Sensor Chip Surface Research Articles

In Vitro Performance of a Long-Range Surface Plasmon Hydrogel Aptasensor for Continuous and Real-Time Vancomycin Measurement in Human Serum.

This study investigated the suitability of surface modification for a long-range surface plasmon (LRSP) aptasensor using two different hydrogels, aiming at real-time monitoring of vancomycin (VCM) in undiluted serum and blood. Three different layer structures were formed on a gold surface of LRSP sensor chip using poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-N-methacryloyl-(L)-tyrosinemethylester (MAT)] (PMM) and poly[MPC-co-2-ethylhexyl methacrylate (EHMA)-co-MAT] (PMEM). The peptide aptamer for VCM was immobilized in PMM and PMEM via MAT. Among four differently prepared sensor chips, the LRSP hydrogel aptasensor with PMM, referred to as the PMM hydrogel, exhibited the highest sensor output and superior antifouling properties. Following the optimization of the PMM hydrogel preparation conditions, the shelf life of the PMM hydrogel was determined to exceed 2 weeks, and the same sensor chip could be used for 102 days without significant performance deterioration. The PMM hydrogel was then applied for VCM measurement in undiluted serum in vitro, where it demonstrated a limit of detection of 0.098 μM and a dynamic range of 0.18-100 μM, covering the therapeutic range. Additionally, the PMM hydrogel enabled the continuous measurement of various VCM concentrations in serum without rinsing and showed a concentration-dependent output in undiluted blood. These findings underscore the potential of the PMM hydrogel for real-time and direct monitoring of VCM in body fluids.

The investigation of the interaction between fluorescent carbon dots labeling silk fibroin using a fluorescence microscope–surface plasmon resonance system

The use of fluorescent carbon dots (CDs) as highly precise biolabeling probes has been widespread in the fields of live cell imaging and protein labeling due to their small size and excellent photoluminescence ability to accurately target specific molecules with surface chemical properties. However, there was a lack of research on the interaction between CDs and labeled molecules. In this work, we presented a novel investigation strategy, the fluorescence microscopy-surface plasmon resonance (FM-SPR) system, which combined the use of fluorescence microscopy and wavelength modulation surface plasmon resonance to study the interaction between CDs and labeled molecules in real-time. Using this system, simultaneously recorded the SPR signals and the fluorescence images on the surface of the FM-SPR sensor chip. We observed the dynamic curve and fluorescence images of the interaction between green emissive nitrogen-doped carbon dots (N-CDs) and silk fibroin (SF) in real-time. The kinetic parameters, the quantitative analysis, and the investigation of the binding could be achieved. The results showed a strong linear relationship between the change in SPR signals and the concentration of N-CDs, with a linear coefficient of 0.99913. The linear detection range was 2.5 μg/mL–100 μg/mL, and the real lowest detection limit reached 0.5 μg/mL. Additionally, the green fluorescence points in the imaging region on the FM-SPR sensor chip increased with the concentration of N-CDs, which was consistent with the change in SPR signals. Using this system we also acquired the association rate and dissociation rate of N-CDs to SF which were 2.65 × 10−5/s and 1.52 × 10−5/s, respectively. This demonstrated the effectiveness of our method in quantitatively analyzing SF labeled with N-CDs.

Gold nanoparticle-powered screening of membrane protein-specific lipids from complex lipid mixtures

Membrane proteins (MPs) are affected by binding of specific lipids. We previously developed a methodology for systematically analyzing MP-lipid interactions leveraging surface plasmon resonance (SPR). In this method, the gold sensor chip surface was modified with a self-assembled monolayer (SAM), which allowed for a larger amount of MP-immobilization. However, the laborious lipid purification step remained a bottleneck. To address this issue, a new strategy has been developed utilizing gold nanoparticles (AuNPs) instead of the gold sensor chip. AuNPs were coated with SAM, on which MP was covalently anchored. The MP-immobilized AuNPs were mixed with a lipid mixture, and the recovered lipids were quantified by LC–MS. Bacteriorhodopsin (bR) was used as an MP to demonstrate this concept. We optimized immobilization conditions and confirmed the efficient immobilization of bR by dynamic light scattering and electron micrographs. Washing conditions for pulldown experiments were optimized to efficiently remove non-specific lipids. A new binding index was introduced to qualitatively reproduce the known affinity of lipids for bR. Consequently, the low-abundant and least-studied lipid S-TeGD was identified as a candidate for bR-specific lipids. This technique can skip the laborious lipid purification process, accelerating the screening of MP-specific lipids from complex lipid mixtures.

