Biosensing Measurements Research Articles

Interaction of Pseudomonas aeruginosa with surface-modified silica studied by ultra-high frequency acoustic wave biosensor

Aim: This study aimed to examine the amount of surface non-specific adsorption, or fouling, observed by Pseudomonas aeruginosa (P. aeruginosa) on a quartz crystal based acoustic wave biosensor under different flow conditions with and without an anti-fouling layer. Methods: An electromagnetic piezoelectric acoustic sensor (EMPAS) based on electrode free quartz crystals was used to perform the analysis. Phosphate buffered saline (PBS) was flowed over the crystal surface at various flow rates from 50 μL/min to 200 μL/min, with measurements being taken at the 43rd harmonic (~864 MHz). The crystal was either unmodified, or modified with a monoethylene glycol [2-(3-silylpropyloxy)-hydroxy-ethyl (MEG-OH)] anti-fouling layer. Overnight culture of P. aeruginosa PAO1 (PAO1) in lysogeny broth (LB) was injected into the system, and flow maintained for 30 min. Results: The frequency change of the EMPAS crystal after injection of bacteria into the system was found to change based on the flow rate of buffer, suggesting the flow rate has a strong effect on the level of non-specific adsorption. The MEG-OH layer drastically reduced the level of fouling observed under all flow conditions, as well as reduced the amount of variation between experiments. Flow rates of 150 μL/min or higher were found to best reduce the level of fouling observed as well as experimental variation. Conclusions: The MEG-OH anti-fouling layer is important for accurate and reproducible biosensing measurements due to the reduced fouling and variation during experiments. Additionally, a flow rate of 150 μL/min may prove better for measurement compared to the current standard of 50 μL/min for this type of instrument.

Mechanistic Evaluation of Antimicrobial Lipid Interactions with Tethered Lipid Bilayers by Electrochemical Impedance Spectroscopy

There is extensive interest in developing real-time biosensing strategies to characterize the membrane-disruptive properties of antimicrobial lipids and surfactants. Currently used biosensing strategies mainly focus on tracking membrane morphological changes such as budding and tubule formation, while there is an outstanding need to develop a label-free biosensing strategy to directly evaluate the molecular-level mechanistic details by which antimicrobial lipids and surfactants disrupt lipid membranes. Herein, using electrochemical impedance spectroscopy (EIS), we conducted label-free biosensing measurements to track the real-time interactions between three representative compounds—glycerol monolaurate (GML), lauric acid (LA), and sodium dodecyl sulfate (SDS)—and a tethered bilayer lipid membrane (tBLM) platform. The EIS measurements verified that all three compounds are mainly active above their respective critical micelle concentration (CMC) values, while also revealing that GML induces irreversible membrane damage whereas the membrane-disruptive effects of LA are largely reversible. In addition, SDS micelles caused membrane solubilization, while SDS monomers still caused membrane defect formation, shedding light on how antimicrobial lipids and surfactants can be active in, not only micellar form, but also as monomers in some cases. These findings expand our mechanistic knowledge of how antimicrobial lipids and surfactants disrupt lipid membranes and demonstrate the analytical merits of utilizing the EIS sensing approach to comparatively evaluate membrane-disruptive antimicrobial compounds.

HRP modified pulse laser ablated ZnO nanostructure for H2O2 sensing

Biosensor for the detection of hydrogen peroxide (H2O2) has been prepared by immobilizing horseradish peroxidase (HRP) enzyme using physical adsorption technique on zinc oxide (ZnO) nanostructures. The (002) oriented ZnO nanostructures as confirmed by X-ray diffraction, were successfully grown on indium tin oxide (ITO) coated glass substrate by pulsed laser ablation (PLA) without using any catalyst. The Nafion solution was added onto HRP/ ZnO/ ITO bio-electrode to form a tight membrane on the surface before carrying out bio-sensing measurements by electrochemical analyzer. The electrochemical studies reveal that the prepared bio-electrode HRP/ZnO/ITO is highly sensitive to the detection of H2O2 over a wide range of concentration with a linear range from 2.5 μM to 100 μM with the limit of detection 0.2 μM and sensitivity of 0.034 µA/ µM cm2. The higher sensitivity attributed to larger surface area of ZnO nanostructure for effective loading of HRP besides its high electron communication capability. A relatively low value of the enzyme’s kinetic parameter (Michaelis-Menten constant, Km) of 0.166 μM indicates enhanced enzyme affinity of HRP to H2O2. The reported biosensor may be useful for various applications in bio-sensing, clinical, food and beverage industry.

