Article

Biofunctional polyelectrolytes assembling on biosensors – A versatile surface coating method for protein detections

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Abstract

This paper reports a surface functionalization strategy for protein detections based on biotin-derivatized poly(L-lysine)-grafted oligo-ethylene glycol (PLL-g-OEGx-Biotin) copolymers. Such strategy can be used to attach the biomolecule receptors in a reproducible way simply by incubation of the transducer element in a solution containing such copolymers which largely facilitated the sensor functionalization at an industrial scale. As the synthesized copolymers are cationic in physiology pH, surface biotinylation can be easily achieved via electrostatic adsorption on negatively charged sensor surface. Biotinylated receptors can be subsequently attached through well-defined biotin-streptavidin interaction. In this work, the bioactive sensor surfaces were applied for mouse IgG and prostate specific antigen (PSA) detections using quartz crystal microbalance (QCM), optical sensor (BioLayer Interferometry) and conventional ELISA test (colorimetry). A limit of detection (LOD) of 0.5 nM was achieved for PSA detections both in HEPES buffer and serum dilutions in ELISA tests. The synthesized PLL-g-OEGx-Biotin copolymers with different OEG chain length were also compared for their biosensing performance. Moreover, the surface regeneration was achieved by pH stimulation to remove the copolymers and the bonded analytes, while maintaining the sensor reusability as well. Thus, the developed PLL-g-OEGx-Biotin surface assembling strategy is believed to be a versatile surface coating method for protein detections with multi-sensor compatibility.

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... Hereto, PLL was graed with OEG units and either methyl-tetrazine (Tz) or dibenzocyclooctyl (DBCO) functional groups and adsorbed onto surfaces for the subsequent immobilization of, respectively, TCO or azido-modied biomolecules by fast and catalyst-free bioorthogonal click reactions. 21 The different PLLs were then adsorbed onto activated substrates, thus displaying the respective functional groups at the interface. ...
... Phosphate-buffered saline tablets (PBS, pH 7.4), and poly-L-lysine-HBr (PLL-HBr)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30), DMSO $99.9% anhydrous, sodium dodecyl sulfate (SDS) $98.5%, and DBCO-OEG 4 -NHS were purchased from Sigma Aldrich and used without further purication. Methyl-OEG 4 -NHS was purchased from ThermoFischer Scientic, while methyltetrazine-OEG 4 -NHS was purchased from Click chemistry tools. ...
Article
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Biomolecules are immobilized onto surfaces employing the fast and stable adsorption of poly-L-lysine (PLL) polymers and the versatile copper-free click chemistry reactions. This method provides the combined advantages of versatile surface adsorption with density control using polyelectrolytes and of the covalent and orthogonal immobilization of biomolecules with higher reaction rates and improved yields of click chemistry. Using DNA attachment as a proof of concept, control over the DNA probe density and applicability in electrochemical detection are presented.
... Poly-L-lysine grafted with oligoethylene glycol (PLL-OEG, MW = 15−30 kDa) is synthesized in our lab according to procedures described in previous publications. 37,38 HEPES (N-2-hydroxyethylpiperazine-N-ethane-sulfonicacid, 10 mM, pH = 7) containing 0.2% v/v Tween (Sigma) as the surfactant was used as the solvent. Full serum (F2442, MFCD00132239) was purchased from Sigma. ...
... This is achieved through coating the chips with PLL-OEG. 37,38 Figure 2c presents the principles of the microbeads trap and release with MW-MFC, which forms the basis of the microbeads array. Since the size of the particle and microwell is rather large, a cellphone camera connected to a standard portable microscope is used to acquire the images of the arrayed microbeads by directly attaching the camera to the objective lens of the microscope (Figure 2d). Figure 2e presents a comparison of the MW-MFC images obtained via a professional microscope (top image) and the cellphone (bottom image). ...
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Quantitative biomarker detection methods featured with rapidity, high accuracy, and label-free are demonstrated for the development of point-of-care (POC) technologies or "beside" diagnostics. Microbead aggregation via protein-specific linkage provides an effective approach for selective capture of biomarkers from the samples, and can directly readout the presence and amount of the targets. However, sensors or microfluidic analyzers that can accurately quantify the microbead aggrega-tion are scared. In this work, we demonstrate a microwell-based microbeads analyzing system, by which online manipula-tions of microbeads including trapping, arraying, and rotations can be realized, providing a series of microfluidic approaches to layout the aggregated microbeads for further convenient characterizations. Prostate specific antigen is detected using the proposed system, demonstrating the limit of detection as low as 0.125 ng/mL (3.67 pM). A two-step reaction kinetics model is proposed for the first time to explain the dynamic process of microbeads aggregation. The developed microbeads aggrega-tion analysis system has the advantages of label-free detection, high throughput, and low cost, showing great potential for portable biomarker detection.
... As biotin-SAv complex has been widely used in biosensing 61,62 or molecule interaction studies 63 , we first applied biotin-coated nanoparticles to capture and enrich SAv molecules. The approach is schematically shown in Figure 5a. ...
Article
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We present a nanoscale acoustofluidic trap (AFT) which manipulates nanoparticles in a microfluidic system actuated by a gigahertz acoustic resonator. The AFT generates independent standing closed vortices with high-speed rotation. By carefully designing and optimizing the geometric confinements, the AFT is able to effectively capture and enrich sub-100 nm nanoparticles with low power consumption (0.25~5 μW/μm2) and rapid trapping (within 30 s), showing greatly enhanced particle operating ability towards its acoustic and optical counterparts. Using specifically functionalized nanoparticles (SFNPs) to selectively capture target molecules from the sample, the AFT produces a molecular concentration enhancement of ~200 times. We investigated the feasibility of the SFNPs-assisted AFT preconcentration method for biosensing applications, and successfully demonstrated its capability for serum prostate specific antigen (PSA) detection. The AFT is prepared with a fully CMOS-compatible process, and thus can be conveniently integrated on a single chip, with potential for “lab-on-a-chip” or point-of-care (POC) nanoparticle-based biosensing applications.