Facile Purification and Use of Tobamoviral Nanocarriers for Antibody-Mediated Display of a Two-Enzyme System.

Immunosorbent turnip vein clearing virus (TVCV) particles displaying the IgG-binding domains D and E of Staphylococcus aureus protein A (PA) on every coat protein (CP) subunit (TVCVPA) were purified from plants via optimized and new protocols. The latter used polyethylene glycol (PEG) raw precipitates, from which virions were selectively re-solubilized in reverse PEG concentration gradients. This procedure improved the integrity of both TVCVPA and the wild-type subgroup 3 tobamovirus. TVCVPA could be loaded with more than 500 IgGs per virion, which mediated the immunocapture of fluorescent dyes, GFP, and active enzymes. Bi-enzyme ensembles of cooperating glucose oxidase and horseradish peroxidase were tethered together on the TVCVPA carriers via a single antibody type, with one enzyme conjugated chemically to its Fc region, and the other one bound as a target, yielding synthetic multi-enzyme complexes. In microtiter plates, the TVCVPA-displayed sugar-sensing system possessed a considerably increased reusability upon repeated testing, compared to the IgG-bound enzyme pair in the absence of the virus. A high coverage of the viral adapters was also achieved on Ta2O5 sensor chip surfaces coated with a polyelectrolyte interlayer, as a prerequisite for durable TVCVPA-assisted electrochemical biosensing via modularly IgG-assembled sensor enzymes.

Investigating membrane-binding properties of lipoxygenases using surface plasmon resonance

Lipoxygenases (LOXs) catalyze the oxidation of polyunsaturated fatty acids and synthesize oxylipin products that drive important cellular signaling processes in plants and animals. While there has been indirect evidence presented for the interaction of mammalian LOXs with membranes, a quantitative study of the molecular details of LOX-membrane interactions is lacking. Here, we mimicked biological membranes using surface plasmon resonance (SPR) sensor chips derivatized with 2-D planar lipophilic anchors (2D LP) to capture liposomes of varying phospholipid compositions that self-assemble into lipid bilayers on the SPR chip. The sensor chip surfaces were then used to investigate the membrane-binding properties of model LOX enzymes. SPR binding assays displayed reproducible and stable liposome capture to the sensor chip surface that allowed for the detailed characterization of LOX-membrane interactions. Our studies demonstrate a calcium-dependence for the membrane binding activities of coral 8R-LOX and human 15-LOX-2. Furthermore, our data confirm the importance of key membrane insertion loop residues in each of these LOX enzymes for membrane binding activity. Experiments utilizing model plant and human LOXs reveal differences in membrane-binding specificities. Our study establishes and validates a robust SPR-based platform using 2D LP sensor chips that allows for the detailed study of LOX-membrane interactions under different experimental conditions, including altered membrane compositions. Collectively, this investigation improves our overall understanding of LOX-membrane interaction properties, and our SPR-based approach holds potential for future use in the development of LOX-based therapeutics.

Localized surface plasmon resonance biosensor chip surface modification and signal amplifications toward rapid and sensitive detection of COVID-19 infections

We developed a multi-pronged approach to enhance the detection sensitivity of localized surface plasmon resonance (LSPR) sensor chips to detect SARS-CoV-2. To this end, poly(amidoamine) dendrimers were immobilized onto the surface of LSPR sensor chips to serve as templates to further conjugate aptamers specific for SARS-CoV-2. The immobilized dendrimers were shown to reduce surface nonspecific adsorptions and increase capturing ligand density on the sensor chips, thereby improving detection sensitivity. To characterize the detection sensitivity of the surface-modified sensor chips, SARS-CoV-2 spike protein receptor-binding domain was detected using LSPR sensor chips with different surface modifications. The results showed that the dendrimer-aptamer modified LSPR sensor chip exhibited a limit of detection (LOD) of 21.9 pM, a sensitivity that was 9 times and 152 times more sensitive than the traditional aptamer- or antibody-based LSPR sensor chips, respectively. In addition, detection sensitivity was further improved by combining rolling circle amplification product and gold nanoparticles to further amplify the detection signals by increasing both the target mass and plasmonic coupling effects. Using pseudo SARS-CoV-2 viral particles as detection targets, we demonstrated that this combined signal intensification approach further enhanced the detection sensitivity by 10 folds with a remarkable LOD of 148 vp/mL, making it one of the most sensitive SARS-CoV-2 detection assays reported to date. These results highlight the potential of a novel LSPR-based detection platform for sensitive and rapid detection of COVID-19 infections, as well as other viral infections and point-of-care applications.

