Hydrogel Microarrays Research Articles

Development of peptide-functionalized synthetic hydrogel microarrays for stem cell and tissue engineering applications.

In this manuscript, we described the development of a robust approach to prepare peptide-functionalized synthetic hydrogel microarrays. Combined with bioinformatics, this technology enables us to rapidly identify novel biological ligands for the development of the next generation of functional biomaterials for stem cell and tissue engineering applications.

Simultaneous drug resistance detection and genotyping of Mycobacterium tuberculosis using a low-density hydrogel microarray.

Nucleic acid amplification tests are widely used in TB diagnostics. Priority tasks in their development consist of increasing the specificity and sensitivity of the detection of resistance to a wide spectrum of anti-TB drugs. We developed a multiplexed assay allowing the detection of 116 drug resistance-determining mutations in the rpoB, katG, inhA, ahpC, gyrA, gyrB, rrs, eis and embB genes in the Mycobacterium tuberculosis complex genome and six SNPs to identify the main lineages circulating in Russia. The assay is based on the amplification of 17 fragments of the genome using the universal primer adapter technique and heat pulses at the elongation step, followed by hybridization on a microarray. The method was evaluated using 264 pairs of clinical samples and corresponding isolates. A significant proportion (25%) of smear-negative samples were correctly analysed by microarray analysis in addition to 96% of smear-positive samples. The sensitivity and specificity of the assay exceeded 90% for rifampicin, isoniazid, ofloxacin and second-line injection drugs. In agreement with previous studies, the specificity of ethambutol resistance was as low as 57%, while the sensitivity was 89.9%. Strong association of the Beijing lineage with a resistant phenotype was observed. Euro-American lineage strains, excluding Ural and LAM, were predominantly associated with the susceptible phenotype. The developed test has a high sensitivity and specificity and can be directly applied to clinical samples. The combination of mutation-based drug resistance profiling and basic genotyping could be useful for clinical microbiology studies and epidemiological surveillance of the M. tuberculosis complex.

Bioprinting 3D cell-laden hydrogel microarray for screening human periodontal ligament stem cells response to extracellular matrix

Bioprinting 3D cell-laden hydrogel microarray for screening human periodontal ligament stem cells response to extracellular matrix

Bioprinting 3D cell-laden hydrogel microarray for screening human periodontal ligament stem cell response to extracellular matrix

Periodontitis is an inflammatory disease negatively affecting up to 15% of adults worldwide. Periodontal ligament stem cells (PDLSCs) hold great promises for periodontal tissue regeneration, where it is necessary to find proper extracellular matrix (ECM) materials (e.g., composition, concentration). In this study, we proposed a bioprinting-based approach to generate nano-liter sized three-dimensional (3D) cell-laden hydrogel array with gradient of ECM components, through controlling the volume ratio of two hydrogels, such as gelatin methacrylate (GelMA) and poly(ethylene glycol) (PEG) dimethacrylate. The resulting cell-laden array with a gradient of GelMA/PEG composition was used to screen human PDLSC response to ECM. The behavior (e.g., cell viability, spreading) of human PDLSCs in GelMA/PEG array were found to be depended on the volume ratios of GelMA/PEG, with cell viability and spreading area decreased along with increasing the ratio of PEG. The developed approach would be useful for screening cell-biomaterial interaction in 3D and promoting regeneration of functional tissue.

Photopatterning of Hydrogel Microarrays in Closed Microchips.

To date, optical lithography has been extensively used for in situ patterning of hydrogel structures in a scale range from hundreds of microns to a few millimeters. The two main limitations which prevent smaller feature sizes of hydrogel structures are (1) the upper glass layer of a microchip maintains a large spacing (typically 525 μm) between the photomask and hydrogel precursor, leading to diffraction of UV light at the edges of mask patterns, (2) diffusion of free radicals and monomers results in irregular polymerization near the illumination interface. In this work, we present a simple approach to enable the use of optical lithography to fabricate hydrogel arrays with a minimum feature size of 4 μm inside closed microchips. To achieve this, we combined two different techniques. First, the upper glass layer of the microchip was thinned by mechanical polishing to reduce the spacing between the photomask and hydrogel precursor, and thereby the diffraction of UV light at the edges of mask patterns. The polishing process reduces the upper layer thickness from ∼525 to ∼100 μm, and the mean surface roughness from 20 to 3 nm. Second, we developed an intermittent illumination technique consisting of short illumination periods followed by relatively longer dark periods, which decrease the diffusion of monomers. Combination of these two methods allows for fabrication of 0.4 × 10(6) sub-10 μm sized hydrogel patterns over large areas (cm(2)) with high reproducibility (∼98.5% patterning success). The patterning method is tested with two different types of photopolymerizing hydrogels: polyacrylamide and polyethylene glycol diacrylate. This method enables in situ fabrication of well-defined hydrogel patterns and presents a simple approach to fabricate 3-D hydrogel matrices for biomolecule separation, biosensing, tissue engineering, and immobilized protein microarray applications.