... It is important to detect disease biomarkers for early diagnosis, prognosis tracking and therapeutic evaluation. Numerous methods have been reported to detect target proteins, such as enzyme-linked immunosorbent assay (ELISA), electrochemical immunology sensors and mass spectra analysis [7][8][9][10][11][12]. However, these methods have some limitations; for example, most involve multistep and time-consuming procedures and often cannot be well applied to intracellular detection. ...
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The detection of proteins is of great biological significance as disease biomarkers in early diagnosis, prognosis tracking and therapeutic evaluation. Thus, we developed a simple, sensitive and universal protein-sensing platform based on peptide and graphene oxide (GO). The design consists of a fluorophore (TAMRA, TAM), a peptide containing eight arginines and peptide ligand that could recognize the target protein, and GO used as a quencher. To demonstrate the feasible use of the sensor for target detection, Bcl-xL was evaluated as the model target. The sensor was proved to be sensitive and applied for the detection of the target proteins in buffer, 2% serum and living cells.
... Polyelectrolytes are a wide class of compounds used as coating materials as well as immobilization matrices for some biomolecules in the development of biosensors [16,17]. The deposition of the polyelectrolyte layer on the electrode surface increases the active area of the electrode, and due to the electrostatic interaction between the analyte and the polyelectrolyte, leads to its accumulation on the electrode surface that can enhance the electrochemical detection of the analyte. ...
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Herein, we report on a new type of ethanol biosensor based on a screen-printed electrode modified with poly(allylamine hydrochloride). The alcohol dehydrogenase was immobilized on the surface of the sensor using the sol–gel matrix. Working parameters such as applied potential, pH, NAD+ concentration, storage conditions were optimized. A response range between 0.05 and 2 mM was found with a sensitivity of 13.45 ± 0.67 µA/mM·cm2 and a detection limit of 20 µM. The developed biosensor was used to detect ethanol in commercial beverages with good accuracy.
... 34 At physiological pH, the cationic PLL can be deposited on negatively charged surfaces 35,36 and allows designing monolayers with different functionalities by ensuring critical control over the biosensing interface features. 37,38 PLL can be modified with various functional groups by introducing neutral or charged side chains, 39−41 thus offering the possibility to fabricate multifunctional polymeric structures. The main drawback of multifunctional polymers integrating antifouling materials with recognition elements for target capturing is the limited number of target molecules that can access highly packed brush polymers, thereby hampering the biosensing response. ...
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Standard protocols for the analysis of circulating tumor DNA (ctDNA) include the isolation of DNA from the patient’s plasma and its amplification and analysis in buffered solutions. The application of such protocols is hampered by several factors, including the complexity and time-constrained preanalytical procedures, risks for sample contamination, extended analysis time, and assay costs. A recently introduced nanoparticle-enhanced surface plasmon resonance imaging-based assay has been shown to simplify procedures for the direct detection of tumor DNA in the patient’s plasma, greatly simplifying the cumbersome preanalytical phase. To further simplify the protocol, a new dual-functional low-fouling poly-l-lysine (PLL)-based surface layer has been introduced that is described herein. The new PLL-based layer includes a densely immobilized CEEEEE oligopeptide to create a charge-balanced system preventing the nonspecific adsorption of plasma components on the sensor surface. The layer also comprises sparsely attached peptide nucleic acid probes complementary to the sequence of circulating DNA, e.g., the analyte that has to be captured in the plasma from cancer patients. We thoroughly investigated the contribution of each component of the dual-functional polymer to the antifouling properties of the surface layer. The low-fouling property of the new surface layer allowed us to detect wild-type and KRAS p.G12D-mutated DNA in human plasma at the attomolar level (∼2.5 aM) and KRAS p.G13D-mutated tumor DNA in liquid biopsy from a cancer patient with almost no preanalytical treatment of the patient’s plasma, no need to isolate DNA from plasma, and without PCR amplification of the target sequence.
... In the past two decades, the polyelectrolytes have gained increasing importance in the development of electrochemical biosensors, due to the fact that they are flexible materials that can be tailored for different purposes [23]. The literature reveals that polyelectrolytes are mainly used as coating-materials or as immobilization matrices for biomolecules [24]. The polyelectrolyte layer ensures a high active area, and the electrostatic interaction between NADH and polyelectrolyte could facilitate its accumulation at the electrode surface, resulting in the enhancement of the analytical signal [25]. ...
Article
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This work describes a novel L-lactate biosensor based on the immobilization of L-lactate dehydrogenase enzyme on the screen-printed electrode modified with a ternary composite based on gold nanoparticles, electrochemically-reduced graphene oxide, and poly (allylamine hydrochloride). The enzyme was stabilized by crosslinking with glutaraldehyde. Applied working potential, pH and NAD+ concentration were optimized. The biosensor reports a specific sensitivity of 1.08 µA/mM·cm2 in a range up to 3 mM L-lactic acid with a detection limit of 1 µM. The operational and long-term stability as well as good selectivity allowed the L-lactic acid measurement in dairy products and wine samples.
