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Functionalized Polyelectrolytes Assembling on Nano-BioFETs for Biosensing Applications
Advanced Functional Materials
Abstract
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.
... In this respect, an optimal balance between a high density of reactive groups and positive charges of unfunctionalized PLL moieties is required to obtain both an efficient surface functionalization and bonding efficiency. [21] After adsorption of the appropriately functionalized PLLs, the functionalized sides of the surfaces are put in contact with each other, and pressure is applied at room temperature in order to obtain stable bonding of the two device parts. The application of pressure ensures an increase of the contact area between the two non-conformal substrate surfaces, thus increasing the probability of successful click reaction at the interface. ...
... By tuning the molar ratios of the reactive moieties versus lysine monomer in the reaction mixture, various degrees of functionalization of PLL can be achieved. [21] In this work, 1 H-NMR was used to characterize the formation of the copolymers and to calculate the exact degree of PLL functionalization (Figures S1-S9, Table S1, Supporting Information). We initially aimed for high degrees of functionalization to achieve stable bonding. ...
... In previous studies, the optimal surface functionalization using biotin-modified PLLs (PLL-OEG-biotin) was reported to be achieved with a degree of functionalization of up to ≈40% of the lysines. [21] Nevertheless, while 35% of PLL grafting was successfully achieved for PLL-N 3 (Figures S1 and S2, Supporting Information), a maximum of 23% was obtained with PLL-DBCO ( Figures S3 and S4, Supporting Information). The lower yield obtained for PLL-DBCO can be attributed to the larger steric hindrance of the DBCO moieties in comparison to N 3 , which can inhibit a higher degree of functionalization of the polymer. ...
The production of a new class of microfluidic devices built from thermoplastic materials has recently aroused interest in a high‐volume and cost‐effective fabrication of sensing devices. During device formation, the bonding of two slides, one containing the microchannels and another used as a non‐modified capping layer, is generally performed at high temperature, high pressure, and/or employing organic solvents. Such bonding procedures, however, are not compatible with the use of slides pre‐functionalized with biomolecules. Here a low‐temperature, UV‐light‐free, and solvent‐free bonding method for polymeric devices, based on poly‐l‐lysine (PLL) polymers modified with click‐chemistry moieties is presented. The advantages obtained from the use of PLL and the appended complementary click‐chemistry groups enable a fast adsorption of polymers onto the substrates at room temperature, followed by a fast and stable bonding between two complementarily functionalized slides under mild conditions. Bonded fluidic devices show a resistance to high fluid pressures well above those needed for practical application. The optimal density (2–5%) of reactive groups appended to PLL is assessed using lap shear tests. The here developed method achieves bonding at low temperature, which promises fabrication of microfluidic devices functionalized with biomolecules prior to the sealing process, applicable to a wide range of polymeric materials. Microfluidic devices pre‐functionalized with sensitive (bio)molecules need careful device bonding. Easy functionalization of polymeric substrates with click moiety‐functionalized poly‐l‐lysine polymers permits rapid and low‐temperature bonding, which provides sufficient strength for microfluidic applications.
... 41 Duan et al. reported silicon nano-BioFET biosensors covered by a uniform layer of PLL cografted with short oligo(ethylene glycol) (OEG 4 ) and OEG 4 -biotin moieties, retaining the antifouling properties and strong surface adhesion. 44 Recently, we have demonstrated that PLL polymers with customizable fractions of OEG and maleimide moieties can be easily adsorbed onto both gold and silicon dioxide surfaces, while controlling the type and the density of probe molecules at the interface in the preceding synthetic step. 45 Here, we report two approaches to functionalize different types of plastic surfaces commonly used in biosensing applications, such as COP, Ormostamp, and PDMS, by exploiting the adsorption of PLL grafted with OEG 4 -biotin or OEG 4 -maleimide for fast, efficient, and selective immobilization of biomolecules. ...
