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► IO devices made with silicon based technologies are at the core of biosensors development. ► Surface biofunctionalization is crucial to reach competitive performance. ► Main ...achievements reached in real sample biosensing are critically discussed.
Increasing interest has been paid to label-free biosensors in recent years. Among them, refractive index (RI) optical biosensors enable high density and the chip-scale integration of optical components. This makes them more appealing to help develop lab-on-a-chip devices. Today, many RI integrated optical (IO) devices are made using silicon-based materials. A key issue in their development is the biofunctionalization of sensing surfaces because they provide a specific, sensitive response to the analyte of interest. This review critically discusses the biofunctionalization procedures, assay formats and characterization techniques employed in setting up IO biosensors. In addition, it provides the most relevant results obtained from using these devices for real sample biosensing. Finally, an overview of the most promising future developments in the fields of chemical surface modification and capture agent attachment for IO biosensors follows.
Hydrogel-based holographic sensors consist of a holographic pattern in a responsive hydrogel that diffracts light at different wavelengths depending on the dimensions and refractive index changes in ...the material. The material composition of hydrogels can be designed to be specifically responsive to different stimuli, and thus the diffraction pattern can correlate with the amount of analyte. According to this general principle, different approaches have been implemented to achieve label-free optical sensors and biosensors, with advantages such as easy fabrication or naked-eye detection. A review on the different approaches, sensing materials, measurement principles, and detection setups, and future perspectives is offered.
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In the literature, there are reports of the utilization of various hydrogels to create generic platforms for protein microarray applications. Here, a novel strategy was developed to obtain ...high-performance microarrays. In it, a dextran hydrogel is used to covalently immobilize oligonucleotides and proteins. This method employs aqueous solutions of dextran methacrylate (Dx-MA), which is a biocompatible photopolymerizable monomer. Capture probes are immobilized inside the hydrogel via a light-induced thiol–acrylate coupling reaction at the same time as the dextran polymer is formed. Hydrogel microarrays based on this technique were prepared on different surfaces, such as a Blu-ray Disk and polycarbonate or alkene-functionalized glass slides, and these systems showed high probe-loading capabilities and good biorecognition yields. This methodology presents advantages such as a low cost, a short analysis time, a low limit of detection, and multiplexing capabilities, among others. Confocal fluorescence microscopy analysis demonstrated that in these hydrogel-based microarrays, receptor immobilization and the biorecognition event occurred within the hydrogel and not merely on the surface.
Responsive hydrogel-based surface relief gratings have demonstrated great performances as transducers in optical sensing. However, novel and smart designs of hydrogels are needed for the appropriate ...detection of analytes and biomolecules since the existing materials are very limited to specific molecules. In this work, a biosensing system based on surface relief gratings made of bioresponsive hydrogels has been developed. In particular, the hydrogel contains phosphocholine moieties to specifically recognize C-Reactive protein (CRP). The CRP-Sensing hydrogel capacity to selectively detect CRP was fully demonstrated. Using Direct Laser Interference Patterning, micro-gratings were created on CRP-Sensing hydrogel substrates and applied for the label-free sensing of CRP using a simple laser-based homemade optical setup. Limits of detection (LOD) and quantification (LOQ) in human serum dilutions of 1.07 and 8.92 mg L−1, respectively, were reached. These results demonstrate that the biosensing system allows the selective label-free detection of CRP within concentration ranges around those related to risks of cardiovascular diseases and sepsis. Besides, amplification strategies have been carried out improving the sensitivity, widening the linear range, and reaching better LOD and LOQ (0.30 mg L−1 and 4.36 mg L−1). Finally, all the approaches were tested for the quantification of CRP in certified human serum with recoveries of around 100%.
•Novel label-free biosensing system based on responsive hydrogels surface relief gratings developed for determination of CRP.•Label-free sensing of CRP at clinical concentration range achieved using a homemade measurement setup.•Amplification strategies were applied which improved the analytical performance of the sensor.•The approach was successfully applied to determine CRP in certified human serum sample sensitively and specifically.