... In the experiment, first the sensor with a bare gold surface is immersed in a 4-(2-hydroxyethyl)-1-piperazineetha nesulfonic acid (HEPES) buffer solution to obtain a baseline. Then the sensor is immersed in a (poly-L-lysine)-(polyethylene glycol)biotin (PPB) solution to form an antifouling layer by electrostatic forces, in order to reduce the nonspecific binding [77,78]. Next, a layer of streptavidin (SA) is added on top of the PPB through biotin-SA binding. ...
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Integrating surface plasmon resonance (SPR) devices upon single-mode fiber (SMF) end facets renders label-free sensing systems that have a simple dip-and-read configuration, a small form factor, high compatibility with fiber-optic techniques, and invasive testing capability. Such devices are not only low cost replacement of current equipments in centralized laboratories, but also highly desirable for opening paths to new applications of label-free optical sensing technologies, such as point-of-care immunological tests and intravascular ultrasound imaging. In this paper, we explain the requirements and challenges for such devices from the perspectives of biomolecule and ultrasound detection applications. In such a context, we review our recent work on SMF end-facet SPR cavities. This include a glue-and-strip fabrication method to transfer a nano-patterned thin gold film to the SMF end-facet with high yield, high quality and high alignment precision, the designs of distributed Bragg reflector (DBR) and distributed feedback (DFB) SPR cavities that couple efficiently with the SMF guided mode and reach quality factors of over 100, and the preliminary results for biomolecule interaction sensing and ultrasound detection. The particular advantages and potential values of these devices have been discussed, in terms of sensitivity, data reliability, reproducibility, bandwidth, etc.
... Investigating the biosensing capability with QCM, interferometry, and ELISA techniques, nM LODs were reported for PSA in serum and IgG in buffer for PLL-OEG-biotin with the longer OEG spacers. 294 In a follow-up study, the detection of IgG in serum and PSA in buffer was enhanced by using an acoustic nano-electrochemical system (NEMS) resonator as a biomolecular concentrator at the biorecognition spot (Figure 13b). 295 A 10-and 200-fold decrease of the LOD was found for IgG and PSA, respectively, demonstrating the NEMS resonator applicability with different target molecules and surfacebased biosensing techniques. ...
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The possibility of tuning the chemical moieties and their density plays a fundamental role in targeting surface-confined molecular structures and their functionalities at macro and nanoscale levels. Such interfacial control is crucial for engineered coating formation and biorecognition purposes, where the type and density of ligands/receptors at the surface affect the overall binding affinities and the device performance. Together with the well-established self-assembled monolayers, a surface modification approach based on polyelectrolytes (PEs) has gained importance to provide desired characteristics at the substrate interface. This review presents the innovations of functional PEs, modified in a preceding synthetic step, and their wide applicability in functional (a)biotic substrates. Examples of 2D and 3D architectures made by modified PEs are reviewed in relation with the reactive groups grafted to the PE backbones. The main focus lies on the strategy to use modified PEs to form bioengineered coatings for orthogonally anchoring biological entities, manufacturing biocidal/antifouling films, and their combinations in functional biosensing applications.
... Overall, the designed template contains parallel nanogrooves with a cross section of 100 nm (width) Â 60 nm (depth) and the spacing between the neighboring nanogrooves is 3 mm and 5 mm. To enable bio-functionalization, here, a mixed conductive polymer aqueous ink is designed by mixing a positively charged polymer (PLL-g-OEG4-biotin) with the negatively charged PEDOT : PSS at a typical mixing ratio of 1 : 9. 41,52 The molecular structure of PEDOT:PSS doped with PLL-g-OEG4-biotin is given in Fig. S2a (ESI †). Such molecular design ensures the good conductivity of the nanowires and direct doping with biotin. ...
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Microwave sensors based on microstrip antennas are promising as wearable devices because of their flexibility and wireless communication compatibility. However, their sensitivity is limited due to the reduced sensor size and the potential of biochemical monitoring need to be explored. In this work, we present a new concept to enhance the microwave signals using of nanostrip-based metamaterials. The introduction of the nanostrip structures were achieved by theory and simulations. Experiments proof their enhancement to the electric field and sensing response in the characteristic gigahertz (GHz) wave band. Ordered nanostrips were fabricated on plastic substrate through simple nanoscale printing approach. Glucose oxidase is directly doped into the nanostrips, which enables a flexible wearable enzymatic biosensor for glucose sensing. Sensing experiments demonstrated that the nanostrip biosensor gives excellent performance for glucose detection, including high sensitivity, fast response, low detection limit, high affinity, and low power consumption. The applicability of the nanostrip-based sensor as wearable epidermal device for real-time noninvasive monitoring of glucose in sweat is verified as well.
... The total grafting density of functionalized lysine side chains was kept below 35−40% to ensure strong adsorption to the surface. 57,58 The azido-PNA probe was synthesized as previously reported. 59 As a proof of concept, the surface functionalization processes of modified-PLL deposition, azido-PNA immobilization, and consecutive cDNA and rDNA-Fc hybridization steps were followed on a flat substrate by quartz crystal microbalance with dissipation (QCM-D) monitoring ( Figure 3). ...
