Concentration Of Streptavidin Research Articles

Specific, sensitive, and reliable sensing of cancer cells based on quantum dot/hydrogel-coated double optical fiber ball

For clinical applications, a sensor of cancer cells should exhibit high sensitivity, have a small and robust sensing structure, and exhibit good anti-interference. This paper presents a novel biosensor for the recognition and concentration detection of biotin receptor (BR)-overexpressed cancer cells. This biosensor uses a hydrogel composed of biotin-quantum dots (BQDs) grafted onto oxidized sodium alginate with carboxymethyl chitosan (BQDs-g-OSA/CMCS) and a double optical fiber ball (DOFB) sensing structure. The developed BQDs-g-QSA/CMCS hydrogel exhibited specific recognition capabilities for cancer cells and an excellent fluorescence stability. Meanwhile, the proposed DOFB structure was small and presented a strong robustness and integrated fluorescence excitation and reception, which are favorable for in vivo sensing. In addition, in-depth study of the sensing performance of the sensor was conducted by utilizing streptavidin (SV) protein and BR-overexpressed cancer cells (leukemia cells, K562). The experimental results revealed a linear relationship between the photoluminescence intensity of the sensor and concentrations of SV and K562, with minimum detection concentrations of 1.62 nM and 1.0 × 103 cells/mL, respectively. In addition, the specificity of the sensor was confirmed through investigation of the its response to another BR-overexpressed cancer cell type (lung cancer cells, A549) and normal cells (human gastric mucosal epithelial, GES-1). The sensor exhibited similar responses to A549 as to K562 cells but not to GES-1 cells, which indicates its specificity toward cancer cells with BR overexpression. In summary, the proposed sensor offers important potential for applications in cancer diagnosis and treatment.

Estimation of biomolecule amount by analyzing magnetic nanoparticle cluster distributions from alternating current magnetization spectra for magnetic biosensing

The hydrodynamic size distribution of magnetic nanoparticle (MNP) clusters is crucial for determining the amounts of biomolecules by analyzing the distributions of the Brownian relaxation time in the corresponding alternating current (AC) magnetization spectra. This study proposes a method for estimating the distribution of MNP clusters from recorded AC magnetization spectra using an experimental-based model. The proposed model is used to form a matrix equation that comprises the distribution and AC magnetization spectra of MNP clusters, and the MNP–biomolecule cluster distribution is obtained by solving an inverse problem. The results show that the variations in the MNP clusters distribution estimated from the model correspond to those measured by dynamic light scattering. The MNP cluster distributions estimated using the experimental model were used to estimate streptavidin concentrations in the range of 0.19–3.38 μM. The estimated values agree accurately with the true values, demonstrating the feasibility of the proposed method for biosensing.

Eco-friendly Synthesis of Silver Nanoparticles and its Application in Hydrogen Photogeneration and Nanoplasmonic Biosensing.

Environmentally friendly methods for silver nanoparticles (AgNPs) synthesis without the use of hazardous chemicals have recently drawn attention. In this work, AgNPs have been synthesized by microwave irradiation using only honey solutions or aqueous fresh pink radish extracts. The concentrations of honey, radish extract, AgNO3 and pH were varied. AgNPs presented mean sizes between 7.0 and 12.8 nm and were stable up to 120 days. The AgNPs were employed as co-catalyst (TiO2 @AgNPs) to increase the hydrogen photogeneration under UV-vis and only visible light irradiation, when compared to pristine TiO2 NPs. The prepared photocatalyst also showed hydrogen generation under visible light. Additionally, AgNPs were used to assemble a nanoplasmonic biosensor for the biodetection of extremely low concentrations of streptavidin, owing to its specific binding to biotin. It is shown here that green AgNPs are versatile nanomaterials, thus being potential candidates for hydrogen photogeneration and biosensing applications.