Photoluminescence-Based Bioassay With Cysteamine-Capped TiO2 Nanoparticles for the Selective Recognition of N-Acyl Homoserine Lactones.

Currently available diagnostic procedures for infections are laborious and time-consuming, resulting in a substantial financial burden by increasing morbidity, increased costs of hospitalization, and mortality. Therefore, innovative approaches to design diagnostic biomarkers are imperative to assist in the rapid and sensitive diagnosis of microbial infections. Acyl homoserine lactones (AHLs) are ubiquitous bacterial signaling molecules that are found to be significantly upregulated in infected sites. In this pioneering work, we have developed a simple photoluminescence-based assay using cysteamine-capped titanium oxide (TiO2) nanoparticles for AHL detection. The PL intensity variation of the oxygen defect state of TiO2 was used for the biosensing measurements. The bioassays were validated using two well-studied AHL molecules (C4-HSL and 3-oxo-C12 HSL) of an important human pathogen, Pseudomonas aeruginosa. The developed system has a maximum relative response of 98%. Furthermore, the efficacy of the system in simulated host urine using an artificial urine medium showed a linear detection range of 10–160 nM. Also, we confirmed the relative response and specificity of the system in detecting AHLs produced by P. aeruginosa in a temporal manner.

Double-Langmuir model for optimized nanohole array-based plasmonic biosensors

The sensing mechanism of plasmonic nanohole arrays is investigated and a novel model is proposed to interpret their optical response over a wide dynamic range of concentrations (10-13-10-5 M), based on a double-Langmuir model. This model describes the signal response of the analyte binding as the sum of two independent contributions which are related to two different surface regions of the biosensor, namely the top gold surface of the nanohole array and the lateral gold area inside the nanoholes. Numerical simulations highlight the different near-field behaviour of these two regions and their very different refractive index sensitivities, which both support the double-Langmuir model. This is corroborated by experimental biosensing measurements with gold nanohole arrays with hexagonal symmetry, synthesized by nanosphere lithography. Their sensing performances are optimized by numerical simulations by changing their geometrical parameters (i.e., lattice constant, nanohole diameter and height) in order to achieve a maximum sensitivity. For the biosensing experiments, the biotin-streptavidin complex is used as a benchmark test for the optimized nanohole array and a robust calibration is provided by the double-Langmuir model obtaining a limit of detection of 0.3 ng/mL, which corresponds to an absolute analyte quantity of 0.02 fmol.

Ultrasensitive Optical Label-Free Biosensing and Fluorescent Measurements using UV Surface Plasmons in Gold Nanolayers Launched with the Support of a Specially Designed Photonic Crystal

Recently, we used Photonic Crystal (PC) - supported surface plasmons (SP) propagating along thin Pd, Co (in red spectral range) and Au (in blue, 405 nm) nanolayers for ultrasensitive gas sensing (Pd, Au) and magnetoplasmonic researches (Co). In all these cases, without the exploitation of a specially designed PC, the propagation length of the plasmons is just of the order of the wavelength hence there would be no reason to speak about SP at all. Here we experimentally show that the same concept is applicable even in UV spectral range at 375 nm despite the extremely non-favorable conditions of SP excitation at bulk gold - dielectric interface: the real part of the dielectric permittivity of gold at this wavelength is minus 1.5, and its imaginary part 5.6. Nevertheless, the use of a specially designed PC, containing seven pairs of transparent Ta2O5/SiO2 layers plus an additional Ta2O5 and 8 nm-thick gold layer, enabled us to attain the angular width of the surface plasmon excitation around 0.250 and thus the UV SP propagation length of tens of microns. This same structure can be used in quite usual fashion as an ultrasensitive optical label-free biosensor. Besides, the short UV wavelength exploited enables also to use it for excitation of fluorescence for very broad class of studied compounds. The perspectives of the application of this approach to deeper UV, at 280 nm, will be discussed. Such structures will enable direct label-free excitation and measurement of fluorescence from many proteins and DNA/RNA molecules/complexes.