Development of a Plasmonic Sensor for a Chemotherapeutic Agent Cabazitaxel.

Drug dosage is a crucial subject in both human and animal treatment. Administering less drug dosage may prevent treatment or make it less effective, and high drug dosage may cause a heightened risk of adverse effects, or in some cases, cost a patient's life. Also, even when the dosage is administered carefully, metabolic differences may cause different effects on different patients. Because of these considerations, monitoring drug dosage in the body is a critical and significant requirement in the health industry. Within the scope of this study, a reusable surface plasmon resonance (SPR) chip with fast response, high selectivity, and no pretreatment is produced for the chemotherapeutic agent cabazitaxel. A cabazitaxel-imprinted nanofilm was synthesized on the sensor chip surface and characterized by atomic force microscopy, ellipsometry, and contact angle measurements. Standard cabazitaxel solution and an artificial plasma sample were used for the kinetic analysis. Docetaxel, methylprednisolone, and dexamethasone were analyzed for their selectivity experiment. In addition, the repeatability and storage durability of the sensor were also evaluated. As a result of the adsorption studies, the limit of detection and limit of quantitation values were found to be 0.012 and 0.036 μg/mL, respectively. High-performance liquid chromatography analysis was used to validate the response of the cabazitaxel-imprinted sensor.

(Invited, Digital Presentation) Development of Multiplex Electrode Array Sensors for Proteases Activity Profiling Toward Cancer Diagnosis

Proteases are a large family of enzymes involved in many important biological processes. Quantitative detection of the activity profile of specific target proteases is in high demand for the diagnosis and treatment monitoring of diseases such as cancers. The overexpression and enhanced activity of proteases were found to correlate well with cancer development. Protease inhibitors are also a class of major anti-cancer drug candidates. However, due to the presence of a large number (over 600) of proteases in human body, their complex interactions with each other, and large influence by the surrounding factors (such as buffer composition, pH value and temperature), it is challenging to accurately detect the activity of specific proteases in complex media. It is even more difficult to measure the activity profiles of a large set of proteases that are relevant to cancers. Here we summarize our studies on the development of a multiplex electrode array biosensor platform toward cancer diagnosis based on rapid profiling of multiple protease activities. Both nano- and micro-electrode arrays have been demonstrated for electrochemical detection of protease activities. The individually addressed 3x3 gold thin-film microelectrode array is particularly attractive for rapid profiling of multiple protease activities.The general sensor scheme involves attaching short peptides with specific sequences of 4 to 8 amino acids on the electrode surface of the electrochemical sensor chip, which serve as the substrates for selective proteolysis by the cognate proteases. The far end is covalently attached with a ferrocene (Fc) group as the redox tag for electrochemical measurements. The quantity of the intact peptides on the electrode surface is measured with an AC voltammetry (ACV) method, which is proportional to the peak current at the specific electrode potential when Fc oxidation is oxidized into ferrocenium (Fc+). As the peptide is cleaved by the cognate protease, the peak current decreases continuously in the continuously repeated ACV curves. The recorded kinetic proteolytic curve can be quantitatively described by the heterogeneous Michaelis-Menten model following an exponential decay. The inverse of exponential decay time constant, i.e., 1/τ, is found to represent the protease activity which equals to [E](kcat /KM), where [E] is the protease concentration and kcat /KM is the specificity constant defined by the kinetic proteolytic reaction constant kcat and the Michaelis equilibrium constant KM. The kcat /KM values have been investigated versus the substrate peptide sequence and length, temperature, and buffer composition to optimize the detection for activity of cathepsin B, a potential cancer biomarker. The limit of detection (LOD) for activated cathepsin B can reach down to 0.57 pM, sufficient to directly detect cathepsin B in diluted human serum.This electrochemical method has been compared and validated with the commonly used affinity assay, i.e., enzyme-linked immunosorbent assay (ELISA). The ELISA measurements were found to be more sensitive to the concentration of proenzyme (zymogen), while the electrochemical method detects the activity of the proteases which reflects the intrinsic biological function. This provides more critical information than the concentration derived from ELISA.Currently, two sets of peptides have been synthesized which are selective for detection of cathepsin B and ADAM-17, respectively. They are functionalized to the 3x3 gold thin-film microelectrode array and used to simultaneously detect these two types of proteases in serum samples from breast cancer patients at different stages. The activity profiles will be correlated to the cancer development. Figure 1