Ag@SiO2-entrapped hydrogel microarray: a new platform for a metal-enhanced fluorescence-based protein assay.

We developed a novel protein-based bioassay platform utilizing metal-enhanced fluorescence (MEF), which is a hydrogel microarray entrapping silica-coated silver nanoparticles (Ag@SiO2). As a model system, different concentrations of glucose were detected using a fluorescence method by sequential bienzymatic reaction of hydrogel-entrapped glucose oxidase (GOX) and peroxidase (POD) inside a hydrogel microarray. Microarrays based on poly(ethylene glycol)(PEG) hydrogels were prepared by photopatterning a solution containing PEG diacrylate (PEG-DA), photoinitiator, enzymes, and Ag@SiO2. The resulting hydrogel microarrays were able to entrap both enzymes and Ag@SiO2 without leaching and deactivation problems. The presence of Ag@SiO2 within the hydrogel microarray enhanced the fluorescence signal, and the extent of the enhancement was dependent on the thickness of silica shells and the amount of Ag@SiO2. Optimal MEF effects were achieved when the thickness of the silica shell was 17.5 nm, and 0.5 mg mL(-1) of Ag@SiO2 was incorporated into the assay systems. Compared with the standard hydrogel microarray-based assay performed without Ag@SiO2, more than a 4-fold fluorescence enhancement was observed in a glucose concentration range between 10(-3) mM and 10.0 mM using hydrogel microarray entrapping Ag@SiO2, which led to significant improvements in the sensitivity and the limit of detection (LOD). The hydrogel microarray system presented in this study could be successfully combined with a microfluidic device as an initial step to create an MEF-based micro-total-analysis-system (μ-TAS).

Spoligotyping of Mycobacterium tuberculosis complex isolates using hydrogel oligonucleotide microarrays

Mycobacterium tuberculosis remains a leading cause of morbidity and mortality worldwide. This circumstance underscores the relevance of population studies of tuberculosis for transmission dynamics control. In this study, we describe a conversion of the spoligotyping of M.tuberculosis complex isolates on a platform of custom designed hydrogel microarrays (biochips). An algorithm of automated data processing and interpretation of hybridization results using online database was proposed. In total, the 445 samples were tested. Initially, 97 samples representing multiple species of M.tuberculosis complex and nontuberculous mycobacteria were used for protocol optimization and cut-off settings. The developed assay was further evaluated on the out-group of the 348 mycobacterial samples. Results showed high concordance with the conventional membrane-based spoligotyping method. Diagnostic sensitivity and diagnostic specificity of the spoligo–biochip assay were 99.1% and 100%, respectively. The analytical sensitivity was determined to be 500 genomic equivalents of mycobacterial DNA. The high sensitivity and specificity, ease of operation procedures, and the automatic processing of measured data make the developed assay a useful tool for the rapid and accurate genotyping of M. tuberculosis.

Long term mesenchymal stem cell culture on a defined synthetic substrate with enzyme free passaging

Mesenchymal stems cells (MSCs) are currently the focus of numerous therapeutic approaches in tissue engineering/repair because of their wide multi-lineage potential and their ability to modulate the immune system response following transplantation. Culturing these cells, while maintaining their multipotency in vitro, currently relies on biological substrates such as gelatin, collagen and fibronectin. In addition, harvesting cells from these substrates requires enzymatic or chemical treatment, a process that will remove a multitude of cellular surface proteins, clearly an undesirable process if cells are to be used therapeutically. Herein, we applied a high-throughput ‘hydrogel microarray’ screening approach to identify thermo-modulatable substrates which can support hES-MP and ADMSC growth, permit gentle reagent free passaging, whilst maintaining multi-lineage potential. In summary, the hydrogel substrate identified, poly(AEtMA-Cl-co-DEAA) cross-linked with MBA, permitted MSCs to be maintained over 10 passages (each time via thermo-modulation), with the cells retaining expression of MSC associated markers and lineage potency. This chemically defined system allowed the passaging and maintenance of cellular phenotype of this clinically important cell type, in the absence of harsh passaging and the need for biological substrates.