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The available active surface area and the density of probes immobilized on this surface are responsible for achieving high specificity and sensitivity in electrochemical biosensors that detect biologically relevant molecules, including DNA. Here, we report the design of gold-coated, silicon micropillar-structured electrodes functionalized with modified poly-L-lysine (PLL) as adhesion layer to concomitantly assess gain in sensitivity by increase of the electrochemical area and control over the probe density. By systematically reducing the center-to-center distance between the pillars (pitch), denser micropillar arrays were formed at the electrode, resulting in a larger sensing area. Azido-modified peptide nucleic acid (PNA) probes were click-reacted onto the electrode interface exploiting PLL with appended oligo(ethylene glycol) (OEG) and dibenzocyclooctyne (DBCO) moieties (PLL-OEG-DBCO), for antifouling and probe binding properties, respectively. The selective electrochemical sandwich assay formation, composed of consecutive hybridization steps of the target complementary DNA (cDNA) and reporter DNA modified with the electroactive ferrocene functionality (rDNA-Fc), was monitored by quartz crystal microbalance. The DNA detection performance of micropillared electrodes with different pitch was evaluated by quantifying the cyclic voltammetric response of the surface-confined rDNA-Fc. By decrease of the pitch of the pillar array, the area of the electrode was enhanced by up to a factor 10.6. Comparison of the electrochemical data with the geometrical area of the pillared electrodes confirmed the validity of the increased sensitivity of the DNA detection by the design of the micropillar array.
... These systems can be placed miniaturized on a microarray chip. In order to analyze the interactions between biomolecules and proteins, several types of protein microarrays with various molecules should immobilize on the substrate (as a capture molecule) [97][98][99]. ...
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Background Multiple Sclerosis (MS) involves an immune-mediated response in which body’s immune system destructs the protective sheath (myelin). Part of the known MS biomarkers are discovered in cerebrospinal fluid like oligoclonal lgG (OCGB), and also in blood like myelin Oligodendrocyte Glycoprotein (MOG). The conventional MS diagnostic methods often fail to detect the disease in early stages such as Clinically Isolated Syndrome (CIS), which considered as a concerning issue since CIS highlighted as a prognostic factor of MS development in most cases. Methods MS diagnostic techniques include Magnetic Resonance Imaging (MRI) of the brain and spinal cord, lumbar puncture (or spinal tap) that evaluate cerebrospinal fluid, evoked potential testing revealing abnormalities in the brain and spinal cord. These conventional diagnostic methods have some negative points such as extensive processing time as well as restriction in the quantity of samples that can be analyzed concurrently. Scientists have focused on developing the detection methods especially early detection which belongs to ultra-sensitive, non-invasive and needed for the Point of Care (POC) diagnosis because the situation was complicated by false positive or negative results. Results As a result, biosensors are utilized and investigated since they could be ultra-sensitive to specific compounds, cost effective devices, body-friendly and easy to implement. In addition, it has been proved that the biosensors on physiological fluids (blood, serum, urine, saliva, milk etc.) have quick response in a non-invasive rout. In general form, a biosensor system for diagnosis and early detection process usually involves; biomarker (target molecule), bio receptor (recognition element) and compatible bio transducer. Conclusion Studies underlined that early treatment of patients with high possibility of MS can be advantageous by postponing further abnormalities on MRI and subsequent attacks. This Review highlights variable disease diagnosis approaches such as Surface Plasmon Resonance (SPR), electrochemical biosensors, Microarrays and microbeads based Microarrays, which are considered as promising methods for detection and early detection of MS.
... Article below 35−40% to have a sufficiently strong surface adhesion as well as enough OEG moieties to avoid nonspecific interactions. 37,46 The results presented in Table S1 indicate a small discrepancy between the maximum theoretical amounts, based on full conversion of the supplied NHS esters of OEG and Mal, and the measured grafting densities observed for both the OEG and Mal groups. Typically about 80−90% of the Mal-NHS derivative was coupled successfully to the PLL. ...
Article
Full-text available
Biosensors and materials for biomedical applications generally require chemical functionalization to bestow their surfaces with desired properties, such as specific molecular recognition and anti-fouling properties. The use of modified poly-L-lysine (PLL) polymers with appended oligo(ethylene glycol) (OEG) and thiol-reactive maleimide (Mal) moieties (PLL-OEG-Mal) offers control over the presentation of functional groups. These reactive groups can readily be conjugated to, for example, probes for DNA detection. Here we demonstrate the reliable conjugation of thiol-functionalized peptide nucleic acid (PNA) probes onto pre-deposited layers of PLL-OEG-Mal and the control over their surface density “a priori” in the preceding synthetic step of the PLL modification with Mal groups. By monitoring the quartz crystal microbalance (QCM) frequency shifts of the binding of complementary DNA versus the density of Mal moieties grafted to the PLL, a linear relationship between probe density and PLL grafting density was found. Cyclic voltammetry experiments using Methylene Blue-functionalized DNA were performed to establish the absolute probe density values at the biosensor surfaces. These data provided a density of 1.2 × 1012 probes per cm2 per % of grafted Mal, thus confirming the validity of the “a priori” density control in the synthetic PLL modification step without the need of further surface characterization.
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We have developed a new three-dimensional (3D) surface for use in biosensors that is based on modified novel thorns-like polyelectrolytes (3D-PETx), which comprises of poly-l-lysine (PLL) appended with multitude oligo (ethylene glycol) (OEG) and biotin moieties. It tethered to the sensor surface by PLL, while the OEG-biotin chains are forced to stretch away from the surface for target detections. Due to its 3D structure, the number of the OEG-biotin per surface unit is markedly increased compared to conventional 2D polyelectrolytes (2D-PET) coating. Their antifouling property and sensing performance for human IgG and PSA were compared with the 2D-PET by BioLayer Interferometry (Blitz), Surface Plasmon Resonance (SPR), microfluidic devices and Enzyme-Linked ImmunoSorbent Assay (ELISA). Experimental results show that 3D-PETx presents higher sensitivity for biomarker detection both in buffer and in serum and provides an almost non-fouling surface even in undiluted serum. In addition, a sensitive PSA detection was achieved in undiluted serum with a LOD down to 0.6 ng/mL. The successful immunosensing in undiluted serum demonstrate the potential of the 3D-PETx coating for real clinical applications.