... PLL polymers (15−30 kDa) were grafted with oligo-(ethylene glycol) (OEG) spacers and either biotin or maleimide (Mal) moieties, following reported procedures. 44,45 All the modified PLLs synthesized, namely, PLL-OEG, PLL-OEG-biotin, and PLL-OEG-Mal, present the OEG functionality to enhance the antifouling properties of the substrate, providing concomitantly a secondary functional group for further selective bio-orthogonal modification, such as biotin or maleimide ( Figure 1d). The total degree of functionalization of the PLL polymers has been determined by 1 H NMR (see Figure S1 and Table S1), and has been intentionally kept between 20% and 40%, to maintain a balance between a stable surface adhesion and antifouling properties. ...
... All modified PLLs were synthesized according to previously reported procedures. 44 Preparation of PDMS Substrates. Poly(dimethylsiloxane) (PDMS) substrates (for use of the stamping device) and molds for MIMIC were prepared as reported previously. ...
The immobilization of biomolecules onto polymeric surfaces employed in the fabrication of biomedical and biosensing devices, is generally a challenging issue, as the absence of functional groups in such materials does not allow the use of common surface chemistries. Here we report the use of modified poly-L-lysine (PLL) as an effective method for the selective modification of polymeric materials with biomolecules. Cyclic olefin polymer (COP), Ormostamp and polydimethylsiloxane (PDMS) surfaces were patterned with modified PLLs displaying either biotin or maleimide functional groups. Different patterning techniques were found to provide faithful microscale pattern formation, including micromolding in capillaries (MIMIC) and a hydrogel-based stamping device with micropores. The surface modification and pattern stability were tested with fluorescence microscopy, contact angle and X-ray photoelectron spectroscopy (XPS), showing an effective functionalization of substrates stable for over 20 days. By exploiting the strong biotin-streptavidin interaction or the thiol-maleimide coupling, DNA and PNA probes were displayed successfully on the surface of the materials, and these probes maintained the capability to specifically recognize complementary DNA sequences from solution. The printing of three different PNA-thiol probe molecules in a microarray fashion allowed selective DNA detection from a mixture of DNA analytes, demonstrating that the modified-PLL methodology can be used for the multiplexed detection of DNA sequences.
... 30,32,33 VandeVondele and Hubbel varied the grafting density of RGD peptide to form PLL-g-PEG-RGD polymers that promote cell adhesion, blocking the nonspecific protein interactions at the same time, 34 while Huang et al. formed a bioaffinity sensor to detect rabbit immunoglobulin (RIgG) changing the graft ratio 36 in combination with OEG 4 -biotin to PLL backbones on silicon nano-BioFETs, forming a biosensing surface with uniform layers, maintaining the strong surface stability and antifouling properties. 37 Here we show the formation of DNA biorecognition surfaces prepared by the deposition of modified poly(L-lysine) polymers with various ratios of OEG and maleimide (Mal) moieties (PLL-OEG-Mal) on surfaces, so that the probe density control is achieved during a preceding and simple synthetic step, where the degree of functionalization is readily analyzed and quantified by 1 H NMR. Afterward, by means of a predictable and straightforward, single-step surface assembly process over gold and silicon oxide, the PLL-OEG-Mal polymers are adsorbed on the sensing surface and coupled subsequently with thiol-modified peptide nucleic acid (PNA) probes, able to detect KRAS sequences of DNA (involved in the early formation of several types of cancer). 38,39 We used PNA sequences as probes to achieve a better affinity for cDNA sequences, compared to DNA probes. ...
... Following a reported procedure, 37 OEG and Mal groups were covalently grafted to the PLL backbone in a one-step, one-pot synthesis by NHS ester coupling, as depicted in Figure 1b, using a mixture of NHS-OEG 4 and NHS-OEG 4 -Mal. The grafted OEG/Mal ratio attached to PLL was calculated taking the proper 1 H NMR integrals, after normalization by the peak at 4.29 ppm (1H, lysine backbone, NH−CH−C(O)−) (see Figure S1). ...
... 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. ...
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.
