the particles could be obtained from sandwich assays on polystyrene, and a strongly amplified response was observed in similar assays based on Surface Plasmon Resonance (SPR): for h-IgG, detection limits below 100 pg/mL could be achieved. Three different polymer lengths and two different particles sizes were compared in sandwich assays performed on polystyrene and gold. The resulting binding curves fitted well to the Langmuir-Freundlich isotherm and the binding constants were in good agreement with the values found in earlier studies. The amplification afforded by the nanoparticles was strongly dependent on the antigen concentration, on the type of polymer and on the particle size. Compared to the direct response of the antigen, amplification factors larger than 100 could be achieved. The study proves that the polymers give stabilised particles, which can be used in highly sensitive sandwich assays.

CMOS-integrated film bulk acoustic resonators for label-free biosensing.

The throughput is an important parameter for label-free biosensors. Acoustic resonators like the quartz crystal microbalance have a low throughput because the number of sensors which can be used at the same time is limited. Here we present an array of 64 CMOS-integrated film bulk acoustic resonators. We compare the performance with surface plasmon resonance and the quartz crystal microbalance and demonstrate the performance of the sensor for multiplexed detection of DNA.

An antibody surface for selective neuronal cell attachment.

An optimal surface for culturing human embryonic stem cell (hESC)-derived neuronal cells is of high interest. In this study, a specific antibody to a neural cell adhesion molecule (NCAM) was immobilised on a solid surface of polystyrene and used as a selective matrix for culturing of hESC-derived neuronal cells. Thereafter, hESC-derived neurospheres were seeded on the matrix. The neurospheres did not attach to the NCAM antibody containing matrix whereas individual neuronal cells did. The neuronal cell attachment was depended on the NCAM antibody concentration. The neuronal cells were viable on the NCAM antibody containing matrix during an 8 day follow-up and exhibited typical bipolar morphology of immature neurons. Specific binding of the NCAM antigen to an immunoglobulin-polymer coated surface was verified by surface plasmon resonance (SPR) measurements. This study is to our knowledge the first demonstrating the use of an antibody layer as a selective surface for hESC-derived neuronal cells.

Functional characterisation of Fab'-fragments self-assembled onto hydrophilic gold surfaces.

Antibody Fab'-fragments have been immobilised on hydrophilic gold by direct self-assembly, and embedded in a matrix of non-ionic hydrophilic polymers, tris(hydroxymethyl)methylacrylamide, carrying lipoate terminal linking groups. Different polymers were synthesised, and co-adsorbed or post-adsorbed between the antibody fragments in order to optimise the antigen binding. Various factors were investigated that influence the activity of the immobilised Fab'-fragments for binding of the antigen, human IgG. The Fab'-fragments were immobilised in dense layers close to monolayer coverage, and the stoichiometric efficiency of immobilisation was up to 30%, with the human IgG also approaching monolayer coverage. The cleaning of the gold surface was a crucial factor in preservation of activity. Besides the usual treatment in hot ammonia/peroxide solution, hot DMSO appeared to be highly effective as a cleaning agent.

A comparative evaluation of molecular recognition by monolayers composed of synthetic receptors or oriented antibodies.

Recombinant anti-morphine Fab' fragments have been immobilised on gold by covalent attachment through the free thiol groups of the fragment. The antibody fragments were intercalated with a non-ionic hydrophilic polymer in order to suppress non-specific binding of interfering substances. The antibodies are oriented on the surface due to the thiol groups of the antibody and the layer shows a high response to antigen. Non-specific binding of bovine serum albumin is moreover very low because of the repellent polymer. Synthetic receptors composed of an imprinted self-assembled monolayer made from lipoates and the template, morphine, exhibit the same binding response to the antigen, morphine as the site-specific oriented antibody monolayer. A similar binding curve could be obtained as that for binding of morphine to an antibody Fab' fragment/polymer layer - indicating that synthetic receptors produced are comparable to those of antibody layers. Concentrations down to 0.1ng/ml have been measured with surface plasmon resonance.

Lipoate-based imprinted self-assembled molecular thin films for biosensor applications.

