Label-free Optical Biosensor Research Articles

Disk-based one-dimensional photonic crystal slabs for label-free immunosensing

One-dimensional photonic crystal slabs are periodic optical nanostructures that produce guided-mode resonance. They couple part of the incident light into the waveguide generating bandgaps in the transmittance spectrum, whose position is sensitive to refractive index variations on their surface. In this study, we present one-dimensional photonic crystal slab biosensors based on the internal nanogrooved structure of Blu-ray disks for label-free immunosensing. We demonstrated that this polycarbonate structure coated with a critical thickness of TiO2 generates guided-mode resonance. Its optical behavior was established comparing it with other compact disk structures. The results were theoretically calculated and experimentally demonstrated, all them being in agreement. The bioanalytical performance of these photonic crystals was experimentally demonstrated in a model assay to quantify IgGs as well as in two immunoassays to determine the biomarkers C-reactive protein and lactate dehydrogenase (detection limits of 0.1, 87, and 13 nM, respectively). The results are promising towards the development of new low-cost, portable, and label-free optical biosensors that join these photonic crystals with dedicated bioanalytical scanners based on compact disk drives.

Detection of naphthalene in sea-water by a label-free plasmonic optical fiber biosensor

In this study we developed an optical fiber biosensor able to detect the presence of naphthalene in sea-water. With this aim, we designed and produced an antibody specific for the naphthalene molecule. The capability of the antibody to bind to naphthalene was characterized by ELISA tests.A surface plasmon resonance (SPR) sensor platform was realized by sputtering a gold layer on a modified plastic optical fiber (POF). The gold surface was derivatizated and functionalized with the produced antibody by using the EDC/NHS amino-coupling immobilization protocol. The obtained results indicated that the POF-biosensor is able to sense the presence of naphthalene in a sea-water solution. The limit of detection (LOD) value was calculated to be 0.76 ng/mL, a value lower than the maximum residue limit value of naphthalene (0.13 µg/mL) referred as the water environmental quality standards (EQS). In addition, to the high sensitivity of the assay, it is remarkable to point out the possibility to monitor the presence of naphthalene in a real sea water solution by exploiting a simple experimental setup with a remote sensing capability offered by the POF-biosensor.

Design of an Aluminum/Polymer Plasmonic 2D Crystal for Label-Free Optical Biosensing.

A design study of a nanostructured two-dimensional plasmonic crystal based on aluminum and polymeric material for label-free optical biosensing is presented. The structure is formed of Al nanohole and nanodisk array layers physically separated by a polymeric film. The photonic configuration was analyzed through finite-difference time-domain (FDTD) simulations. The calculated spectral reflectance of the device exhibits a surface plasmon polariton (SPP) resonance feature sensitive to the presence of a modeled biolayer adhered onto the metal surfaces. Simulations also reveal that the Al disks suppress an undesired SPP resonance, improving the device performance in terms of resolution as compared to that of a similar configuration without Al disks. On the basis of manufacturability issues, nanohole diameter and depth were considered as design parameters, and a multi-objective optimization process was employed to determine the optimum dimensional values from both performance and fabrication points of view. The effect of Al oxidation, which is expected to occur in an actual device, was also studied.

Kinetics and Structure of Self-Assembled Flagellin Monolayers on Hydrophobic Surfaces in the Presence of Hofmeister Salts: Experimental Measurement of the Protein Interfacial Tension at the Nanometer Scale

In the present study, we monitor the adsorption–desorption kinetics and adsorbed layer structure of the bacterial protein flagellin in the presence of Hofmeister salts by a surface sensitive label-free optical biosensor (optical waveguide lightmode spectroscopy, OWLS). The recorded OWLS data were analyzed by a computer code using a set of coupled differential equations modeling the adsorption–desorption process. By supposing reversibly and irreversibly adsorbed protein states with different adsorption footprints, the kinetic data could be perfectly fitted. We revealed that the proteins adsorbing in the presence of kosmotropic salts had smaller footprints, leading to a more oriented and densely packed layer. Kosmotropic salts increased both the adsorption rate constant and the transition rate constants from the reversibly to the irreversibly adsorbed state. In contrast, chaotropic salts increased the desorption rate constant and led to decreased adsorbed mass and a more loosely packed film. Neither circula...

Simultaneous determination of aflatoxin B1, fumonisin B1 and deoxynivalenol in beer samples with a label-free monolithically integrated optoelectronic biosensor.