Label-free detection of glypican-3 using reduced graphene oxide /polyetherimide/gold nanoparticles enhanced aptamer specific sensing interface on light-addressable potentiometric sensor

Glypican-3 (GPC3) protein is considered as an effective and specific biomarker for the accurate diagnosis of hepatocellular carcinoma (HCC), and there is a critical demand to construct strategies with shorter time, lower cost noise and higher sensitivity. In this study, a biosensor for label-free detection of GPC3 was prepared by modifying GPC3 aptamer (GPC3Apt) on the surface of Si-based light addressable potentiometric sensor (LAPS) chip modified by reduced graphene oxide/polyetherimide/gold nanoparticles (rGO/PEI/AuNPs). Among them, GPC3Apt with a low equilibrium dissociation constant was used as a biorecognition molecule to recognize and capture GPC3. The rGO/PEI/AuNPs decorated on the LAPS surface could enlarge the surface area for GPC3Apt immobilization and provide more active sites for GPC3Apt to bind effectively with the target GPC3. When a bias voltage was provided to the LAPS system, the forming GPC3-GPC3Apt complexs on the sensing interface of LAPS would cause the potential of the sensitive membrane surface to change and generate a corresponding offset response in photocurrent-voltage (I-V) curves. The potential shift and GPC3 concentration showed a good linear range from 0.1 to 100.0 μg/mL with a detection limit of 40.0 ng/mL (S/N = 3) and a sensitivity of 1.33638 mV/μg/mL. In addition, the LAPS detection system also has good specificity and stability, which can be used for the detection of GPC3 in spiked serum samples.

Evaluation of Polytyramine Film and 6-Mercaptohexanol Self-Assembled Monolayers as the Immobilization Layers for a Capacitive DNA Sensor Chip: A Comparison.

The performance of a biosensor is associated with the properties of an immobilization layer on a sensor chip. In this study, gold sensor chips were modified with two different immobilization layers, polytyramine film and 6-mercaptohexanol self-assembled monolayer. The physical, electrochemical and analytical properties of polytyramine film and mercaptohexanol self-assembled monolayer modified gold sensor chips were studied and compared. The study was conducted using atomic force microscopy, cyclic voltammetry and a capacitive DNA-sensor system (CapSenze™ Biosystem). The results obtained by atomic force microscopy and cyclic voltammetry indicate that polytyramine film on the sensor chip surface possesses better insulating properties and provides more spaces for the immobilization of the capture probe than a mercaptohexanol self-assembled monolayer. A capacitive DNA sensor hosting a polytyramine single-stranded DNA-modified sensor chip displayed higher sensitivity and larger signal amplitude than that of a mercaptohexanol single-stranded DNA-modified sensor chip. The linearity responses for polytyramine single-stranded DNA- and mercaptohexanol single-stranded DNA-modified sensor chips were obtained at log concentration ranges, equivalent to 10−12 to 10−8 M and 10−10 to 10−8 M, with detection limits of 4.0 × 10−13 M and 7.0 × 10−11 M of target complementary single-stranded DNA, respectively. Mercaptohexanol single-stranded DNA- and polytyramine single-stranded DNA-modified sensor chips exhibited a notable selectivity at an elevated hybridization temperature of 50 °C, albeit the signal amplitudes due to the hybridization of the target complementary single-stranded DNA were reduced by almost 20% and less than 5%, respectively.

Antigen improves binding of IgGs to FcγRs in SPR analysis

FcγR binding characterization is one of the critical attributes during the development of therapeutic antibodies. Here, we report a novel assay format to characterize IgG-FcγR interaction in the presence of antigen using Surface plasmon resonance (SPR). The new assay format was developed by creating stable antigen/antibody immunocomplexes on a sensor chip surface before injection of FcγRs. In this assay format, binding activity of both huIgG1 (including IgG1 Fc fusion Protein) and huIgG2 increased significantly to most activating human FcγRs, especially to FcγRI, FcγRIIa-131H and FcγRIIIa-158F. To our knowledge, this study provides the first set of evidence using a biophysical method to demonstrate antigen binding facilitating IgG-FcγR interaction, especially for huIgG2 where previous studies did not indicate its binding to human FcγRI or FcγRIIIa-158F. Although further studies are needed to investigate the correlation of the binding data with effector function data in vivo, our results suggest that it may be useful to evaluate the IgG-FcγR interaction in the presence of antigen to help design safer and more effective biotherapeutics.