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Sensitive and precise detection of prostate-specific antigen (PSA) is critical for prostate cancer screening and monitoring. Herein, a target-triggered and self-calibration aptasensor based on a core-satellite nanostructure using surface-enhanced Raman spectroscopy (SERS) technology was developed for the sensitive and reliable determination of PSA protein, with a limit of detection of 0.38 ag mL-1 and a dynamic detection range of 10-2 to 10-15 mg mL-1. Furthermore, the proposed approach for the detection of PSA in patient blood samples was performed, and results showed that it is capable of providing comparable detection accuracy associated with a larger dynamic detection range and a lower detection limit as well as less sample requirement (only 5 μL) in comparison with the clinical commonly used method. Therefore, this SERS-based aptasensor for the detection of PSA in human blood samples has promising potential to be an alternative tool for clinical application in the accurate screening of prostate cancer.
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Simultaneous, sensitive and quantitative detection of biomarkers in infectious disease is crucial for guiding antimicrobial treatment and predicting prognosis. This work reported an ultrasensitive and quantitative microfluidic immunoassay combined with the streptavidin-biotin-peroxidase (SA-B-HRP) nanocomplex-signal amplification system (MIS) to detect two inflammatory biomarkers, procalcitonin (PCT, for discriminating bacterial infections from nonbacterial infections) and interleukin-6 (IL-6, for monitoring the kinetics of infectious disease) simultaneously. The amplification system was based on the one step self-assembly of SA and B-HRP to form the SA-B-HRP nanocomplex, which effectively amplified the chemiluminescent signals. The linear ranges for PCT and IL-6 detections by MIS were 250-1.28×10⁵ pg mL⁻¹ and 5-1280 pg mL⁻¹, and the limit of detection (LOD) were 48.9 pg mL⁻¹ and 1.0 pg mL⁻¹, respectively, both of which were significantly improved compared with microfluidic immunoassays without amplification system (MI). More importantly, PCT and IL-6 in human serum could be simultaneously detected in the same run by MIS, which could greatly improve the detection efficiency and reduce the cost. Given the advantages of high sensitivity, multiplex and quantitative detection, MIS could be potentially applied for detection of biomarkers at low concentration in clinical diagnosis.
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A novel biosensor interface exploiting the spontaneous surface assembly of a polycationic, polyethylene glycol (PEG)-grafted, biotinylated copolymer is investigated. By means of optical waveguide lightmode spectroscopy (OWLS), streptavidin and avidin are shown to bind specifically to the biotin-functionalized PEG, while the resistance of the remaining PEG chains to protein absorption yields a high specific binding to nonspecific binding ratio. The various components of this model immunoassay are shown to retain their biological activity and the effects of protein charge and the ionic strength of the buffer is explored.
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Protein biochips are at the heart of many medical and bioanalytical applications. Increasing interest has been focused on surface activation and subsequent functionalization strategies for immobilizing these biomolecules. Different approaches using covalent and noncovalent chemistry are reviewed; particular emphasis is placed on the chemical specificity of protein attachment and on retention of protein function. Strategies for creating protein patterns (as opposed to protein arrays) are also outlined. An outlook on promising and challenging future directions for protein biochip research and applications is also offered.
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A high-capacity system was developed to monitor the expression of many genes in parallel. Microarrays prepared by high-speed robotic printing of complementary DNAs on glass were used for quantitative expression measurements of the corresponding genes. Because of the small format and high density of the arrays, hybridization volumes of 2 microliters could be used that enabled detection of rare transcripts in probe mixtures derived from 2 micrograms of total cellular messenger RNA. Differential expression measurements of 45 Arabidopsis genes were made by means of simultaneous, two-color fluorescence hybridization.
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We report on the design and characterization of a class of biomolecular interfaces based on derivatized poly(l-lysine)-grafted poly(ethylene glycol) copolymers adsorbed on negatively charged surfaces. As a model system, we synthesized biotin-derivatized poly(l-lysine)-grafted poly(ethylene glycol) copolymers, PLL-g-[(PEGm)((1-x)) (PEG-biotin)(x)], where x varies from 0 to 1. Monolayers were produced on titanium dioxide substrates and characterized by x-ray photoelectron spectroscopy. The specific biorecognition properties of these biotinylated surfaces were investigated with the use of radiolabeled streptavidin alone and within complex protein mixtures. The PLL-g-PEG-biotin monolayers specifically capture streptavidin, even from a complex protein mixture, while still preventing nonspecific adsorption of other proteins. This streptavidin layer can subsequently capture biotinylated proteins. Finally, with the use of microfluidic networks and protein arraying, we demonstrate the potential of this class of biomolecular interfaces for applications based on protein patterning.
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A new surface functionalization scheme for nano-Bio field effect transistors (FETs) using biocompatible polyelectrolyte thin films (PET) is developed. PET assemblies on Si nanowires (Si-NWs) are driven by electrostatic interactions between the positively charged polymer backbone and negatively charged Si/SiO2 surface. Such assemblies can be directly coated from PET aqueous solutions and result in a uniform nanoscale thin film, which is more stable compared to the conventional amine silanization. Short oligo-ethylene glycol chains are grafted on the PETs to prevent nonspecific protein binding. Moreover, the reactive groups of the polymer chains can be further functionalized to other chemical groups in specific stoichiometry for biomolecules detection. Therefore, it opens a new strategy to precisely control the functional group densities on various biosensor surfaces at the molecular level. In addition, such assemblies of the polymers together with the bound analytes can be removed with the pH stimulation resulting in regeneration of a bare sensor surface without compromising the integrity and performance of the Si-NWs. Thus, it is believed that the developed PET coating and sensing systems on Si-NW FETs represent a versatile, promising approach for regenerative biosensors which can be applied to other biosensors and will benefit real device applications, enhancing sensor lifetime, reliability, and repeatability.