A label-free optical biosensor for the fast simultaneous determination of three mycotoxins, aflatoxin B1 (AFB1), fumonisin B1 (FB1) and deoxynivalenol (DON), in beer samples is presented. The biosensor is based on an array of ten Mach-Zehnder interferometers (MZIs) monolithically integrated along with their respective broad-band silicon light sources onto a single chip. Multi-analyte determination is accomplished by functionalizing the sensing arms of individual MZIs with mycotoxin-protein conjugates. Assay is performed by pumping over the chip mixtures of calibrators or samples with a mixture of specific monoclonal antibodies, followed by reaction with a secondary anti-mouse IgG antibody. Reactions are monitored in real-time by continuously recording the MZI output spectra, which are then subjected to Discrete Fourier Transform to convert spectrum shifts to phase shifts. The detection limits achieved for AFB1, FB1 and DON were 0.8, 5.6 and 24 ng/ml, respectively, while the assay duration was 12 min. Recovery values ranging from 85 to 115% were determined in beer samples spiked with known concentrations of the three mycotoxins. In addition, beers of different types and origin were analysed with the biosensor developed and the results were compared with those provided by established laboratory methods, further supporting the accuracy of the proposed device.

Interaction of Positively Charged Gold Nanoparticles with Cancer Cells Monitored by an in Situ Label-Free Optical Biosensor and Transmission Electron Microscopy.

Functionalized nanoparticles (NPs) can penetrate into living cells and vesicles, opening up an extensive range of novel directions. For example, NPs are intensively employed in targeted drug delivery and biomedical imaging. However, the real-time kinetics and dynamics of NP-living cell interactions remained uncovered. In this study, we in situ monitored the cellular uptake of gold NPs-functionalized with positively charged alkaline thiol-into surface-adhered cancer cells, by using a high-throughput label-free optical biosensor employing resonant waveguide gratings. The characteristic kinetic curves upon NP exposure of cell-coated biosensor surfaces were recorded and compared to the kinetics of NP adsorption onto bare sensor surfaces. We demonstrated that from the above kinetic information, one can conclude about the interactions between the living cells and the NPs. Real-time biosensor data suggested the cellular uptake of the functionalized NPs by an active process. It was found that positively charged particles penetrate into the cells more effectively than negatively charged control particles, and the optimal size for the cellular uptake of the positively charged particles is around 5 nm. These conclusions were obtained in a cost-effective, fast, and high-throughput manner. The fate of the NPs was further revealed by electron microscopy on NP-exposed and subsequently fixed cells, well confirming the results obtained by the biosensor. Moreover, an ultrastructural study demonstrated the involvement of the endosomal-lysosomal system in the uptake of functionalized NPs and suggested the type of the internalization pathway.

Covalent grafting of graphene oxide on functionalized macroporous silicon

Abstract Graphene oxide (GO) is a single-atom-thick and two-dimensional carbon material that has attracted great attention because of its remarkable electronic, mechanical, chemical and thermal properties. GO could be an ideal substrate for the development of label-free optical biosensors, however, its weak photoluminescence (PL) strongly limits the use for this purpose. In this study, we developed a covalent chemical strategy in order to obtain a hybrid GO-macroporous silicon (PSi) structure for biomedical applications. The realized structure was characterized by atomic force microscopy (AFM), scanning electron microscopy (SEM)water contact angle (WCA) measurements, Fourier transform infrared spectroscopy (FTIR) and label- free optical methods based on spectroscopic reflectometry and PL analysis. Investigations showed that the hybrid structure is suitable as a transducer material for biosensing applications due to its peculiar optical properties resulting from the combination of GO and PSi.

Confinement of Bloch surface waves in a graphene-based one-dimensional photonic crystal and sensing applications**Project supported by the National Natural Science Foundation of China (Grant Nos. 61203211 and 41675154), the Six Major Talent Peak Expert of Jiangsu Province, China (Grant No. 2015-XXRJ-014), and the Natural Science Foundation of Jiangsu Province, China (Grant No. BK20141483).

Bloch surface waves (BSWs) are excited in one-dimensional photonic crystals (PhCs) terminated by a graphene monolayer under the Kretschmann configuration. The field distribution and reflectance spectra are numerically calculated by the transverse magnetic method under transfer-matrix polarization, while the sensitivity is analyzed and compared with those of the surface plasmon resonance sensing method. It is found that the intensity of magnetic field is considerably enhanced in the region of the terminated layer of the multilayer stacks, and that BSW resonance appears only in the interface of the graphene and solution. Influences of the graphene layers and the thickness of a unit cell in PhCs on the reflectance are studied as well. In particular, by analyzing the performance of BSW sensors with the graphene monolayer, the wavelength sensitivity of the proposed sensor is 1040 nm/RIU whereas the angular sensitivity is 25.1°/RIU. In addition, the maximum of figure of merit can reach as high as 3000 RIU−1. Thus, by integrating graphene in a simple Kretschmann structure, one can obtain an enhancement of the light–graphene interaction, which is prospective for creating label-free, low-cost and high-sensitivity optical biosensors.