Polyoxazolines with a Vicinally Double-Bioactivated Terminus for Biomacromolecular Affinity Assessment.

Interactions between proteins and carbohydrates with larger biomacromolecules, e.g., lectins, are usually examined using self-assembled monolayers on target gold surfaces as a simplified model measuring setup. However, most of those measuring setups are either limited to a single substrate or do not allow for control over ligand distance and spacing. Here, we develop a synthetic strategy, consisting of a cascade of a thioesterification, native chemical ligation (NCL) and thiol-ene reaction, in order to create three-component polymer conjugates with a defined double bioactivation at the chain end. The target architecture is the vicinal attachment of two biomolecule residues to the α telechelic end point of a polymer and a thioether group at the ω chain end for fixating the conjugate to a gold sensor chip surface. As proof-of-principle studies for affinity measurements, we demonstrate the interaction between covalently bound mannose and ConA in surface acoustic wave (SAW) and surface plasmon resonance (SPR) experiments.

Rapid detection of enrofloxacin using a localized surface plasmon resonance sensor based on polydopamine molecular imprinted recognition polymer

Due to concerns of antibiotic-resistant bacterial strains from animal-derived food products, different countries or organizations have established their maximum residue limits for antibiotics. Current HPLC and ELISA methods are commonly used in testing antibiotic residues, but have their limitations for in-field rapid detections. Localized surface plasmon resonance (LSPR) sensors have shown great potential in biodetection, due to its sensitive, label-free and real-time features. Therefore, the objective of this project was to develop an LSPR biosensing method for rapid, sensitive and specific detection of enrofloxacin (ENRO), using polydopamine molecular imprinted polymer (PDA-MIP) as the recognition element. The PDA-MIP film was fabricated by polymerization of dopamine and ENRO in Tris buffer on the surface of LSPR sensor chip. After blocking with bovine serum albumin and removal with sodium dodecyl sulphate, the modified sensor chip was used to selectively capture ENRO in a sample. To amplify LSPR detection signals of ENRO, conjugates with protein molecules were synthesized, served as competitors, and reacted with the residual binding sites on the PDA-MIP film. The detection could be done within 20 min, with a detection range of 25–1000 ng/mL and a limit of detection of 61.1 ng/mL. The PDA-MIP film demonstrated a higher binding capacity to ENRO than the corresponding non-imprinted polymer film, and discriminated ENRO among structural analogues. Good reusability was achieved within seven binding-regeneration cycles, with a low relative standard deviation. With high sensitivity, specificity and stability, the developed LSPR/PDA-MIP sensor showed its potential for in-field rapid detection of ENRO residues.

The impact of caffeine on tau-tau interaction: LSPR detection, structural modification and molecular dynamics simulation

Protein-protein interactions are involved in many biological processes and cellular functions and they are interesting therapeutic targets is several diseases for drug design and development. In the present study, the possible effects of caffeine (CF) as mostly consumed psychoactive substances in the modern life on tau-tau interactions were studied by localized surface plasmon resonance (LSPR) method. The obtained data indicated that tau monomers have high affinity to bind to the immobilized tau proteins on the surface of LSPR sensor chip of the gold nanoparticles (LSPR-AuNPs) and the LSPR peak was red-shifted from 598 ± 1.5 nm to 629 ± 0.1 nm. However, it was shown that the binding affinity of CF-tau complex was remarkably reduced. These results revealed that the binding of CF molecules on free tau monomers could potentially interrupt the interaction of tau-tau proteins. Circular dichroism (CD) and FTIR spectroscopy was applied to analyze the secondary structure of tau protein in the absence or presence of different amounts of CF. The results showed that CF can induce more random coil structure to the protein and also increase its structural stability. Docking studies and molecular dynamics simulations elucidated that CF not only can possibly provide a molecular and physical barrier for tau-tau interaction by binding to the microtubule binding domain, but also it decreases the stability of tau dimer, which were supported by experimental data.