Mass fabrication of size-controllable hydrogel microarrays by dip-pen nanolithography with viscosity-tunable ink

A method of mass fabricating poly(ethylene glycol) (PEG) hydrogel microarrays is demonstrated. Microarrays of poly(ethylene glycol) dimethacrylate (PEG-DMA) with photoinitiator were patterned by one-dimensional (1-D) parallel dip-pen nanolithography (DPN), and the microarrays were cross-linked to form PEG hydrogels by UV irradiation in N2 air. As an ink material for DPN printing, solid and liquid phase of PEG-DMA were mixed and prepared to tune viscosity of the ink material by temperature. Thus, the diameter of the microarrays was able to be averagely controlled from 1.7 to 6.2 μm as temperature during printing was increased from 25 °C to 37 °C, respectively. The overall microarrays showed less than 16% coefficient of variation (C.V.). Moreover, small molecules, such as fluorescence dyes, were able to be embedded in the PEG hydrogel microarrays.

Fabricating three-dimensional carbohydrate hydrogel microarray for lectin-mediated bacterium capturing

Herein, a three-dimensional carbohydrate modified polyacrylamide hydrogel microarray (3D carbohydrate hydrogel microarray) has been fabricated and employed as micro-reactor for capturing Escherichia coli (E. coli) by multivalent binding of concanavalin A (Con A) with O-antigen on the cellular surface of E. coli and immobilized monosaccharides on hydrogel spot, and the interactions of type 1 fimbriae of E. coli with immobilized monosaccharides. Because of the transparent performance of polyacrylamide hydrogel, the captured E. coli can be directly observed by a conventional microscope under a bright-field mode. The experimental result demonstrates that α-D-mannopyranoside (Man-α) modified hydrogel surface shows high efficiency of E. coli capturing. The 3D Man-α hydrogel microarray-based assay shows reasonable low detection limit (1.0×104 cells/mL) and large dynamic range (1.0×105 to 1.0×109cells/mL) for detecting E. coli. In addition, bacterial adhesion inhibition assay has been demonstrated by the interactions of E. coli with ten saccharides, and satisfactory results have been obtained.

Easy Daylight Fabricated Hydrogel Array for Colorimetric DNA Analysis

The fabrication of 3D hydrogel microarrays for DNA analytics that allow simple visual signal readout for on-site applications is described. A convenient one-step polymerization of the hydrogel including in situ capture oligonucleotide immobilization is accomplished by using N,N'-dimethylacrylamide/polyethylene glycol (PEG1900 )-bisacrylamide monomers. The implementation of an acylphosphine-oxide photoinitiator even allows polymerization at daylight, whereas other approaches require exposure with light in the UV-range. This minimizes the risk of UV-caused DNA damages within the capture DNA-strand that could adversely affect the subsequent hybridization step. The porous network of these gel segments allows DNA as well as protein penetration. Thus, the successful in-gel DNA hybridization is monitored by the deposition of silver nanoparticles. These metal particles allow naked eye signal readout.

Emerging Applications of Superhydrophilic‐Superhydrophobic Micropatterns

Water on superhydrophilic surfaces spreads or is absorbed very quickly, and exhibits water contact angles close to zero. We encounter superhydrophilic materials in our daily life (e.g., paper, sponges, textiles) and they are also ubiquitous in nature (e.g., plant and tree leaves, Nepenthes pitcher plant). On the other hand, water on completely non-wettable, superhydrophobic surfaces forms spherical droplets and rolls off the surface easily. One of the most well-known examples of a superhydrophobic surface is the lotus leaf. Creating novel superhydrophobic surfaces has led to exciting new properties such as complete water repellency, self-cleaning, separation of oil and water, and antibiofouling. However, combining these two extreme states of superhydrophilicity and superhydrophobicity on the same surface in precise two-dimensional micropatterns opens exciting new functionalities and possibilities in a wide variety of applications from cell, droplet, and hydrogel microarrays for screening to surface tension confined microchannels for separation and diagnostic devices. In this Progress Report, we briefly describe the methods for fabricating superhydrophilic-superhydrophobic patterns and highlight some of the newer and emerging applications of these patterned substrates that are currently being explored. We also give an outlook on current and future applications that would benefit from using such superhydrophilic-superhydrophobic micropatterns.