... (see Scheme S1 †). 22 PLL was dissolved in PBS (pH ¼ 7.2) at a concentration of 10 mg mL À1 . NHS-OEG 4 -methyl and NHS-OEG 4 -methyltetrazine, both dissolved in DMSO at a concentration of 250 mM, were added to the PLL solution simultaneously under nitrogen atmosphere in desired ratios. ...
... 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 purication. Methyl-OEG 4 -NHS was purchased from ThermoFischer Scientic, while methyltetrazine-OEG 4 -NHS was purchased from Click chemistry tools. ...
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.
... Surface functionalization of top-down silicon nanoribbon with SLB To begin, we fabricated top-down silicon nanoribbon devices with windows exposing only the nanoribbon/ oxide surface to solution (figure 1). Figure 1(b) shows an optical image of a 2 μm wide and 20 μm long silicon nanoribbon. p-type Al 2 O 3 -passivated and SiO 2 -passivated devices were fabricated similarly to previously reported recipes [8,9]. The fabrication process was carefully controlled such that the final thicknesses of the silicon nanoribbon and dielectric are 40 nm and 20 nm, respectively. ...
... The data clearly show the effect of a charged polymer on the threshold voltage of the nanoribbons. Although the lipid bilayer threshold voltage shift has not been studied before this work, similar polyelectrolyte results were published on silicon nanowires/ nanoribbons in [9,11]. ...
Using top-down fabricated silicon nanoribbons, we measure the opening and closing of ion channels alamethicin and gramicidin A. A capacitive model of the system is proposed to demonstrate that the geometric capacitance of the nanoribbon is charged by ion channel currents. The integration of top-down nanoribbons with electrophysiology holds promise for integration of electrically active living systems with artificial electronics.
... 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). ...
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.
... Furthermore, these biosensors exhibited analytical sensitivities in the range of 10′s of ng mL −1 , making them unsuitable for the detection of many clinically relevant biomarkers that are present at low concentrations (10′s to 1000′s pg mL −1 ) in bodily fluids. FET-based sensors have also been developed with various reusability strategies, including using sensor coatings with reversible protein binding properties, [23] modifying nano-FET sensing elements with polymers or peptides, [24][25][26] or utilizing aptamers with shape-transitioning properties as biorecognition elements. [27] While these platforms could be reused at least 10 times, the fabrication of FET-based sensors is complicated and expensive, limiting their use to research purposes. ...
The detection and quantification of protein biomarkers in bodily fluids is important for many clinical applications, including disease diagnosis and health monitoring. Current techniques for ultrasensitive protein detection, such as enzyme‐linked immunosorbent assay (ELISA) and electrochemical sensing, involve long incubation times (1.5–3 h) and rely on single‐use sensing electrodes which can be costly and generate excessive waste. This work demonstrates a reusable electrochemical immunosensor employing magnetic nanoparticles (MNPs) and dually labeled gold nanoparticles (AuNPs) for ultrasensitive measurements of protein biomarkers. As proof of concept, this platform is used to detect C‐X‐C motif chemokine ligand 9 (CXCL9), a biomarker associated with kidney transplant rejection, immune nephritis from checkpoint inhibitor therapy, and drug‐associated acute interstitial nephritis, in human urine. The sensor successfully detects CXCL9 at concentrations as low as 27 pg mL⁻¹ within ≈1 h. This immunosensor was also adapted onto a handheld smartphone‐based diagnostic device and used for measurements of CXCL9, which exhibited a lower limit of detection of 65 pg mL⁻¹. Lastly, this work demonstrates that the sensing electrodes can be reused for at least 100 measurements with a negligible loss in analytical performance, reducing the costs and waste associated with electrochemical sensing.
... [84] Moreover, the modified polymer could serve as a blocking layer to prevent nonspecific adsorption, thus enabling the long-term and continuous detection of biomolecules in the complicated biological environment. [29,85] Except for the modification with polymers, lipid layers were also employed to assemble on FET biosensors to overcome the Debye screening effect. [86,87] Attributed to the fluidity and flexibility of the biomimetic lipid layer, the morphology and affinity of bioreceptors could be well preserved. ...