Structure–activity relationship of ipglycermide binding to phosphoglycerate mutases

Catalysis of human phosphoglycerate mutase is dependent on a 2,3-bisphosphoglycerate cofactor (dPGM), whereas the nonhomologous isozyme in many parasitic species is cofactor independent (iPGM). This mechanistic and phylogenetic diversity offers an opportunity for selective pharmacologic targeting of glycolysis in disease-causing organisms. We previously discovered ipglycermide, a potent inhibitor of iPGM, from a large combinatorial cyclic peptide library. To fully delineate the ipglycermide pharmacophore, herein we construct a detailed structure–activity relationship using 280 substituted ipglycermide analogs. Binding affinities of these analogs to immobilized Caenorhabditis elegans iPGM, measured as fold enrichment relative to the index residue by deep sequencing of an mRNA display library, illuminated the significance of each amino acid to the pharmacophore. Using cocrystal structures and binding kinetics, we show that the high affinity of ipglycermide for iPGM orthologs, from Brugia malayi, Onchocerca volvulus, Dirofilaria immitis, and Escherichia coli, is achieved by a codependence between (1) the off-rate mediated by the macrocycle Cys14 thiolate coordination to an active-site Zn2+ in the iPGM phosphatase domain and (2) shape complementarity surrounding the macrocyclic core at the phosphotransferase–phosphatase domain interface. Our results show that the high-affinity binding of ipglycermide to iPGMs freezes these structurally dynamic enzymes into an inactive, stable complex.

Field-Effect Sensors for Virus Detection: From Ebola to SARS-CoV-2 and Plant Viral Enhancers.

Coronavirus disease 2019 (COVID-19) is a novel human infectious disease provoked by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Currently, no specific vaccines or drugs against COVID-19 are available. Therefore, early diagnosis and treatment are essential in order to slow the virus spread and to contain the disease outbreak. Hence, new diagnostic tests and devices for virus detection in clinical samples that are faster, more accurate and reliable, easier and cost-efficient than existing ones are needed. Due to the small sizes, fast response time, label-free operation without the need for expensive and time-consuming labeling steps, the possibility of real-time and multiplexed measurements, robustness and portability (point-of-care and on-site testing), biosensors based on semiconductor field-effect devices (FEDs) are one of the most attractive platforms for an electrical detection of charged biomolecules and bioparticles by their intrinsic charge. In this review, recent advances and key developments in the field of label-free detection of viruses (including plant viruses) with various types of FEDs are presented. In recent years, however, certain plant viruses have also attracted additional interest for biosensor layouts: Their repetitive protein subunits arranged at nanometric spacing can be employed for coupling functional molecules. If used as adapters on sensor chip surfaces, they allow an efficient immobilization of analyte-specific recognition and detector elements such as antibodies and enzymes at highest surface densities. The display on plant viral bionanoparticles may also lead to long-time stabilization of sensor molecules upon repeated uses and has the potential to increase sensor performance substantially, compared to conventional layouts. This has been demonstrated in different proof-of-concept biosensor devices. Therefore, richly available plant viral particles, non-pathogenic for animals or humans, might gain novel importance if applied in receptor layers of FEDs. These perspectives are explained and discussed with regard to future detection strategies for COVID-19 and related viral diseases.

Reusable surface plasmon resonance biosensor chip for the detection of H1N1 influenza virus

We developed a reusable magnetic surface plasmon resonance (SPR) sensor chip for detecting various target molecules repeatedly in a conventional SPR system. Here, ferromagnetic patterns on a SPR sensor chip were utilized to trap a layer of magnetic particles, and they were utilized as a solid substrate for SPR sensing in a conventional SPR system. After a sensing experiment, the used magnetic particles were removed by external magnetic fields, and a new layer of magnetic particles was immobilized to the SPR sensor chip for additional sensing measurements. Since magnetic particles were trapped on the ferromagnetic patterns, we could use our reusable SPR chip for SPR sensing measurements in a traditional SPR system without any applied magnetic fields. Significantly, ferromagnetic patterns on the sensor chip surface deflected the strong external fields, so that the large aggregation of magnetic particles on the sensor surface was reduced. We demonstrated using a single reusable SPR sensor chip to measure the nucleoprotein (NP) of H1N1 influenza virus solution ranging repeatedly for more than 7 times without significant signal degradation. Also, different target molecules could be repeatedly measured in a single SPR chip. Since our reusable SPR sensor chip can be repeatedly used in a conventional SPR system without any chemical processes for refreshment, the cost for SPR sensing should be significantly reduced. In this case, our reusable SPR sensor chip can be a major breakthrough and can be used for versatile practical applications of SPR sensors.