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In this paper, we fabricated a magnetically driven walking device comprising magnetically active layer-by-layer films. By alternating the magnetic fields, the device walked steadily and fast on the substrate like an inchworm, and showed good transportation capability.
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The modification of surfaces by the deposition of a robust overlayer provides an excellent handle with which to tune the properties of a bulk substrate to those of interest. Such control over the surface properties becomes increasingly important with the continuing efforts at down-sizing the active components in optoelectronic devices, and the corresponding increase in the surface area/volume ratio. Relevant properties to tune include the degree to which a surface is wetted by water or oil. Analogously, for biosensing applications there is an increasing interest in so-called "romantic surfaces": surfaces that repel all biological entities, apart from one, to which it binds strongly. Such systems require both long lasting and highly specific tuning of the surface properties. This Review presents one approach to obtain robust surface modifications of the surface of oxides, namely the covalent attachment of monolayers.
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The adsorption of immunoglobulin G (IgG) on chemically modified mica surfaces via physical and covalent immobilisation and the effect of detergent washing have been studied using atomic force microscopy (AFM). The chemical modification was performed by introducing amino groups to the mica surface via chemical vapor deposition of 3-aminopropyltriethoxy silane. The covalent and physical immobilization of IgG on the silanized mica surfaces were carried out with and without the use of a coupling reagent, glutaraldehyde, in three different buffer solutions of pH 7.4, 6.5, and 5.0 and at concentrations of 25 and 100 μg·ml−1. The samples were subsequently subjected to sodium dodecyl sulfate detergent washing. The IgG molecules either covalently attached to the glutaraldehyde/silanized mica surfaces or physically adsorbed onto the silanized mica surfaces before and after the detergent washing were characterized qualitatively and quantitatively using AFM in the corresponding buffer solutions, in terms of surface topography, surface roughness, layer thickness, and adhesive forces. The experimental results show that detergent washing removes loosely adsorbed IgG molecules from the surfaces, reduces the surface roughness and the adhesive force, and, most importantly, improves the resolution of AFM images obtained. Remarkable differences in the AFM topography and data were observed, clearly indicating that (1) nonspecifically adsorbed IgG molecules on the silanized mica form a partial mono- or multilayer of flat orientation and with characteristic strand-like structures and (2) specifically bound IgG molecules build a compact monolayer of random orientations and with representative granular structures. This study demonstrates that AFM has the capability of characterizing protein adsorption qualitatively and quantitatively.
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A supramolecular interface for Si nanowire FETs has been developed with the aim of creating regenerative electronic biosensors. The key to the approach is Si-NWs functionalized with β-cyclodextrin (β-CD), to which receptor moieties can be attached with an orthogonal supramolecular linker. Here we demonstrate full recycling using the strongest biomolecular system known, streptavidin (SAv)-biotin. The bound SAv and the linkers can be selectively removed from the surface through competitive desorption with concentrated β-CD, regenerating the sensor for repeated use. An added advantage of β-CD is the possibility of stereoselective sensors, and we demonstrate here the ability to quantify the enantiomeric composition of chiral targets.
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Layers of the polyelectrolytes poly(allylamine hydrochloride) (PAH, polycationic) and poly(styrene sulfonate) (PSS, polyanionic) are consecutively adsorbed on flat silicon oxide surfaces, forming stable, ultrathin multilayer films. Subsequently, a final monolayer of the polycationic copolymer poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) is adsorbed onto the PSS-terminated multilayer in order to impart protein resistance to the surface. The growth of each of the polyelectrolyte layers and the protein resistance of the resulting [PAH/PPS](n)(PLL-g-PEG) multilayer (n = 1-4) are followed quantitatively ex situ using X-ray photoelectron spectroscopy and in situ using real-time optical-waveguide lightmode spectroscopy. In a second approach, the same type of [PAH/PSS](n)(PLL-g-PEG) multilayer coatings are successfully formed on the surface of colloidal particles in order to produce surface-functionalized, hollow microcapsules after dissolution of the core materials (melamine formaldehyde (MF) and poly(lactic acid) (PLA; colloid diameters: 1.2-20 mu m). Microelectrophoresis and confocal laser scanning microscopy are used to study multilayer formation on the colloids and protein resistance of the final capsule. The quality of the PLL-g-PEG layer on the microcapsules depends on both the type of core material and the dissolution protocols used. The greatest protein resistance is achieved using PLA cores and coating the polyelectrolyte microcapsules with PLL-g-PEG after dissolution of the cores. Protein adsorption from full serum on [PAH/PPS](n)(PLL-g-PEG) multilayers (on both flat substrates and microcapsules) decreases by three orders of magnitude in comparison to the standard [PAH/PPS](n) layer. Finally, biofunctional capsules of the type [PAH/PPS](n)(PLL-g-PEG/PEG-biotin) (top copolymer layer with a fraction of the PEG chains end-functionalized with biotin) are produced which allow for specific recognition and immobilization of controlled amounts of streptavidin at the surface of the capsules. Biofunctional multilayer films and capsules are believed to have a potential for future applications as novel platforms for biotechnological applications such as biosensors and carriers for targeted drug delivery.