Gate-assisted MoSe2 transistor to detect the streptavidin via supporter molecule engineering

Atomically thin two-dimensional (2D) transition metal dichalcogenide (TMDC) semiconductor materials have garnered significant attention due to their remarkable properties surpassing conventional metal-oxide semiconductors. These properties include high carrier mobility, adjustable bandgaps, and exceptional sensitivity, providing them with tremendous potential for various applications. The remarkable characteristics of these 2D semiconductor materials led to fabricating of atomically thin electronic devices including diodes, photodetectors, memristors, and biosensors. Here, we present an atomically thin transistor composed of n-type molybdenum di-selenide (n-MoSe2), exhibiting a large reverse current. The charge transport through the MoSe2 transistor is modulated via back gate voltage, studied at various basing and gate voltages. Finally, the MoSe2 transistor device is utilized as a biosensor to detect the protein (streptavidin) as target analyte at fixed biasing and gate voltages. Using our designed pyrene-based supporter molecule, which is stacked over the MoSe2 surface via π-π stacking, the target protein is identified while avoiding any screening effects like Debye screening. Additionally, the real-time response of the biosensing device is recorded while maintaining a constant biasing voltage of 0.5 V, covering a range of streptavidin concentrations down to the lowest detectable concentration of 1 pM. Such utilization of the MoSe2 device as a biosensor could pave the way for manufacturing cutting-edge electrical devices which can detect a broad range of biomolecules, such as DNA (like ct-DNA) and the COVID-19 spike protein.

A Fluorescent Biosensor for Streptavidin Detection Based on Double-Hairpin DNA-Templated Copper Nanoparticles

In this paper, we developed a sensitive, label-free and facile fluorescent strategy for detecting streptavidin (SA) based on double-hairpin DNA-templated copper nanoparticles (CuNPs) and terminal protection of small molecule-linked DNA. Herein, a special DNA hairpin probe was designed and synthesized, which contained two poly T single-stranded loops and a nick point in the middle of the stem. Inspired by the concept of the terminal protection interaction, the specific binding of SA to the biotinylated DNA probe can prevent the exonuclease degradation and keep the integrity of DNA probe, which can be used for synthesizing fluorescent CuNPs as a template. Conversely, the DNA probe would be digested by exonucleases and therefore, would fail to form CuNPs without SA. After systematic optimization, the detection range of SA concentration is from 0.5 to 150 nM with a low detection limit of 0.09 nM. Additionally, the proposed method was also successfully applied in the biological samples. Finally, the proposed method is sensitive, effective and simple, and can be potentially applied for predicting diseases and discovering new drugs.

Low-Power Negative-Differential-Resistance Device for Sensing the Selective Protein via Supporter Molecule Engineering.

Van der Waals (vdW) heterostructures composed of atomically thin two-dimensional (2D) materials have more potential than conventional metal-oxide semiconductors because of their tunable bandgaps, and sensitivities. The remarkable features of these amazing vdW heterostructures are leading to multi-functional logic devices, atomically thin photodetectors, and negative differential resistance (NDR) Esaki diodes. Here, an atomically thin vdW stacking composed of p-type black arsenic (b-As) and n-type tin disulfide (n-SnS2 ) to build a type-III (broken gap) heterojunction is introduced, leading to a negative differential resistance device. Charge transport through the NDR device is investigated under electrostatic gating to achieve a high peak-to-valley current ratio (PVCR), which improved from 2.8 to 4.6 when the temperature is lowered from 300 to 100K. At various applied-biasing voltages, all conceivable tunneling mechanisms that regulate charge transport are elucidated. Furthermore, the real-time response of the NDR device is investigated at various streptavidin concentrations down to 1pm, operating at a low biasing voltage. Such applications of NDR devices may lead to the development of cutting-edge electrical devices operating at low power that may be employed as biosensors to detect a variety of target DNA (e.g., ct-DNA) and protein (e.g., the spike protein associated with COVID-19).