Promising advances in molecular medicine have promoted the urgent requirement for reliable and sensitive diagnostic tools. Electronic biosensing devices based on field‐effect transistors (FETs) exhibit a wide range of benefits, including rapid and label‐free detection, high sensitivity, easy operation, and capability of integration, possessing significant potential for application in disease screening and health monitoring. In this perspective, the tremendous efforts and achievements in the development of high‐performance FET biosensors in the past decade are summarized, with emphasis on the interface engineering of FET‐based electrical platforms for biomolecule identification. First, an overview of engineering strategies for interface modulation and recognition element design is discussed in detail. For a further step, the applications of FET‐based electrical devices for in vitro detection and real‐time monitoring in biological systems are comprehensively reviewed. Finally, the key opportunities and challenges of FET‐based electronic devices in biosensing are discussed. It is anticipated that a comprehensive understanding of interface engineering strategies in FET biosensors will inspire additional techniques for developing highly sensitive, specific, and stable FET biosensors as well as emerging designs for next‐generation biosensing electronics.
... However, this method's shortcoming is that nanoparticles do not completely cover the surface of the sensing material, so single-stranded target nucleic acid molecules are absorbed on the exposed sensing material through π-π stacking. But there are also methods (such as tween 20 [165,166], PEG [167,168]) to encapsulate the exposed sensing material to overcome the nonspecific adsorption problem. 5. The functionalization method of immobilizing probes based on Carbodiimide (EDC) + N-hydroxysuccinimide ester (NHS) cross-linking [169,170] [172,173]: the pyrene group of PBASE is linked to the graphene or MX 2 surface through the π-π stacking, and the active NHS ester of PBASE is linked to the amino-modified probes through the amide bond (Fig. 6i) [47]. ...
Field-effect transistor (FET) is regarded as the most promising candidate for the next-generation biosensor, benefiting from the advantages of label-free, easy operation, low cost, easy integration, and direct detection of biomarkers in liquid environments. With the burgeoning advances in nanotechnology and biotechnology, researchers are trying to improve the sensitivity of FET biosensors and broaden their application scenarios from multiple strategies. In order to enable researchers to understand and apply FET biosensors deeply, focusing on the multidisciplinary technical details, the iteration and evolution of FET biosensors are reviewed from exploring the sensing mechanism in detecting biomolecules (research direction 1), the response signal type (research direction 2), the sensing performance optimization (research direction 3), and the integration strategy (research direction 4). Aiming at each research direction, forward perspectives and dialectical evaluations are summarized to enlighten rewarding investigations.
... The 2D-PET is assembled onto the negatively charged surface to form an anchored module via electrostatic adsorption. The surface density of the side PEG-biotin chains is around 40% to guarantee the adsorption stability between 2D-PET and surface [43,44], as well as sufficient binding sites for subsequent growable module. ...
Nanochannel-based resistive pulse sensing (nano-RPS) system is widely used for the high-sensitive measurement and characterization of nanoscale biological particles and biomolecules due to its high surface to volume ratio. However, the geometric dimensions and surface properties of nanochannel are usually fixed, which limit the detections within particular ranges or types of nanoparticles. In order to improve the flexibility of nano-RPS system, it is of great significance to develop nanochannels with tunable dimensions and surface properties. In this work, we proposed a novel multi-module self-assembly (MS) strategy which allows to shrink the geometric dimensions and tune surface properties of the nanochannels simultaneously. The MS-tuned nano-RPS device exhibits an enhanced signal-to-noise ratio (SNR) for nanoparticle detections after shrunk the geometric dimensions by MS strategy. Meanwhile, by tuning the surface charge, an enhanced resolution for viral particles detection was achieved with the MS-tuned nano-RPS devices by analyzing the variation of pulse width due the tuned surface charge. The proposed MS strategy is versatile for various types of surface materials and can be potentially applied for nanoscale surface reconfiguration in various nanofluidic devices.