Impedimetric Biosensor for Monitoring Complementary DNA from Hepatitis B Virus Based on Gold Nanocrystals

A highly sensitive label free electrochemical impedance biosensor based on gold nanocrystals (AuNCs) for hepatitis B virus (HBV) detection in blood serum was reported. A functional platform for measuring the geno-biosensor sensitivity for detecting HBV DNA based on AuNCs electrode was materialized. The adjusted morphologies of 50–100 nm beaded AuNCs on the In2O3 support layer were materialized and characterized by a field emission scanning electron microscope (FESEM) and a high resolution transmission electron microscope (HRTEM). The bio-sensing measurements were conducted by electrochemical impedance spectroscopy (EIS) approach under redox reactions process. The HBV DNA probe (ssDNA) was immobilized on the AuNCs surface by forming a special bond between ssDNA and gold. The DNA target was diagnosed using ssDNA/AuNCs biosensor through EIS measurements in concentration ranges from 0.1 pM to 0.1 μM with a limit of detection (LOD) of 0.1 fM. The selectivity property of the biosensor was investigated and it could distinctively distinguish the complementary DNA target from the non-complementary DNA, 1-, 2- and 3-mismatch targets. Finally, the capability of the electrode for detection of the HBV in blood serum samples was explored and biosensor showed interesting results in the HBV DNA sensing as a potential candidate for practical applications.

A Compact TE-Notch Filter With Simultaneous Capability of TM-Suppression and Sensing Applications

An optical notch filter has been designed based on a silicon-slab Photonic Crystal (PhC) W1 waveguide. Birefringence originated usual performance degradation of notch filters has been eliminated by optimizing the slab-thickness and radius of the holes to embed a TE pass filter in the same structure. This resulted in TE and TM transmittances as $\approx$ $\mathrm{-1.5}$ dB and $\approx$ $\mathrm{-28}$ dB respectively at the wavelength $\mathrm{1550}$ nm. Structural parameters of the device are represented by normalizing them with respect to the lattice constant such that the structure can be scaled based on the application wavelength. Thereafter, generation of the mini stop band (MSB), i.e. notch-band, has been studied for various relative longitudinal-shifts of the lateral cladding for different filling fractions. Two different longitudinal-shifts are combined in a footprint of $\mathrm{9.8 \times 7.35}$ ${\mu \rm {m}^{2}}$ to realize a $\mathrm{10}$ nm notch-band having a notch suppression of $\approx$ $\mathrm{27}$ dB. Application of the notch-filter as a refractive index sensor has been investigated that exhibits its sensitivity as $\mathrm{500}$ nm/RIU. The potentiality of the device in applications like - bio-sensing (e.g., detection of morphological forms of certain bacteria, and some typical viral particles) and industrial measurements (typically, sensing of Ethyl Alcohol percentage in aqueous solution) has also been explored.

Water-Induced Fused Silica Glass Surface Alterations Monitored Using Long-Period Fiber Gratings

Long-period gratings (LPGs) induced in optical fibers show exceptional refractive index (RI) sensitivity and thus they are often applied for label-free biosensing. However, during measurements at changing environmental conditions, the surface of the fiber made of fused silica is subjected to stresses that can cause alterations in its structure. The problem is particularly important when long-term or biosensing measurements are considered and when sensor surface may be exposed to subsequent drying and immersion in aqueous solutions. The aim of this paper was to investigate the influence of sequential drying in air and soaking in water of the fused silica fiber cladding surface on the optical response of LPGs. We demonstrated that transmission spectrum of LPG measured in water before and after drying in air differed significantly and further changed with subsequent drying steps indicating increase of the local RI. All these changes correspond to the alterations in the cladding surface, i.e., its fracturing and pores rearrangement, caused by the stress induced during drying and then immersing in water, as well as possible corrosion and formation of nanosized objects on the cladding surface. The effect can be additionally influenced by washing the samples in organic solvents. We confirmed that measurements done in a flow-cell system, where the sensor was kept wet during the sensing experiment may eliminate the water-induced fiber surface alteration effect and thus minimize the amount of false results. Described findings are highly important for biosensing applications of any optical-fiber-based devices.