Fabrication of scalable poly(N-isopropylacrylamide)/gold nanoparticles composite ring array as LSPR sensor for label-free biosensor application

Self-organization facilitates the formation of specific structures as a result of constituent interaction. In this study, the bottom of a 500-nm hole array photoresist template, which was deposited with a hydrophobic atom transfer radical polymerization (ATRP) initiator, was wetted by treatment with oxygen plasma. After the removal of the photoresist template, ring patterns of the ATRP initiator were formed at the interface between the hydrophobic and wetting regions. Poly(N-isopropylacrylamide) (PNIPAAm) was grafted from the ring array of the initiator to immobilize gold nanoparticles (AuNPs) as a uniform ring array on the silicon substrate via repeated swelling/shrinking cycles. The localized surface plasmon resonance (LSPR) peaks of the biotin-anchored AuNP–PNIPAAm composite ring (APCR) were employed to detect streptavidin as biosensors. The coupling of biotin and streptavidin led to asymptotical convergence from rings to disks as well as blue-shifts of the LSPR peaks. Linear correlation between the blue-shift and streptavidin concentration showed a limit of detection of ∼10 nM and a linear range of 10–150 nM for the detection of streptavidin within 30 min. The simple method of combining lithography and plasma technology provides a versatile platform for developing the scalable ring structure of AuNP for highly sensitive and sensitive biosensing.

Highly sensitive plasmonic nanorod hyperbolic metamaterial biosensor

Plasmonic sensing based on nanostructures is a powerful analytical tool for ultrasensitive label-free biomolecule detection that holds great potential in the field of clinical diagnostics and biomedical research. Here, we report the fabrication, the characterization, and the principle of operation of gold nanorod hyperbolic metamaterials (NHMMs) along with ultrasensitive bulk refractive index and label-free biomolecular detection. By combining electron-beam lithography and nanoscale electroplating, we demonstrate the fabrication of a highly ordered, height-controllable, and vertical array of nanorods. By exciting the bulk plasmon–polariton mode in the NHMM using a prism-coupling technique and integrating the sensor in microfluidics, we demonstrate that the bulk sensitivity and figure of merit of our device could reach 41,600 nm/RIU and 416 RIU-1, respectively. The physical mechanism of this high bulk sensitivity is revealed through theoretical and experimental studies. Moreover, by bio-functionalizing the surface of the NHMM sensor, monitoring the binding of streptavidin at dilute concentrations is performed in real time. We test different concentrations of streptavidin ranging from 200 to 5 µg/mL, and the NHMM biosensor exhibits a 1 nm wavelength shift for a 5 µg/mL streptavidin detection. By fitting the Hill equation of the NHMM biosensor and taking into account the level of noise (0.05 nm) as the minimum wavelength shift of the detectable limit, the limit of detection of the NHMM biosensor to streptavidin can be estimated to be 0.14 µg/mL (2.4 nm). As a direct comparison, a 0.5 nm wavelength shift for 20 µg/mL of streptavidin is reported when using a conventional gold film sensor under identical experimental conditions. The developed plasmonic NHMM sensor shows tremendous potential for highly sensitive bulk solutions and biomolecule detection and provides a promising avenue for free-label biosensing applications in the future.

Ultrasensitive SEIRA detection using gold nanobipyramids: Toward efficient multimodal immunosensor

Surface enhanced spectroscopies are proven to be highly efficient in biosensing applications. Their implementation as analytical tools in the detection of medically relevant target analytes demonstrated their ability to considerably decrease the minimum detected analyte concentration, thus showing improved sensitivity compared to commercially available detection kits. In this work, we propose the employment of Surface Enhanced Infrared Absorption (SEIRA), a less exploited analysis tool, for the efficient, specific, and ultrasensitive detection of the biotin-streptavidin recognition interaction using a gold nano-bipyramid-based (AuBPs) immunosensor. The unique optical properties of the AuBPs, such as tunability of the LSPR band ranging from the visible to NIR regions of the spectrum, great absorption capabilities and locally enhanced electromagnetic field at their tips and edges, enable an improved efficiency of the IR absorption of the target analytes. To the as-synthesized AuBPs, a chemically formed p-ATP@biotin recognition element was grafted due to the strong Au-S interaction, followed by the exposure of the obtained biotinylated nanosystem to the streptavidin target analyte. FT-IR experimental measurements as well as theoretical simulations confirm both the successful grafting of the p-ATP@biotin onto the Au surface and successful capture of the target streptavidin protein. A limit of detection of 10−12 M streptavidin concentration was determined, thus standing as a “proof-of-concept” for the development of efficient ultrasensitive SEIRA biosensing devices for more complex biologically relevant applications.