Label-free optical biosensor for real-time monitoring the cytotoxicity of xenobiotics: A proof of principle study on glyphosate

Rapid and inexpensive biosensor technologies allowing real-time analysis of biomolecular and cellular events have become the basis of next-generation cell-based screening techniques. Our work opens up novel opportunities in the application of the high-throughput label-free Epic BenchTop optical biosensor in cell toxicity studies. The Epic technology records integrated cellular responses about changes in cell morphology and dynamic mass redistribution of cellular contents at the 100–150 nm layer above the sensor surface. The aim of the present study was to apply this novel technology to identify the effect of the herbicide Roundup Classic, its co-formulant polyethoxylated tallow amine (POEA), and its active ingredient glyphosate, on MC3T3-E1 cells adhered on the biosensor surface. The half maximal inhibitory concentrations of Roundup Classic, POEA and glyphosate upon 1 h of exposure were found to be 0.024%, 0.021% and 0.163% in serum-containing medium and 0.028%, 0.019% and 0.538% in serum-free conditions, respectively (at concentrations equivalent to the diluted Roundup solution). These results showed a good correlation with parallel end-point assays, demonstrating the outstanding utility of the Epic technique in cytotoxicity screening, allowing not only high-throughput, real-time detection, but also reduced assay run time and cytotoxicity assessment at end-points far before cell death would occur.

Ultrasensitive label-free optical microfiber coupler biosensor for detection of cardiac troponin I based on interference turning point effect

Sensitive detection of cardiac biomarkers is critical for clinical diagnostics of myocardial infarction (MI) while such detection is quite challenging due to the ultra-low concentration of cardiac biomarkers. In this work, a label-free immunosensor based on optical microfiber coupler (OMC) has been developed for the ultrasensitive detection of cardiac troponin I (cTnI), a selective and highly sensitive biomarker of acute myocardial infarction (AMI). CTnI monoclonal antibodies were immobilized on the surface of the fiber through polyelectrolyte layer using layer-by-layer deposition technique. For refractive index sensing characterization, an ultra-high sensitivity of 91777.9 nm/RIU was achieved when the OMC works around the dispersion turning point, which is the highest experimental demonstration in the field of fiber-optic evanescent biosensors. For biosensing, the immunosensor with good specificity showed a linear wavelength shift in the range of 2–10 fg/mL and an ultra-low detection limit of 2 fg/mL. Such immunosensors have huge application potential for the detection of cardiac biomarkers of myocardial infarction due to simple detection scheme, quick response time, ease of handling and miniaturation.

Mining the Potential of Label-Free Biosensors for In Vitro Antipsychotic Drug Screening.

The pharmaceutical industry is facing enormous challenges due to high drug attribution rates. For the past decades, novel methods have been developed for safety and efficacy testing, as well as for improving early development stages. In vitro screening methods for drug-receptor binding are considered to be good alternatives for decreasing costs in the identification of drug candidates. However, these methods require lengthy and troublesome labeling steps. Biosensors hold great promise due to the fact that label-free detection schemes can be designed in an easy and low-cost manner. In this paper, for the first time in the literature, we aimed to compare the potential of label-free optical and impedimetric electrochemical biosensors for the screening of antipsychotic drugs (APDs) based on their binding properties to dopamine receptors. Particularly, we have chosen a currently-used atypical antipsychotic drug (Buspirone) for investigating its dopamine D3 receptor (D3R) binding properties using an impedimetric biosensor and a nanoplasmonic biosensor. Both biosensors have been specifically functionalized and characterized for achieving a highly-sensitive and reliable analysis of drug-D3R binding. Our biosensor strategies allow for comparing different affinities against the D3R, which facilitates the identification of strong or weak dopamine antagonists via in vitro assays. This work demonstrates the unique potential of label-free biosensors for the implementation of cost-efficient and simpler analytical tools for the screening of antipsychotic drugs.

On Chip Protein Pre-Concentration for Enhancing the Sensitivity of Porous Silicon Biosensors.