Multiresonant plasmonic nanostructure for ultrasensitive fluorescence biosensing

Abstract A novel metallic nanostructure for efficient plasmon-enhanced fluorescence readout of biomolecular binding events on the surface of a solid sensor chip is reported. It is based on gold multiperiod plasmonic grating (MPG) that supports spectrally narrow plasmonic resonances centered at multiple distinct wavelengths. They originate from diffraction coupling to propagating surface plasmons (SPs) forming a delocalized plasmonic hotspot associated with enhanced electromagnetic field intensity and local density of optical states at its surface. The supported SP resonances are tailored to couple with the excitation and emission transitions of fluorophores that are conjugated with the biomolecules and serve as labels. By the simultaneous coupling at both excitation and emission wavelengths, detected fluorescence intensity is enhanced by the factor of 300 at the MPG surface, which when applied for the readout of fluorescence immunoassays translates to a limit of detection of 6 fM within detection time of 20 min. The proposed approach is attractive for parallel monitoring of kinetics of surface reactions in microarray format arranged on a macroscopic footprint. The readout by epi-fluorescence geometry (that inherently relies on low numerical aperture optics for the imaging of the arrays) can particularly take advantage of the reported MPG. In addition, the proposed MPG nanostructure can be prepared in scaled up means by UV-nanoimprint lithography for future practical applications.

Surface plasmon resonance-based aptasensor for direct monitoring of thrombin in a minimally processed human blood

Optical affinity biosensors are pursued for timely monitoring of thrombin in human blood, which is of urgent need in tailored anticoagulation therapies. However, the unspecific deposition of molecules, cells, and aggregates from the blood at their surface (also termed fouling) severely hinders their development and impedes the deploying of this technology to everyday clinical practice. We addressed this challenge by designing surface plasmon resonance (SPR) sensor chip with an antifouling polymer brush architecture and incorporated thrombin aptamer bioreceptors. Poly[(N-(2-hydroxypropyl)-methacrylamide)-co-(carboxybetaine methacrylamide)] brushes were synthesized on gold sensor chip surface via photoinduced single-electron transfer living radical polymerization and postmodified with three thrombin aptamers (HD1 short, HD1 and HD22). The affinity interaction of the aptamer bioreceptors with thrombin (as well as with other molecules present in the blood) was investigated and changes in their performance when incorporated into the polymer brushes were characterized. The combination of brushes and aptamer bioreceptors allowed for the analysis of medically relevant concentrations of thrombin in the 10 % blood by direct SPR detection format. This is the first time that the optical affinity biosensor is demonstrated for label-free analysis of biomarkers in a minimally processed human blood without a need for pre-separation steps. We believe that this system constitutes a basis for the future affinity biosensor applications that are suitable for the clinical settings and can be readily adapted to detect a range of important biological markers.

Detection of Pesticide via Nanoparticle Based Quartz Crystal Microbalance Sensor

In this study, Chlorpyrifos (Cps) imprinted nanoparticles based QCM sensor were prepared for detection of chlorpyrifos pesticide. Cps imprinted poly(ethylene glycol dimethacrylate- N-metacryloyl-(L)-tryptophan) (PEDMATrp) nanoparticles were prepared and then these nanoparticles were attached to the surface of QCM sensor chip. Also, non-imprinted PEDMATrp QCM sensor was prepared without using the Cps molecule to evaluate the imprinting efficiency. Cps imprinted and non-imprinted nanoparticles were characterized by zeta-sizer and Fourier transform infrared spectrophotometer-attenuated total reflection (FTIR-ATR) spectrophotometer. Cps imprinted and non-imprinted QCM sensors were also characterized by atomic force microscopy, ellipsometer, and contact angle measurements. The prepared sensors were applied for selective Cps detection in aqueous solution for the range of 0.015-2.9 nM. The selectivity studies of the prepared PEDMATrp quartz crystal microbalance sensor were examined by using competitive pesticide molecules such as Diazinon and Parathion (0.75 nM), which are similar to Cps in size and shape. The reusability studies of the prepared sensors were investigated by applying the same pesticide concentration (1.45 nM), four times consecutively.