Fluorometric Detection of Streptavidin with a Cationic Conjugated Polymer and Hairpin DNA Probe

AbstractA simple and rapid method for streptavidin (SA) detection is developed based on a cationic conjugated polymer (CCP)‐mediated fluorescence resonance energy transfer (FRET) between poly [(9,9‐bis(6′‐N, N, N‐trimethylammonium) hexyl) fluorenylene phenylene (PFP) and SYBR Green I (SG I). A hairpin DNA labeled with biotin at the 3′ terminus was designed for the binding of SA and SYBR Green I (SG I) so that an amplified signal of emission fluorescence was detected as the CCP‐mediated FRET was induced in the presence of SA. The calibration curve exhibited a linear correlation between fluorescence intensity and SA concentration from 0 to 40 nM, and the linear regression equation was y=0.0467x+0.5814, with a correlation coefficient (R2) of 0.9927, and the detection limit was 0.5 nM. Further investigations showed that the method exhibited a significantly high specificity for the target protein and a satisfactory practical performance when human serum samples spiked with SA were tested. The results suggest that this strategy is practically applicable as it may enable an effective tool for studies on the ligand‐protein interactions as well as the qualitative and quantitative detection of proteins.

Optical Detection of Streptavidin-Biotin Binding on Nanoporous Anodic Alumina Pore Surface

Nanoporous anodic alumina (NAA) is a material with a high interest in nanotechnology[1]. Its physical and chemical properties combined with a cost-effective and scalable production process make it a good candidate in many nanotechnologically-related applications. Its high and tuneable surface-to-volume ratio as well as its interesting optical properties[2] have been used as the basis of different biosensing schemes[3]. Biosensing, and specifically optical biosensing is of great interest in health and environmental applications. The reflection interference spectroscopy (RIfS) method based on NAA has demonstrated its ability in detecting many kinds of molecules (proteins, antibodies[4]).One of the molecular systems with interest in biotechnological applications is the streptavidin-biotin system. The high affinity between both molecules is exploited in many applications for the binding of different biological species. For this reason, we have studied the streptavidin-biotin binding event with NAA-based RIfS, as part of a biosensing process. In our study we assess the optimal conditions for real time monitoring of the different steps (covalent streptavidin bonding, surface passivation, biotin complexation). In order to establish such optimal conditions, we have chosen the protein thrombin as a model molecule to be detected.We prepared NAA with the usual anodization conditions under oxalic acid electrolyte to obtain porous layers with uniform pore diameter and with a thickness that permits the measurement with RIfS in a flow-cell where the different species in solution can be introduced. The experiments consisted of monitoring the change in effective optical thickness (EOT, the quantitative characteristic parameter obtained from RIfS) in the different steps of the biosensing process. The NAA pore surfaces were initially functionalized with (3-aminopropyl)triethoxysilane (APTES) with the carboxyl groups activated in order to form later a peptide bond with the streptavidin.One of the parameters to be optimized was the pore diameter. Furthermore, in order to study the ability of RIfS to detect the covalent streptavidin binding to the NAA pore surface, different concentrations of streptavidin were tested. Finally, the biosensing capabilities were evaluated by detecting biotinylated thrombin at different concentrations.An example of a complete experiment with all the steps in the streptavidin-biotinylated molecule attachment detection can be seen in Figure 1. After establishing a baseline with a PBS flow, streptavidin at 50 µg/ml is introduced in the RIfS setup and its covalent attachment to APTES is detected as an increase in EOT. Following this first step, PBS is introduced to wash away the non-specifically bound molecules and a 3% Bovine Serum Albumin (BSA) solution is flowed to passivate the remaining activated carboxyl groups of APTES and the remaining hydroxyl groups on the NAA pore surface. Such steps are detected by the RIfS procedure. Then, after a subsequent PBS wash, biotinylated thrombin at a concentration of 20 µg/ml is introduced causing a steady increase in EOT reaching a value of 3.5 nm above the initial one. The experiment in Figure 1 was repeated for different NAA pore sizes revealing a higher change in EOT for bigger pore sizes.An important parameter in the detection of the biotinylated molecules binding is the concentration at the streptavidin immobilization step. Thus, different streptavidin concentrations were tested in 60 nm pore diameter NAA. Results are summarized in Figure 2: for the highest concentrations (50 µg/ml) the attachment of streptavidin shows a two-phase curve with an initial phase with a faster increase in EOT and a second phase with a slower increase after an inflection point in the EOT versus time curve. Instead, for a small concentration (1 µg/ml) an initial fast increase is followed by a stabilization of the EOT signal. These two behaviors are associated with the attachment of streptavidin to the activated carboxyl of APTES for the initial fast EOT increase, while the subsequent slower increase can be explained by the aggregation of streptavidin through amine group binding.Finally, in order to demonstrate the capability of quantifying the attachment of biotinylated molecules to the NAA surface, the process was carried out using thrombin as the model molecule and for increasing concentrations. Figure 3 shows the change in EOT as a function of the thrombin concentration. It can be seen that RIfS based on NAA sensing platforms is able to quantify such attachment.[1] Josep Ferré-Borrull, et al., Nanomaterials, vol. 7, p. 5225 (2014).[2] Josep Ferré-Borrull et al., in NANOPOROUS ALUMINA: FABRICATION, STRUCTURE, PROPERTIES AND APPLICATIONS, Springer Series in Materials Science, vol. 219, p. 185 (2015).[3] Chris Eckstein, ACS Applied Materials and Interfaces, vol. 10, p.10571 (2018).[4] Gerard Macías, Analyst, vol. 140, p. 4848 (2015). Figure 1