Porous silicon (PSi) nanomaterials have been widely studied as label-free optical biosensors for protein detection. However, these biosensors' performance, specifically in terms of their sensitivity (which is typically in the micromolar range), is insufficient for many applications. Herein, we present a proof-of-concept application of the electrokinetic isotachophoresis (ITP) technique for real-time preconcentration of a target protein on a PSi biosensor. With ITP, a highly concentrated target zone is delivered to the sensing area, where the protein target is captured by immobilized aptamers. The detection of the binding events is conducted in a label-free manner by reflective interferometric Fourier transformation spectroscopy (RIFTS). Up to 1000-fold enhancement in local concentration of the protein target and the biosensor's sensitivity are achieved, with a measured limit of detection of 7.5 nM. Furthermore, the assay is successfully performed in complex media, such as bacteria lysate samples, while the selectivity of the biosensor is retained. The presented assay could be further utilized for other protein targets, and to promote the development of clinically useful PSi biosensors.

A differential weak measurement system based on Sagnac interferometer for self-referencing biomolecule detection

A differential weak measurement system was presented, exhibiting the self-referencing function for biomolecule real time detection as a label-free optical biosensor. We built a Sagnac interferometer, which limited horizontal (H) and vertical (V) polarization to propagating along the common path but in opposite directions to realize weak measurements with two measuring channels installed in two corners of this Sagnac interferometer. By introducing two half wave plates into the system alternately with the two channels to convert between H and V polarization, we obtained a differential measurement for phase delay, which could quantitatively characterize the refractive index change corresponding to the concentration of samples in the channels. With this system, a real time monitor of molecule concentration in the dialysis process was accomplished, demonstrating the function of self-referencing, which is important for optical label-free molecule detection in a complex biological sample solution.

New Generation of Ultrasensitive Label-Free Optical Si Nanowire-Based Biosensors

We demonstrate the realization of the first label-free optical biosensor based on the room temperature luminescence of silicon nanowires (NWs) tested for the selective detection of C-reactive protein in human serum. High aspect ratio Si NW arrays used as sensing interface, are synthesized by a fast, low cost and Si industrially compatible approach. Si NW optical biosensors are fast and offer a broad concentration dynamic range that can be tuned according to different applications. Moreover, the platform is endowed with a high selectivity toward the target analyte and a sensitivity down to the fM limit of detection, opening the route toward noninvasive analysis in biofluids such as saliva.

A label-free optical biosensor based on nanoporous anodic alumina for tumour necrosis factor-alpha detection in chronic wounds

Tumour necrosis factor-alpha (TNF-α) is a pro-inflammatory cytokine important to wound healing. In non-healing wounds, it has been suggested that the expression of TNF-α is prolonged and elevated which contributes to impaired healing responses. Hence it is of great interest to develop biosensors towards the detection of TNF-α in non-healing wounds. In this study, we have developed a label-free optical TNF-α biosensor based on interferometric reflectance spectroscopy (IRS) technique using a functionalized nanoporous anodic alumina (NAA) thin film transducer. The biosensor is fabricated by functionalizing NAA pore walls with anti-TNF-α antibodies using silanization chemistry. Binding of TNF-α to the bioreceptors within the pores causes a change in effective optical thickness (EOT) of the NAA thin film. Thus, analyte detection is achieved by monitoring EOT evolution with time. Label-free detection of TNF-α was demonstrated in buffer solution and in complex media such as simulated wound fluid. A limit of detection of 0.13μg/mL was achieved. This study provides proof-of-concept evidence which sets foundations for further development of biosensors as point-of-care (POC) diagnostic tools for chronic wound care.

Fabrication of ordered metallic glass nanotube arrays for label-free biosensing with diffractive reflectance

In this study, a photoresist template with well-defined contact hole array was fabricated, to which radio frequency magnetron sputtering process was then applied to deposit an alloyed Zr55Cu30Al10Ni5 target, and finally resulted in ordered metallic glass nanotube (MGNT) arrays after removal of the photoresist template. The thickness of the MGNT walls increased from 98 to 126nm upon increasing the deposition time from 225 to 675s. The wall thickness of the MGNT arrays also increased while the dimensions of MGNT reduced under the same deposition condition. The MGNT could be filled with biomacromolecules to change the effective refractive index. The air fraction of the medium layer were evaluated through static water contact angle measurements and, thereby, the effective refractive indices the transverse magnetic (TM) and transverse electric (TE) polarized modes were calculated. A standard biotin–streptavidin affinity model was tested using the MGNT arrays and the fundamental response of the system was investigated. Results show that filling the MGNT with streptavidin altered the effective refractive index of the layer, the angle of reflectance and color changes identified by an L*a*b* color space and color circle on an a*b* chromaticity diagram. The limit of detection (LOD) of the MGNT arrays for detection of streptavidin was estimated as 25nM, with a detection time of 10min. Thus, the MGNT arrays may be used as a versatile platform for high-sensitive label-free optical biosensing.

Label-Free and High-Throughput Detection of Biomolecular Interactions Using a Flatbed Scanner Biosensor.