Non-Invasive Temperature-Feedback SERS with all-Dielectric Resonant Nanostructures

All-dielectric resonant micro-and nanostructures emerge as a promising platform, which can complement the metal-based counterparts in routine biosensing measurements based on surface-enhanced Raman scattering (SERS). However, lack of in-situ temperature control limits performance of nanostructures for precise SERS-based applications. Here, we present an approach for SERS measurement with simultaneous temperature control and employ Raman spectroscopy to mapping of temperature-dependent Raman signal distribution. We attest a chemically inert black silicon (b-Si) substrate for a non-invasive (chemically non-perturbing) SERS identification of the molecular fingerprints at low concentrations. Additional studies of the slow daylight-driven para-aminothiophenol (PATP)-to-4,4'-dimercaptoazobenzene (DMAB) catalytic conversion in the aqueous methanol solution loaded with colloidal silver nanoparticles confirm the non-invasive SERS performance of the all-dielectric crystalline b-Si substrate. Proposed SERS substrates can be fabricated using easy-to-implement scalable technology of plasma etching making such inexpensive all-dielectric substrates promising for routine SERS applications, where the temperature-feedback and the noninvasiveness are of mandatory importance.

Decreasing the Limits of Detection and Analysis Time of Ion Current Rectification Biosensing Measurements via a Mechanically Applied Pressure Differential

Improving on the analytical capabilities of a measurement is a fundamental challenge with all assays, particularly decreasing the limit of detection while maintaining a practical associated analysis time. Of late, ion current rectification (ICR) biosensing measurements have received a great deal of attention as an analyte-specific, label-free assay. In ICR biosensing, a nanopore coated with an analyte specific binding molecule (e.g., an antibody, an aptamer, etc.) is used to detect a target analyte based on the ability of the target analyte to alter the ICR response of the nanopore upon it binding to the aperture interior. This binding changes the local surface charge and/or size of the nanopore aperture, thus altering its ICR response in a time dependent manner. Here, we report the ability to enhance the transport of a target analyte molecule to and through the aperture of an antibody modified glass nanopore membrane (AMGNM) with the application of a mechanically applied pressure differential. We demonstrate that there is an optimal pressure that balances the flux of the target analyte through the AMGNM aperture with its ability to be bound and detected. Applying the optimal pressure differential allows for picomolar concentrations of the cleaved form of synaptosomal-associated protein 25 (cSNAP-25) to be detected within the same analysis time as micromolar concentrations detected without the use of the pressure differential. The methodology presented here significantly expands the utility of ICR biosensing measurements for detecting low-abundance biomolecules by lowering the limit of detection and reducing the associated analysis time.

Antigen detection via the rate of ion current rectification change of the antibody-modified glass nanopore membrane.

Ion current rectification (ICR), defined as an increase in ion conduction at a given polarity and a decrease in ion conduction for the same voltage at the opposite polarity, i.e., a deviation from a linear ohmic response, occurs in conical shaped pores due to the voltage dependent solution conductivity within the aperture. The degree to which the ionic current rectifies is a function of the size and surface charge of the nanopore, with smaller and more highly charged pores exhibiting greater degrees of rectification. The ICR phenomenon has previously been exploited for biosensing applications, where the level of ICR for a nanopore functionalized with an analyte-specific binding molecule (e.g., an antibody, biotin, etc.) changes upon binding its target analyte (e.g., an antigen, streptavidin, etc.) due to a resulting change in the size and/or charge of the aperture. While this type of detection measurement is typically qualitative, for the first time, we demonstrate that the rate at which the nanopore ICR response changes is dependent on the concentration of the target analyte introduced. Utilizing a glass nanopore membrane (GNM) internally coated with a monoclonal antibody specific to the cleaved form of synaptosomal-associated protein 25 (cSNAP-25), creating the antibody-modified glass nanopore membrane (AMGNM), we demonstrate a correlation between the rate of ICR change and the concentration of introduced cSNAP-25, over a range of 500 nM–100 μM. The methodology presented here significantly expands the applications of nanopore ICR biosensing measurements and demonstrates that these measurements can be quantitative in nature.