An aptasensor using ceria electrodeposited-screen-printed carbon electrode for detection of epithelial sodium channel protein as a hypertension biomarker.

Epithelial sodium channel (ENaC) is a transmembrane protein that has an essential role in maintaining the levels of sodium in blood plasma. A person with a family history of hypertension has a high enough amount of ENaC protein in the kidneys or other organs, so that the ENaC protein acts as a marker that a person is susceptible to hypertension. An aptasensor involves aptamers, which are oligonucleotides that function similar to antibodies, as sensing elements. An electrochemical aptasensor for the detection of ENaC was developed using a screen-printed carbon electrode (SPCE) which was modified by electrodeposition of cerium oxide (CeO2). The aptamer immobilization was via the streptavidin–biotin system. The measurement of changes in current of the active redox [Fe(CN)6]3−/4− was carried out by differential pulse voltammetry. The surfaces of SPCE and SPCE/CeO2 were characterized using scanning electron microscopy, voltammetry and electrochemical impedance spectroscopy. The Box–Behnken experimental optimization design revealed the streptavidin incubation time, aptamer incubation time and streptavidin concentrations were 30 min, 30 min and 10.8 µg ml−1, respectively. Various concentrations of ENaC were used to obtain the linearity range of 0.05–3.0 ng ml−1, and the limits of detection and quantification were 0.012 ng ml−1 and 0.038 ng ml−1, respectively. This aptasensor method has the potential to measure the ENaC protein levels in urine samples as well as to be a point-of-care device.

Demonstration of a Label-Free and Low-Cost Optical Cavity-Based Biosensor Using Streptavidin and C-Reactive Protein.