Spatially controlled anchoring of NA probes onto microscope glass slides by a novel fluor-thiol coupling reaction is performed. By this UV-initiated reaction, covalent immobilization in very short ...times (30 s at 254 nm) is achieved with probe densities of up to 39.6 pmol/cm2. Modulating the surface hydrophobicity by combining a hydrophobic silane and a hydrophilic silane allows the fabrication of tuned surfaces where the analyte approaches only the anchored probe, which notably reduces nonspecific adsorption and the background. The generated substrates have proven clear advantages for discriminating single-base-pair mismatches, and for detecting bacterial PCR products. The hybridization sensitivity achieved by these high-performance surfaces is about 1.7 pM. Finally, this anchoring reaction is demonstrated using two additional surfaces: polytetrafluoroethylene (PTFE) and polyvinylidene fluoride (PVDF) membranes. This provides a very interesting pathway for anchoring thiolated biomolecules onto surfaces with C–F motifs via a quick clean UV reaction.
Analyte-sensitive DNA-based hydrogels find multiple applications in the field of biosensors due to their adaptable nature. Here, the design of DNA-based hydrogel and its application as sensing ...platform for the detection of a specific target sequence are presented. DNA-functionalized hydrogel structures were formed via a free radical co-polymerization process. A simple one-step probe immobilization procedure is reported: DNA probe molecules are added to the photoactive polymer mixture, dispensed onto a solid support, or a mold, and covalently attached while the hydrogel is formed through UV light exposure. Such hydrogels can be synthesized with desired recognition ability through the selection of a certain nucleotide sequence. Here we show the application of DNA-based hydrogel to detect the target with high performance in fluorescence microarray format and, additionally, to fabricate holographic surface relief gratings for label-free sensing assays.
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•One-step hydrogel synthesis and functionalization with acrydite-modified DNA by UV.•Development of DNA-based hydrogel microarray with high target sensitivity.•Diffractive surface relief biogratings successfully fabricated made of DNA-functionalized hydrogel.
A rapid strategy for the covalent immobilization of DNA onto silicon-based materials using the UV-initiated radical thiol–ene reaction is presented in this study. Following this approach, thiol- and ...alkene-modified oligonucleotide probes were covalently attached in microarray format, reaching immobilization densities around 6 pmol·cm–2. The developed methodology presents the advantages of spatially controlled probe anchoring (using a photomask), direct attachment without using cross-linkers (one-pot fashion), and short irradiation times (20 min). Using the described strategy, hybridization efficiencies up to 65% with full complementary strands were reached. The approach was evaluated by scoring single-base pair mismatches with discrimination ratios around 15. Moreover, the efficacy of the proposed DNA detection scheme is further demonstrated through the assay on a genomic target of bacterial Escherichia coli.
The present research is focused on the development of a biofunctionalized hydrogel with a surface diffractive micropattern as a label-free biosensing platform. The biosensors described in this paper ...were fabricated with a holographic recording of polyethylene terephthalate (PET) surface micro-structures, which were then transferred into a hydrogel material. Acrylamide-based hydrogels were obtained with free radical polymerization, and propargyl acrylate was added as a comonomer, which allowed for covalent immobilization of thiolated oligonucleotide probes into the hydrogel network, via thiol-yne photoclick chemistry. The comonomer was shown to significantly contribute to the immobilization of the probes based on fluorescence imaging. Two different immobilization approaches were demonstrated: during or after hydrogel synthesis. The second approach showed better loading capacity of the bioreceptor groups. Diffraction efficiency measurements of hydrogel gratings at 532 nm showed a selective response reaching a limit of detection in the complementary DNA strand of 2.47 µM. The label-free biosensor as designed could significantly contribute to direct and accurate analysis in medical diagnosis as it is cheap, easy to fabricate, and works without the need for further reagents.
Modulation of support wettability used for microarray format biosensing has led to an improvement of results. Hydrophobicity of glass chips was set by derivatizing with single vinyl organosilanes of ...different chain length and silane mixtures. Thiol-ene photochemical linking has been used as effective chemistry for covalent anchoring of thiolated probes. Lowest unspecific binding and highest signal intensity and SNR were obtained with large hydrocarbon chain (C
22
) silanes or a shorter one (C
10
) containing fluorine atoms. SNR resulting values are improved, reaching levels higher than 1500 in some cases, when using vinyl silanes modified with 1% C
10
alkyl fluorinated one, because mild hydrophobicity was achieved (water contact angle ca. 110°) for all silanes, including the short C
2
and C
3
, thus giving rise to smaller and better defined array spots. In addition, unspecific binding of reagents and targets was totally withdrawn. Hence, good-performing surfaces for biosensing applications can be built using appropriate organosilane reagent selection, including fluorinated ones.
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