Fluorescence based microarray detection systems provide sensitive measurements; however, variation of probe immobilization and poor repeatability negatively affect the final readout, and thus quantification capability of these systems. Here, we demonstrate a label-free and high-throughput optical biosensor that can be utilized for calibration of fluorescence microarrays. The sensor employs a commercial flatbed scanner, and we demonstrate transformation of this low cost (∼100 USD) system into an Interferometric Reflectance Imaging Sensor through hardware and software modifications. Using this sensor, we report detection of DNA hybridization and DNA directed antibody immobilization on label-free microarrays with a noise floor of ∼30 pg/mm2, and a scan speed of 5 s (50 s for 10 frames averaged) for a 2 mm × 2 mm area. This novel system may be used as a standalone label-free sensor especially in low-resource settings, as well as for quality control and calibration of microarrays in existing fluorescence-based DNA and protein detection platforms.

An optical waveguide sensor based on mesoporous silica films with a comparison to surface plasmon resonance sensors

Mesoporous silica films (MSF) with large surface areas and high stability were fabricated by a simple Stöber solution growth method. The sensitivity and limit of detection (LOD) for an optical waveguide transverse electric mode (TE-OWG) sensor are 62.869 deg/RIU and 5.8250 × 10−6 RIU, respectively. The sensitivity and LOD for a surface plasma resonance (SPR) sensor are 131.58 deg/RIU and 1.0551 × 10−5 RIU, respectively. The MSF sensor can detect both small and large molecules. In the detection of non-adsorbing glucose, a LOD of 40.416 mg/L was recorded, which resulted from the change of the refractive index of the solution. For the detection of CTAB, which may adsorb onto the film, the LOD significantly decreases to 1.6315 nM. Although, large molecules, such as BSA, cannot diffuse into the mesoporous structure of MSF to adsorb onto the inner pore walls, physical adsorption on the outer surface of MSF still leads to a LOD of 84.209 pM. Fresnel calculations suggested higher potential sensitivity of the current system compared to that of the conventional SPR sensors. Thus, the MSF may be used as a versatile platform for high-sensitive label-free optical biosensing due to the high performance and the large potential of the surface functionality.

Antibiotic-induced release of small extracellular vesicles (exosomes) with surface-associated DNA

Recently, biological roles of extracellular vesicles (which include among others exosomes, microvesicles and apoptotic bodies) have attracted substantial attention in various fields of biomedicine. Here we investigated the impact of sustained exposure of cells to the fluoroquinolone antibiotic ciprofloxacin on the released extracellular vesicles. Ciprofloxacin is widely used in humans against bacterial infections as well as in cell cultures against Mycoplasma contamination. However, ciprofloxacin is an inducer of oxidative stress and mitochondrial dysfunction of mammalian cells. Unexpectedly, here we found that ciprofloxacin induced the release of both DNA (mitochondrial and chromosomal sequences) and DNA-binding proteins on the exofacial surfaces of small extracellular vesicles referred to in this paper as exosomes. Furthermore, a label-free optical biosensor analysis revealed DNA-dependent binding of exosomes to fibronectin. DNA release on the surface of exosomes was not affected any further by cellular activation or apoptosis induction. Our results reveal for the first time that prolonged low-dose ciprofloxacin exposure leads to the release of DNA associated with the external surface of exosomes.

Label-free detection of bovine serum albumin based on an in-fiber Mach-Zehnder interferometric biosensor.

We propose and experimentally verify an innovative label-free optical fiber biosensor based on a Mach-Zehnder interferometer for bovine serum albumin (BSA) concentration detection. The proposed fiber biosensor utilized a micro-cavity within a single-mode fiber to induce Mach-Zehnder interference. A remarkable feature of this biosensor is that external media can directly interact with the fiber core signal through microfluidic channels connected to the micro-cavity and sensor surface. The device was fabricated by means of femtosecond laser micromachining and chemical etching. A fiber interferometer of this type exhibits an ultrahigh refractive index sensitivity of -10,055 nm/RIU and a detection limit of 3.5 × 10-5 RIU. Different concentrations of BSA with an infinitesimally small refractive index difference can be clearly differentiated in situ by the interferential spectra of the structure. Experiments demonstrated the biosensor exhibited a BSA solution concentration sensitivity of -38.9 nm/(mg/mL) and a detection limit of 2.57 × 10-4 mg/mL, respectively. Moreover, this biosensor is a sub-microliter dose and ultrasensitive at the low concentrations detected in BSA, which make it a promising for biochemical applications such as DNA hybridization, cancer screenings, medicine examination and environmental engineering, etc.