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Celiac disease is a condition associated with the ingestion of gluten by genetically susceptible individuals. Measurement of serum antigliadin antibodies is a diagnostic tool also used as a means of monitoring a patient's compliance to a gluten-free diet. In this work, we demonstrate the applicability of an electrochemical supramolecular platform based on cyclodextrin-modified gold surfaces to detect antigliadin antibodies in real serum samples. Several support layer-biorecognition element combinations were tested in order to maximize the electrochemical response, and the assay was optimized in terms of incubation times and resistance to nonspecific interactions. The developed supramolecular biosensor was then applied to the amperometric detection of antigliadin IgA and IgG autoantibodies in real samples of celiac disease patients under follow-up treatment; the results were compared with a commercial enzyme linked immunosorbent assay (ELISA) test, and an excellent correlation was observed between both methods.
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The efficiency of a pre-absorbed bovine serum albumin (BSA) layer in blocking the non-specific adsorption of different proteins on hydrophobic and hydrophilic surfaces was evaluated qualitatively and quantitatively using infrared reflection spectroscopy supported by spectral simulations. A BSA layer with a surface coverage of 35% of a close-packed monolayer exhibited a blocking efficiency of 90-100% on a hydrophobic and 68-100% on a hydrophilic surface, with respect to the non-specific adsorption of concanavalin A (Con A), immunoglobulin G (IgG), and staphylococcal protein A (SpA). This BSA layer was produced using a solution concentration of 1 mg/mL and 30 min incubation time. BSA layers that were adsorbed at conditions commonly employed for blocking (a 12 h incubation time and a solution concentration of 10 mg/mL) exhibited a blocking activity that involved competitive adsorption-desorption. This activity resulted from the formation of BSA-phosphate surface complexes, which correlated with the conformation of adsorbed BSA molecules that was favourable for blocking. The importance of optimisation of the adsorbed BSA layer for different surfaces and proteins to achieve efficient blocking was addressed in this study.
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Reversible and oriented immobilization of proteins in a functionally active form on solid surfaces is a prerequisite for the investigation of molecular interactions by surface-sensitive techniques. We demonstrate a method generally applicable for the attachment of proteins to oxide surfaces. A nitrilotriacetic acid group serving as a chelator for transition metal ions was covalently bound to the surface via silane chemistry. Reversible binding of the green fluorescent protein, modified with a hexahistidine extension, was monitored in situ using total internal reflection fluorescence. The association constant and kinetic parameters of the binding process were determined. The reversible, directed immobilization of proteins on surfaces as described here opens new ways for structural investigation of proteins and receptor-ligand interactions.
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The structure of an intact monoclonal antibody for phenobarbital, subclass IgG1, has been determined to 3.2 A resolution by X-ray crystallography. The molecule was visualized in a monoclinic unit cell having an entire immunoglobulin as the asymmetric unit. The two Fab segments, both with elbow angles of 155 degrees , were related by a rotation of 179.7 degrees plus a translation along the approximate dyad of 9 A. This is the first observation of such an Fab translation in a structurally defined antibody. The approximate 2-fold of the Fc was independent of that relating Fabs, making an angle of 107 degrees with the Fab dyad. The angle between long axes of the Fabs was 115 degrees, the most acute angle yet observed, yielding a distorted Y shaped molecule. This is in contrast to the distorted T shape of the only other intact IgG (2a) whose complete structure is known. Primary lattice interactions arise through formation of VH antiparallel beta ribbons whose strands are contributed by pseudo dyad related H2, and by L3 hypervariable loops from neighboring molecules. While one CH2 domain was mobile, Fabs and three domains of the Fc were well defined, as were hinge polypeptides connecting Fabs to the Fc, and the covalently attached oligosaccharides. Direct interactions are observed between hinge polypeptides, the glycosylated loop of one CH2 domain, and the oligosaccharide. Lattice interactions clearly influence, perhaps even determine the overall conformation of the antibody observed in this crystal. Comparison of this IgG1 with previously determined intact antibody structures extends the conformational range arising from segmental flexibility.
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Biosensors for organophosphates in solution may be constructed by monitoring the activity of acetylcholinesterase (AChE) or organophosphate hydrolase (OPH) immobilized to a variety of microsensor platforms. The area available for enzyme immobilization is small (< 1 mm2) for microsensors. In order to construct microsensors with increased surface area for enzyme immobilization, we used a sol-gel process to create highly porous and stable silica matrices. Surface porosity of sol-gel coated surfaces was characterized using scanning electron microscopy; pore structure was found to be very similar to that of commercially available porous silica supports. Based upon this analysis, porous and non-porous silica beads were used as model substrates of sol-gel coated and uncoated sensor surfaces. Two different covalent chemistries were used to immobilize AChE and OPH to these porous and non-porous silica beads. The first chemistry used amine-silanization of silica followed by enzyme attachment using the homobifunctional linker glutaraldehyde. The second chemistry used sulfhydryl-silanization followed by enzyme attachment using the heterobifunctional linker N-gamma-maleimidobutyryloxy succinimide ester (GMBS). Surfaces were characterized in terms of total enzyme immobilized, total and specific enzyme activity, and long term stability of enzyme activity. Amine derivitization followed by glutaraldehyde linking yielded supports with greater amounts of immobilized enzyme and activity. Use of porous supports not only yielded greater amounts of immobilized enzyme and activity, but also significantly improved long term stability of enzyme activity. Enzyme was also immobilized to sol-gel coated glass slides. The mass of immobilized enzyme increased linearly with thickness of coating. However, immobilized enzyme activity saturated at a porous silica thickness of approximately 800 nm.