Lipoate derivatives were used for the formation of imprinted self-assembled molecular thin films for the recognition of morphine. A large collection of lipoate derivatives was screened by molecular dynamics simulations in various solvents. A set of ligands showing favourable interactions with morphine in aqueous environment was selected for synthesis. Morphine-imprinted layers were then produced on gold substrates in mixed monolayers with morphine added as the template. The binding of ligands and morphine to gold, as well as the association/dissociation of morphine to the formed layers were studied with Surface Plasmon Resonance. Imprinted factors were highly variable and were dependent on ligand/morphine mixing ratio, lipoate derivative and monolayer preparation method. The imprinted factors were as high as 100 and 600 for one of the ligands. The results show that the simulations are able to provide correct information of the relative strengths of the molecular interactions between the ligand and morphine molecules in different solutions. The liquid phase simulations are, however, not able to predict the imprinted factors (i.e. distinguish between specific and non-specific binding), because the specificity is not formed before self-assembly on the surface.

Hybridisation of surface-immobilised single-stranded oligonucleotides and polymer monitored by surface plasmon resonance.

We have investigated the hybridisation of thiol-modified single-stranded DNA embedded in a polyacrylamide layer through the technique of surface plasmon resonance (SPR). Kinetic studies were carried out by two different immobilisation methods: (a) SH-ssDNA was firstly attached on gold and the remaining free space was filled with polymer and (b) SH-ssDNA and the polymer was attached onto the surface from the same solution. The immobilisation methods were compared for various concentrations of SH-ssDNA. Hybridisation was dependent on both the immobilisation method and the concentration of the components. The highest hybridisation was obtained when SH-ssDNA and the polymer was immobilised from the same solution at low SH-ssDNA concentration or when high concentrations of oligos were spread onto the surface and the surface was post-treated with polymer. The target response corresponded to a surface coverage of 100+/-15 ng/cm2. The same surface coverage on hybridisation was also obtained when low concentration of SH-ssDNA and polymer was attached onto the surface from the same solution. The non-specific binding of sample DNA was very low at optimal concentrations due to the polymer and the hybridisation was linearly dependent on target concentration.

Site-directed immobilisation of antibody fragments for detection of C-reactive protein.

C-reactive protein, CRP antibody Fab'-fragments have been attached on pre-cleaned gold slides and protein repellent polymers have been used to block the remaining free space in between the antibody fragments. At optimal conditions the antibody fragments are site-directly immobilised on the surface and non-specific binding is reduced. The amount of Fab'-fragments in the polymer host monolayer has been optimised for various buffers. Binding of CRP to Fab'-fragment/polymer layers produced in phosphate buffered saline decreased with NaCl salt concentration. In a 1M NaCl phosphate buffer, the antibodies seem to be randomly oriented on the surface with a similar response to CRP as that of an antibody F(ab)(2)-fragment layer. In a 150 mM NaCl phosphate buffer, on the other hand, the fragments seem to be site-directly oriented and the response to CRP was fivefold. The highest response to CRP was obtained to a layer with a Fab'-fragment concentration of 60 microg/ml. CRP could be detected in a concentration range of 1 ng/ml to 50 microg/ml from a standard solution in phosphate buffer and in a range of 4 ng/ml to 50 microg/ml from serum/PBS. CRP was, moreover, successfully detected in patient samples with good reproducibility. The layer would thus be sensitive enough to analyse the CRP concentration in human serum for predicting cardiovascular disease.

Immunosensing based on site-directed immobilization of antibody fragments and polymers that reduce nonspecific binding.

Antibody Fab'-fragments can be directly coupled onto gold, and the space between the fragments can be filled with protein repellent disulfide bearing polymers. Coupling of the antibody Fab'-fragments, and thus both the amount of nonspecific binding and antigen binding but also the ability to regenerate the layer, is dependent on the immobilization procedure. First, the immobilization has taken place by coupling the Fab'-fragments to the surface and thereafter attaching the polymer in the remaining space between the antibodies. Second, the Fab'-fragments have been added after the surface has been coated by polymer. Third, the Fab'-fragments and polymer have been added onto the surface from the same solution. Up to 80% of the antigen could be removed during regeneration, if proper concentrations of polymer and Fab'-fragments were immobilized onto the gold surface. Only about 60% of the antigen could be removed, when the fragments were coupled directly onto a clean Au surface before the polymer or if low concentrations of polymer were attached onto gold before the Fab'-fragments. The first immobilization method, however, showed the highest response to antigen.