Fabricating three-dimensional hydrogel oligonucleotide microarrays to detect single nucleotide polymorphisms

Here, a three-dimensional (3D) oligonucleotide microarray-based fluorescent assay has been developed for single nucleotide polymorphisms (SNPs) and allele frequency determination. The 3D oligonucleotide microarray is fabricated on a home-made polyacrylamide hydrogel microarray which is generated by UV light photopolymerization of tiny acrylamide monomer droplets on a PEG terminated glass slide. After activation by glutaraldehyde (GA), the polyacrylamide hydrogel is able to react with NH2-modified oligonucleotides with high efficiency to generate a high signal-to-background ratio without the conventional blocking step. We demonstrate that the assay is able to detect complimentary 25 mer oligonucleotide target (fragment of a tumor suppressor gene P53) down to 100 pM and SNP with true-to-false ratios (the ratio of fluorescence signal from perfect matched DNA strands to that from the one-base mismatched DNA strands) above 20. Taking advantage of the high specificity of the assay, allele frequency as low as 2% can be accurately determined.

Microfluidic bioassay system based on microarrays of hydrogel sensing elements entrapping quantum dot–enzyme conjugates

This paper presents a simple method to fabricate a microfluidic biosensor that is able to detect substrates for H2O2-generating oxidase. The biosensor consists of three components (quantum dot–enzyme conjugates, hydrogel microstructures, and a set of microchannels) that were hierarchically integrated into a microfluidic device. The quantum dot (QD)–enzyme conjugates were entrapped within the poly(ethylene glycol) (PEG)-based hydrogel microstructures that were fabricated within the microchannels by a photopatterning process. Glucose oxidase (GOX) and alcohol oxidase (AOX) were chosen as the model oxidase enzymes, conjugated to carboxyl-terminated CdSe/ZnS QDs, and entrapped within the hydrogel microstructures, which resulted in a fluorescent hydrogel microarray that was responsive to glucose or alcohol. The hydrogel-entrapped GOX and AOX were able to perform enzyme-catalyzed oxidation of glucose and alcohol, respectively, to produce H2O2, which subsequently quenched the fluorescence of the conjugated QDs. The fluorescence intensity of the hydrogel microstructures decreased as the glucose and alcohol concentrations increased, and the detection limits of this system were found to be 50μM of glucose and 70μM of alcohol. Because each microchannel was able to carry out different assays independently, the simultaneous detection of glucose and alcohol was possible using our novel microfluidic device composed of multiple microchannels.

Application of 3D hydrogel microarrays in molecular diagnostics: advantages and limitations

Keywords:: 3D-hydrogelbiomarkerbiomoleculecoatinggel matriximmobilizationmicroarraymolecular diagnostics

Light-directed, spatially addressable oxygen detection in a hydrogel microarray based on phase-based lifetime detection using a digital micromirror device

A digital light modulation system that utilizes a modified commercial digital micromirror device (DMD) projector, which is equipped with a UV LED as a light modulation source, has been developed to spatially direct excitation light toward a hydrogel microarray to detect local oxygen via phase-based phosphorescence lifetime detection. Oxygen-sensitive luminescent microspheres that were encapsulated into the hydrogel microarray were used to detect the local O 2 concentration by sequentially addressing the modulated UV light to each hydrogel microsensing element. Two different hydrogels of alginate and polyethylene glycol diacrylate (PEGDA) were used as the supporting matrices to encapsulate the oxygen-sensitive luminescent microspheres within the hydrogels as microsensing elements. By controlling the illumination pattern on the DMD, the modulated excitation light can be spatially projected to only excite the microsensing element of interest, which can significantly decrease background noise and improve sensitivity. Gaseous oxygen, dissolved oxygen (DO), and glucose sensors that monitored the DO concentrations in the presence of oxygen and oxidase enzymes were characterized using phase-based lifetime detection; the measurements were consistent with the linear Stern–Volmer relationship. In addition, the sensitivity of the alginate supporting matrix was found to be higher than that of PEGDA due to its higher oxygen permeability in the alginate matrix structures. The proposed hydrogel microarray could easily produce large-scale cell-trapped hydrogel microarray. Moreover, the proposed DMD-based digital light modulation system is especially suited to these large-scale cell-trapped hydrogel microarrays in living cell assays.