An optical cavity-based biosensor (OCB) has been developed for point-of-care (POC) applications. This label-free biosensor employs low-cost components and simple fabrication processes to lower the overall cost while achieving high sensitivity using a differential detection method. To experimentally demonstrate its limit of detection (LOD), we conducted biosensing experiments with streptavidin and C-reactive protein (CRP). The optical cavity structure was optimized further for better sensitivity and easier fluid control. We utilized the polymer swelling property to fine-tune the optical cavity width, which significantly improved the success rate to produce measurable samples. Four different concentrations of streptavidin were tested in triplicate, and the LOD of the OCB was determined to be 1.35 nM. The OCB also successfully detected three different concentrations of human CRP using biotinylated CRP antibody. The LOD for CRP detection was 377 pM. All measurements were done using a small sample volume of 15 µL within 30 min. By reducing the sensing area, improving the functionalization and passivation processes, and increasing the sample volume, the LOD of the OCB are estimated to be reduced further to the femto-molar range. Overall, the demonstrated capability of the OCB in the present work shows great potential to be used as a promising POC biosensor.

Metasurface‐Enhanced Lab‐on‐Fiber Biosensors

AbstractThe integration of metasurfaces on the tip of optical fibers enables advanced wavefront manipulations in Lab‐on‐Fiber application scenarios, and brings about new degrees of freedom that can be exploited for optimizing the surface sensitivity to local variations of the refractive index. Here, a novel biosensing platform is reported based on the integration of a phase‐gradient plasmonic metasurface on the tip of an optical fiber, able to detect biomolecular interactions with very high sensitivity. Specifically, the capability of the proposed platform to detect very low concentrations of streptavidin in running buffer solutions, with a limit of detection of the order of a few ng mL−1, is demonstrated. In addition, its inherent integrability within medical catheters/needles renders it potentially very attractive for application scenarios of real‐time diagnosis via liquid biopsy at precise locations inside the human body.

Extended-Gate Amorphous InGaZnO Thin Film Transistor for Biochemical Sensing

We report on a stable and sensitive biochemical sensor based on amorphous indium-gallium-zinc-oxide (a-IGZO) thin-film transistors (TFTs) with a versatile extended-gate electrode used as the sensing membrane. The extended-gate allows the active transistor channel not to be exposed to the ionic analyte and the gate surface may be modified with chemicals or affinity reagents without affecting the operation of the active sensor. The extended-gate a-IGZO TFT was able to detect the pH levels of various solutions based on protonation and deprotonation at the surface of the indium-tin-oxide (ITO) extended-gate electrode achieving a pH dependent threshold voltage shift of 22.8 mV/pH. Highly sensitive and specific protein sensing was achieved for both electrical polarities according to pH values of solutions using the biotin-streptavidin interaction. The extended-gate TFT sensor demonstrated a detection limit for streptavidin down to 10 fM, using the biotin-streptavidin binding on the extended-gate surface. It was also possible to detect changes in the streptavidin concentration in real-time with specificity and repeatability. Since the extended-gate can be easily replaced without affecting the performance of the a-IGZO TFT, this sensor configuration may be utilized for future multifunctional biomolecular sensing and analysis.

(Invited) Optical Detection of Streptavidin-Biotin Binding on Nanoporous Anodic Alumina Pore Surface