Article
Antibody microarrays have the potential to revolutionize protein expression profiling. The intensity of specific signal produced on a feature of such an array is related to the amount of analyte that is captured from the biological mixture by the immobilized antibody (the "capture agent"). This in turn is a function of the surface density and fractional activity of the capture agents. Here we investigate how these two factors are affected by the orientation of the capture agents on the surface. We compare randomly versus specifically oriented capture agents based on both full-sized antibodies and Fab' fragments. Each comparison was performed using three different antibodies and two types of streptavidin-coated monolayer surfaces. The specific orientation of capture agents consistently increases the analyte-binding capacity of the surfaces, with up to 10-fold improvements over surfaces with randomly oriented capture agents. Surface plasmon resonance revealed a dense monolayer of Fab' fragments that are on average 90% active when specifically oriented. Randomly attached Fab's could not be packed at such a high density and generally also had a lower specific activity. These results emphasize the importance of attaching proteins to surfaces such that their binding sites are oriented toward the solution phase.
Article
Multilayer films have been prepared by the sequential electrostatic adsorption of poly(L-lysine) and hyaluronic acid onto charged silicon surfaces from dilute aqueous solutions under various pH conditions. Microelectrophoresis was used to examine the local acid-base equilibria of the polyelectrolytes within the films as a function of the total number of layers in the film and the assembly solution pH. The acid-base dissociation constants were observed to deviate significantly from dilute solution values upon adsorption, to be layer dependent only within the first 3-4 layers, and to show sensitivity to the assembly solution pH. As a result, some of the physicochemical properties of the films have also been found to exhibit pH-responsive behavior. For example, the thickest films result when at least one of the polyelectrolytes is only partially dissociated in solution. As well, the pH-dependent degree of dissociation of the surface functional groups can be used to vary the contact angle of a water droplet by as much as 25 degrees and the coefficient of friction by up to an order of magnitude. In addition, the extent to which PLL/HA films can be made to swell in solution can be varied by a factor of 7 depending on the assembly solution and swelling solution pH. The anomalies found in the dissociation constants account for the trends in these pH-dependent properties. Here, we demonstrate that knowledge of the acid-base dissociation behavior in multilayer films is key to understanding and controlling the physical properties of the films, particularly surface friction and wettability, which are fundamentally important factors for many biomaterials applications. For example, we outline a mechanism whereby biopolymer thin films can be electrostatically adsorbed under highly charged "sticky" conditions and then quickly transformed into stable low-friction films simply by altering the pH environment.
Article
An overview is given of the preparation, formation, structure, and applications of self-assembled monolayers (SAMs) formed from alkanethiols (and derivatives of alkanethiols) on gold, silver, copper, palladium, platinum, mercury, and alloys of these metals. Emphasis is on advances made in this area over the past five years (1999-2004). First, the structure and mechanism of formation of SAMs formed by adsorption of n-alkanethiols on metals are described. Following this, the applications of SAMs where they act as nanostructures themselves, enable other nanosystems, interact with biological nanostructures, and form patterns on surfaces with critical dimensions below 100 nm are outlined. Furthermore, an attempt is made to outline what is not understood about these SAMs and which of their properties are not yet controlled. Finally, some of the important opportunities that still remain for future progress in research involving SAMs are sketched.
Article
Thin films of 3-aminopropyltriethoxysilane (APTES) are commonly used to promote adhesion between silica substrates and organic or metallic materials with applications ranging from advanced composites to biomolecular lab-on-a-chip. Unfortunately, there is confusion as to which reaction conditions will result in consistently aminated surfaces. A wide range of conflicting experimental methods are used with researchers often assuming the creation of smooth self-assembled monolayers. A range of film morphologies based on the film deposition conditions are presented here to establish an optimized method of APTES film formation. The effect of reaction temperature, solution concentration, and reaction time on the structure and morphology was studied for the system of APTES on silica. Three basic morphologies were observed: smooth thin film, smooth thick film, and roughened thick film.
Article
Orientation of reagents is a key step in the construction of immunosensors. When the immunoreagent is a recombinant protein, this can be achieved by employing hexahistidine tags. The orientation of recombinant histidine-tagged Fab fragments of monoclonal anti-pneumolysin antibodies on gold films is evaluated. Using histidine as a chelator of Ni or employing an anti-polyhistidine antibody for capturing the His6 residue is considered. Measurements are based in the signal of indigo, which comes from the hydrolysis of 3-indoxylphosphate by alkaline phosphatase (AP). The attachment of the enzyme occurs through the interaction of biotin with AP-labelled streptavidin or employing AP-conjugated immunoreagents. In the case of the interaction Ni-histidine, for the study of the self-assembling process a His-tagged and biotinylated protein (His6-GST-B) was employed. General conditions were studied and non-specific adsorption was avoided with the use of 1-hexanethiol. Improvements of the signal compared with the direct adsorption were only achieved by the use of histidine capturing antibodies. With an optimised ratio anti-polyhis:His6-Fab the signal increases approximately a 100%. Precision is adequate and the response is linear with the concentration of pneumolysin between 0.1 and 10 ng/mL.
Article
Antibody immobilization on a solid support is an essential process for the development of most immune-based assay systems. The choice of the immobilization method greatly affects antibody-antigen interactions on the assay surface. For the past several years, numerous strategies have been reported to control antibody immobilization, mainly by directing the orientation, stability, and density of bound antibodies on different assay platforms. Here we discuss recent developments in antibody immobilization methods with a particular focus on the strengths and limitations of reported approaches, and thereby provide a useful guideline for the selection of suitable antibody coupling procedures.