Fabrication of poly(ethylene glycol)‐based hydrogels entrapping enzyme‐immobilized silica nanoparticles

AbstractIn this study, we immobilized enzymes by combining covalent surface immobilization and hydrogel entrapment. A model enzyme, glucose oxidase (GOX), was first covalently immobilized on the surface of silica nanoparticles (SNPs) via 3‐aminopropyltriethoxysilane (APTES), and the resultant SNP‐immobilized enzyme was physically entrapped within photopolymerized hydrogels prepared from two different molecular weights (MWs) (575 and 8000 Da) of poly(ethylene glycol)(PEG). The hydrogel entrapment resulted in a decrease in reaction rate and an increase in apparent Km of SNP‐immobilized GOX, but these negative effects could be minimized by using hydrogel with a higher MW PEG, which provides higher water content and larger mesh size. The catalytic rate of the PEG 8000 hydrogel was about ten times faster than that of the PEG 575 hydrogel because of enhanced mass transfer. Long‐term stability test demonstrated that SNP‐immobilized GOX entrapped within hydrogel maintained more than 60% of its initial activity after a week, whereas non‐entrapped SNP‐immobilized GOX and entrapped GOX without SNP immobilization maintained less than 20% of their initial activity. Incorporation of SNPs into hydrogel enhanced the mechanical strength of the hydrogel six‐fold relative to bare hydrogels. Finally, a hydrogel microarray entrapping SNP‐immobilized GOX was fabricated using photolithography and successfully used for quantitative glucose detection. Copyright © 2009 John Wiley & Sons, Ltd.

Temperature and Time-Resolved Total Internal Reflectance Fluorescence Analysis of Reusable DNA Hydrogel Chips

Total internal reflection fluorescence (TIRF) coupled with hydrogel-DNA droplet microarrays covalently bound on PMMA substrates presents a reusable, sensitive platform for evaluating DNA hybridization and for rapid biochip development. Hydrogel microarrays, which contain covalently bound DNA probes, are created via a simple printing and photocross-linking process. TIRF measurements of the arrays display robust reusability, show linear sensitivity down to 5 fmol of fluorescently labeled target DNA, and are sensitive to single basepair mismatches. Additionally, the ability to interrogate larger DNA is shown through studies with PCR amplification hybridization. We conclusively demonstrate an efficient, reproducible, low cost platform for DNA hybridization studies that could be used for fast high-throughput diagnostics as well as biochip development.

Phenol biosensor based on hydrogel microarrays entrapping tyrosinase and quantum dots

This paper describes the use of microarray-based biosensor system for the determination of phenol. Microarrays based on poly(ethylene glycol)(PEG) hydrogel were prepared by photopatterning of a solution containing PEG diacrylate (PEG-DA), photoinitiator, tyrosinase, and CdSe/ZnS quantum dots (QDs). During photo-induced crosslinking, tyrosinase and QDs were entrapped within the hydrogel microarrays, making the hydrogel microarray fluorescent and responsive to phenol. The entrapped tyrosinase could carry out enzyme-catalyzed oxidation of phenol to produce quinones, which subsequently quenched the fluorescence of QDs within hydrogel microarray. The fluorescence intensity of the hydrogel microarrays decreased linearly according to phenol concentration and the detection limit of this system was found to be 1.0 μM. The microarray system presented in this study could be combined with a microfluidic device as an initial step to create a phenol-detecting "micro-total-analysis-system (μ-TAS)".

A microfluidic chip with poly(ethylene glycol) hydrogel microarray on nanoporous alumina membrane for cell patterning and drug testing

In this paper, a microfluidic silicon chip with poly(ethylene glycol) (PEG) hydrogel microarray on the nanoporous anodized aluminum oxide (AAO) membrane was fabricated to form cell based microarray with controlled drug delivery. The PEG hydrogel microstructures were fabricated using photolithography on nanoporous alumina surface modified with a 3-(trimethoxysilyl)propyl methacrylate (TPM) monolayer. During the photopolymerization reaction, 10 × 10 hydrogel microwell arrays were covalently bonded to the substrate via the TPM monolayer for cell patterning. Human KYSE-30 esophageal squamous epithelial cancer cells were shown to selectively adhere on the TPM modified alumina surface inside the microwells. The diffusion studies for anti-cancer drug molecule of cisplatin were carried out for the microfabricated membrane array using a mini-diffusion chamber. The diffusion curves for pore size of 25 nm and 55 nm showed different release profiles. Then, the drug testing experiment with cisplatin was explored using the microfabricated membrane array with pore size of 55 nm for testing the drug effects on the KYSE-30 cancer cells. The cytotoxic effects of cisplatin on cancer cell number and cell morphology within microwells were explored based on this cell microarray.