Naturally oxidized synthesis of ZnO dahlia-flower nanoarchitecture

Three dimensional ZnO dahlia-flowers have been engineered at room temperature relying on natural oxidation based aqueous chemical synthetic approach. Glycine abetted multicomponent isotropic morphology has been synthesized through the conglomeration of thin nanopetals as building blocks with highly large surface area to volume ratio. Highly controllable morphology of thin nanopetals is achieved by stabilizing their polar faces through the adsorption of reactive hydroxyl and amide functions of glycine. Fourier transform infrared conclusions also exemplify good corroboration towards the use of organic additives. Moreover, the synthesized ZnO flowers have been utilized to fabricate cholesterol biosensor and biosensing measurements which have been performed over the cholesterol concentration range of 1×10−6M to 1×10−3M.

Silica Nanowire Arrays for Diffraction‐Based Bioaffinity Sensing

Arrays of electrodeposited silica nanowires (SiO2 NWs) have been fabricated over large areas (cm(2)) on fluoropolymer thin films attached to glass substrates by a combination of photolithography and electrochemically triggered sol-gel nanoscale deposition. Optical and scanning electron microscopy (SEM) measurements revealed that the SiO2 NW arrays had an average spacing of ten micrometers and an average width of 700 nm with a significant grain structure that was a result of the sol-gel deposition process. The optical diffraction properties at 633 nm of the SiO2 NW arrays were characterized when placed in contact with solutions by using a prism-coupled total internal reflection geometry; quantification of changes in these diffraction properties was applied in various sensing applications. Bulk refractive index sensing by using the SiO2 NW grating was demonstrated with a sensitivity of 1.30×10(-5) RIU. Toposelectively chemically modified SiO2 NW arrays were used for diffraction biosensing measurements of surface binding events, such as the electrostatic adsorption of gold nanoparticles and the bioaffinity adsorption of streptavidin onto a biotin monolayer. Finally, the application of the SiO2 NW arrays for practical medical-diagnostic applications was demonstrated by monitoring the diffraction of SiO2 NW arrays functionalized with a single-stranded (ss)DNA aptamer to detect human α-thrombin from solutions at sub-pathologic nanomolar concentrations.

On-Chip Synthesis of Protein Microarrays from DNA Microarrays via Coupled In Vitro Transcription and Translation for Surface Plasmon Resonance Imaging Biosensor Applications

Protein microarrays are fabricated from double-stranded DNA (dsDNA) microarrays by a one-step, multiplexed enzymatic synthesis in an on-chip microfluidic format and then employed for antibody biosensing measurements with surface plasmon resonance imaging (SPRI). A microarray of dsDNA elements (denoted as generator elements) that encode either a His-tagged green fluorescent protein (GFP) or a His-tagged luciferase protein is utilized to create multiple copies of mRNA (mRNA) in a surface RNA polymerase reaction; the mRNA transcripts are then translated into proteins by cell-free protein synthesis in a microfluidic format. The His-tagged proteins diffuse to adjacent Cu(II)-NTA microarray elements (denoted as detector elements) and are specifically adsorbed. The net result is the on-chip, cell-free synthesis of a protein microarray that can be used immediately for SPRI protein biosensing. The dual element format greatly reduces any interference from the nonspecific adsorption of enzyme or proteins. SPRI measurements for the detection of the antibodies anti-GFP and antiluciferase were used to verify the formation of the protein microarray. This convenient on-chip protein microarray fabrication method can be implemented for multiplexed SPRI biosensing measurements in both clinical and research applications.

Design and characterization of a novel sensor combined imaging and zinc ion sensing

A novel fluorescent sensor for Zn(II) detection and imaging is developed by incorporating 1-(2-pyridylazo)-2-naphthol (PAN) and Rhodamine B (RB) in a sol–gel film on an end face of a gradient index (GRIN) lens rod which is tightly connected with an imaging fiber. This technique has high-quality imaging capabilities for observing remote sample while measuring Zn(II). The RB and PAN employed in combination as the indicator for zinc-sensing is much cheaper than that of the other zinc-sensitive fluorescent dyes. The sol is prepared by base catalyzed hydrolysis of tetraethylorthosilicate (TEOS) with the addition of 3-glycidyloxypropyltrimethoxysilane (GLYMO) and 3-aminopropyltriethoxysilane (APTES), which is more suitable for Zn(II) detection. The sensor has the linear range of Zn(II) from 0.1 to 1mmol/L. Simultaneously, it has the capabilities of real-time imaging with the whole system’s resolution of 18.73 line pairs/mm. This bi-functional sensor has potential applications in in situ biosensing and other remote measurements.