Nanoporous anodic alumina (NAA) is a material with a high interest in nanotechnology[1]. Its physical and chemical properties combined with a cost-effective and scalable production process make it a good candidate in many nanotechnologically-related applications. Its high and tunable surface-to-volume ratio as well as its interesting optical properties[2] have been used as the basis of different biosensing schemes[3]. Biosensing, and specifically optical biosensing is of great interest in health and environmental applications. The reflection interference spectroscopy (RIfS) method based on NAA has demonstrated its ability in detecting many kinds of molecules (proteins, antibodies[4]). One of the molecular systems with interest in biotechnological applications is the streptavidin-biotin system. The high affinity between both molecules is exploited in many applications for the binding of different biological species. For this reason, we have studied the streptavidin-biotin binding event with NAA-based RIfS, as part of a biosensing process. In our study we assess the optimal conditions for real time monitoring of the different steps (covalent streptavidin bonding, surface passivation, biotin complexation). In order to establish such optimal conditions, we have chosen the protein thrombin as a model molecule to be detected. We prepared NAA with the usual anodization conditions under oxalic acid electrolyte to obtain porous layers with uniform pore diameter and with a thickness that permits the measurement with RIfS in a flow-cell where the different species in solution can be introduced. The experiments consisted of monitoring the change in effective optical thickness (EOT, the quantitative characteristic parameter obtained from RIfS) in the different steps of the biosensing process. The NAA pore surfaces were initially functionalized with (3-aminopropyl) triethoxysilane (APTES) with the carboxyl groups activated in order to form later a peptide bond with the streptavidin. One of the parameters to be optimized was the pore diameter. Furthermore, in order to study the ability of RIfS to detect the covalent streptavidin binding to the NAA pore surface, different concentrations of streptavidin were tested. Finally, the biosensing capabilities were evaluated by detecting biotinylated thrombin at different concentrations. An example of a complete experiment with all the steps in the streptavidin-biotinylated molecule attachment detection can be seen in Figure 1. After establishing a baseline with a PBS flow, streptavidin at 50 µg/ml is introduced in the RIfS setup and its covalent attachment to APTES is detected as an increase in EOT. Following this first step, PBS is introduced to wash away the non-specifically bound molecules and a 3% Bovine Serum Albumin (BSA) solution is flowed to passivate the remaining activated carboxyl groups of APTES and the remaining hydroxyl groups on the NAA pore surface. Such steps are detected by the RIfS procedure. Then, after a subsequent PBS wash, biotinylated thrombin at a concentration of 20 µg/ml is introduced causing a steady increase in EOT reaching a value of 3.5 nm above the initial one. The experiment in Figure 1 was repeated for different NAA pore sizes revealing a higher change in EOT for bigger pore sizes. An important parameter in the detection of the biotinylated molecules binding is the concentration at the streptavidin immobilization step. Thus, different streptavidin concentrations were tested in 60nm pore diameter NAA. Results are summarized in Figure 2: for the highest concentrations (50 µg/ml) the attachment of streptavidin shows a two-phase curve with an initial phase with a faster increase in EOT and a second phase with a slower increase after an inflection point in the EOT versus time curve. Instead, for a small concentration (1 µg/ml) an initial fast increase is followed by a stabilization of the EOT signal. These two behaviors are associated with the attachment of streptavidin to the activated carboxyl of APTES for the initial fast EOT increase, while the subsequent slower increase can be explained by the aggregation of streptavidin through amine group binding. Finally, in order to demonstrate the capability of quantifying the attachment of biotinylated molecules to the NAA surface, the process was carried out using thrombin as the model molecule and for increasing concentrations. Figure 3 shows the change in EOT as a function of the thrombin concentration. It can be seen that RIfS based on NAA sensing platforms is able to quantify such attachment. [1] Josep Ferré-Borrull, et al., Materials, vol. 7, p. 5225 (2014). [2] Josep Ferré-Borrull et al., in NANOPOROUS ALUMINA: FABRICATION, STRUCTURE, PROPERTIES AND APPLICATIONS, Springer Series in Materials Science, vol. 219, p. 185 (2015). [3] Chris Eckstein, et al. ACS Appl Mater Interfaces, vol. 10, p.10571 (2018). [4] Gerard Macías, et al. Analyst, vol. 140, p. 4848 (2015). Figure 1

Surface lattice resonance of line array of poly (glycidyl methacrylate) with CdS quantum dots for label-free biosensing

One dimensional plasmonic grating is a kind of resonant electromagnetic wave absorber with a characteristic wavelength. This study focusses on one-dimensional plasmonic grating consisting of poly (glycidyl methacrylate) (PGMA) brushes and CdS quantum dots (CdQDs) fabrication and PGMA chains grafted on a primary substrate in a line array continued by the immobilization of biotin-modified CdQDs. PGMA brush line array (PBLA) of plasmonic grating exhibited an absorptance at 441 nm while at the same time, CdQDs immobilized with PBLA showed characteristic absorbance at 396 nm. The blue-shift from 441 nm matches the absorbance peak of biotin-modified CdQDs resulting in the enhancement of photoluminescence emission of CdQDs. With streptavidin incubation to assemble CdQDs at 50 nM, the significant decrease in grating height resulted in the red-shift of the absorbance peak to 536 nm. Due to the deviation in absorbance, the intensity of the PL emission decreased gradually with the increase in concentration of streptavidin. In addition, our results showed that streptavidin incubation altered the color reflected from the surface due to effective changes in the refractive index of the layer as well. The limit of detection of the grating for streptavidin detection was determined to be 50 nM. Thus, PBLA-CdQD has the potential to act as a highly-sensitive, label-free optical biosensor.