Non-steady response of BOD biosensor for the determination of biochemical oxygen demand in wastewater

A biochemical oxygen demand (BOD) biosensor for effective and expeditious BOD(7) estimations was constructed and the non-steady phase of the output signal was extensively studied. The modelling approach introduced allows response curve reconstruction and a curve fitting procedure of good quality, resulting in parameters indicating the relationship between response and organic substrate concentration and stability properties of the BOD biosensor. Also, the immobilization matrixes of different thicknesses were characterized to determine their suitability for bio-sensing measurements in non-stationary conditions, as well as for the determination of the mechanical durability of the BOD biosensor in time. The non-steady response of the experimental output of the BOD biosensor was fitted according to the developed model that enables to determine the stability of the biosensor output and dependency on biodegradable organic substrate concentration. The calibration range of the studied BOD biosensor in OECD synthetic wastewater was 15-110 mg O(2) L(-1). Repeatability tests showed relative standard deviation (RSD) values of 2.8% and 5.8% for the parameter τ(d), characterizing the transient output of the amperometric oxygen sensor in time, and τ(s), describing the dependency of the transient response of the BOD biosensor on organic substrate concentration, respectively. BOD biosensor experiments for the evaluation of the biochemical oxygen demand of easily degradable and refractory municipal wastewater showed good concurrence with traditional BOD(7) analysis.

Magneto-optical phase modulation in integrated Mach–Zehnder interferometric sensors

The integrated Mach–Zehnder interferometric biosensors are one of the most promising optical biosensors due to their extreme sensitivity and possibility of integration in a lab-on-a-chip. However, the periodic response of the interferometric devices complicates the interpretation of experimental results due to the ambiguity and fading of the signal. To overcome these problems we present a phase modulation system based on the introduction of a periodic phase shift in the reference arm of the interferometers and the Fourier analysis of the output signal. This system allows the direct and unambiguous detection of the phase changes induced by the biosensing measurement. As phase modulation mechanism, we propose and theoretically analyze two different magneto-optic (MO) methods compatible with the standard microelectronic processes. The phase modulators are based on silicon-on-insulator (SOI) waveguides and yttrium iron garnets (YIG) as magnetic material. The first one exploits the non-reciprocal phase shifts induced in the TM guided modes when the orientation of the magnetization of an YIG layer is rotated within the plane of the layer. On the other hand, the MO phase modulation can also rely on the birefringence induced in magnetic liquids under an external magnetic field. We demonstrate that the MO phase modulation is compatible with SOI waveguides showing very high surface sensitivity in the biosensing measurements. In addition, the MO phase modulation can be achieved by using MO interaction lengths of only a few millimetres, facilitating the integration within the interferometric biosensors.

Cytotoxicity and DNA binding characteristics of dextran-conjugated doxorubicins

The antitumor antibiotic doxorubicin was conjugated with polymeric dextrans of various molecular weights and the cytotoxicity of the conjugates against human carcinoma KB-3-1 cells and its multidrug-resistant subclone KB-V-1 cells was measured by tetrazolium salt MTT assay. The conjugates were much less toxic to the KB-3-1 cells than the free doxorubicin but exhibited similar toxicity to the KB-V-1 cells. The conjugate-DNA interactions were monitored in real-time using an optical biosensor based on evanescent wave detection to obtain the association ( k a) and dissociation ( k d) rate constants as well as the equilibrium binding constants ( K A) of the bindings. Both k a and k d values for the conjugates are more than three magnitudes smaller than those for free doxorubicin, while the K A values of the conjugate-DNA complexes are only about 10 times smaller than that of the free doxorubicin–DNA complex. The results indicate that the cytotoxicity and the DNA-binding kinetics of doxorubicin may be modified with dextran conjugation. The K A values obtained from the biosensor measurements were in close agreement with those determined in solution by fluorescent titration method, verifying the utility of the label-free biosensing measurements as an efficient method for studying ligand–DNA interactions.