Real-Time Monitoring of Biotinylated Molecules Detection Dynamics in Nanoporous Anodic Alumina for Bio-Sensing.

The chemical modification, or functionalization, of the surfaces of nanomaterials is a key step to achieve biosensors with the best sensitivity and selectivity. The surface modification of biosensors usually comprises several modification steps that have to be optimized. Real-time monitoring of all the reactions taking place during such modification steps can be a highly helpful tool for optimization. In this work, we propose nanoporous anodic alumina (NAA) functionalized with the streptavidin-biotin complex as a platform towards label-free biosensors. Using reflective interferometric spectroscopy (RIfS), the streptavidin-biotin complex formation, using biotinylated thrombin as a molecule model, was monitored in real-time. The study compared the performance of different NAA pore sizes in order to achieve the highest response. Furthermore, the optimal streptavidin concentration that enabled the efficient detection of the biotinylated thrombin attachment was estimated. Finally, the ability of the NAA-RIfS system to quantify the concentration of biotinylated thrombin was evaluated. This study provides an optimized characterization method to monitor the chemical reactions that take place during the biotinylated molecules attachment within the NAA pores.

PEGylated surfaces for the study of DNA-protein interactions by atomic force microscopy.

DNA-protein interactions are vital to cellular function, with key roles in the regulation of gene expression and genome maintenance. Atomic force microscopy (AFM) offers the ability to visualize DNA-protein interactions at nanometre resolution in near-physiological buffers, but it requires that the DNA be adhered to the surface of a solid substrate. This presents a problem when working in biologically relevant protein concentrations, where proteins may be present in large excess in solution; much of the biophysically relevant information can therefore be occluded by non-specific protein binding to the underlying substrate. Here we explore the use of PLLx-b-PEGy block copolymers to achieve selective adsorption of DNA on a mica surface for AFM studies. Through varying both the number of lysine and ethylene glycol residues in the block copolymers, we show selective adsorption of DNA on mica that is functionalized with a PLL10-b-PEG113/PLL1000-2000 mixture as viewed by AFM imaging in a solution containing high concentrations of streptavidin. We show - through the use of biotinylated DNA and streptavidin - that this selective adsorption extends to DNA-protein complexes and that DNA-bound streptavidin can be unambiguously distinguished in spite of an excess of unbound streptavidin in solution. Finally, we apply this to the nuclear enzyme PARP1, resolving the binding of individual PARP1 molecules to DNA by in-liquid AFM.

Effect of the nonspecific binding in differential impedance biosensing.

The detection limits of impedance biosensors are dictated by the presence of background nonspecific binding, yet almost all the detection limits reported in the literature are determined using a clean buffer solution without confronting this real challenge. In this work, the authors employed the simplest "differential" impedance biosensor, composed of poly-l-lysine-polyethylene glycol-biotin-coated gold electrodes for the detection of streptavidin in the presence of 0.1% fetal calf serum, and studied the effect of the nonspecific binding on the performance of the differential impedance biosensing. The lowest streptavidin concentration detected by the system (5 μg/ml) was 1 order of magnitude higher (worse) than that from a previously demonstrated impedance biosensor where avidin was detected in the absence of background proteins. Interestingly, the origin of the differential signal was not due to the electrochemical properties of streptavidin itself but was that of the serum, where the coverage of the electrode by streptavidin indirectly modulated the electrical signal by suppressing the accessibility of the serum to the electrode.