... In general, proteins can be immobilized by a wide variety of approaches, [59][60][61][62] but a detailed description of the protocols is outside the scope of this article. The second step is the actual binding of hmDNA to the MBD2-modified surface. ...
The preselection of hypermethylated DNA (hmDNA) from liquid biopsy samples is key to enable early‐stage cancer diagnostics. Due to limited selectivity of the existing preselection approaches, however, wide integration in the clinic is currently prohibited. Here, it is argued that an affinity method on a surface, such as used in affinity chromatography, can be significantly improved by employing the principles of multivalency and superselectivity. In the here proposed method, a methyl binding domain (MBD) protein immobilized at a surface is used as a receptor for (hyper)methylated DNA. By the organization of multiple MBDs on a surface, a multivalent binding platform is achieved. The MBD surface receptor density on that platform is key to increase the enrichment selectivity of hmDNA as a single MBD protein binds both methylated and non‐methylated DNA with a small difference in affinity. When the receptor density is varied, multivalent analyte binding typically responds in a non‐linear fashion, which phenomenon is called superselectivity. By careful tuning of the MBD density, it is envisaged that the selectivity for methylated over non‐methylated DNA can be optimized. Strong applicability is foreseen in a medical setting by implementing such an enrichment step in an analytical process or a lab‐on‐a‐chip device. By using the principles of multivalency and superselectivity, enrichment of the cancer biomarker hypermethylated DNA from liquid biopsies can be greatly improved. This concept proposes optimization of the surface receptor density of methyl binding domain proteins on the device surface to achieve enrichment suited for use in medical diagnostics.
... Several research groups reported hybrid surface peptide-based modifiers consisting of a poly-L-lysine (PLL) polypeptide backbone partially grafted with different antifouling molecules, like PEG side-chains, through amine residues [93]. PLL is a versatile polymer composed of positively charged lysine amino acid as a repeat unit. ...
Strategies to develop antifouling surface coatings are crucial for surface plasmon resonance (SPR) sensing in many analytical application fields, such as detecting human disease biomarkers for clinical diagnostics and monitoring foodborne pathogens and toxins involved in food quality control. In this review, firstly, we provide a brief discussion with considerations about the importance of adopting appropriate antifouling materials for achieving excellent performances in biosensing for food safety and clinical diagnosis. Secondly, a non-exhaustive landscape of polymeric layers is given in the context of surface modification and the mechanism of fouling resistance. Finally, we present an overview of some selected developments in SPR sensing, emphasizing applications of antifouling materials and progress to overcome the challenges related to the detection of targets in complex matrices relevant for diagnosis and food biosensing.
... 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. ...
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.
... 25 times higher than those of a monolayer of PLL-OEG(29.7)-DBCO(1.6) (a larger amount of OEG was required to avoid non-specific interaction of DNA to the PLL backbone in this case, 68 as shown in the left part of Figure S8). In the case of a linearly grown PEM, the frequency changes recorded at the QCM for the same DNA probe and cDNA incorporation were only a factor of 5.5 higher. ...
Surface-based biosensing devices benefit from a dedicated design of the probe layer present at the transducing interface. The layer architecture, its physicochemical properties, and the embedding of the receptor sites affect the probability of binding the analyte. Here, the enhancement of the probe density at the sensing interface by tuning the exponential growth regime of polyelectrolyte multilayers (PEMs) is presented. PEMs were made of poly-l-lysine (PLL), with appended clickable dibenzocyclooctyne (DBCO) groups and oligo(ethylene glycol) chains, and poly(styrene sulfonate) (PSS). The DNA probe loading and target hybridization efficiencies of the PEMs were evaluated as a function of the PLL layer number and the growth regime by a quartz crystal microbalance (QCM). An amplification factor of 25 in the target DNA detection was found for a 33-layer exponentially grown PEM compared to a monolayer. A Voigt-based model showed that DNA probe binding to the DBCO groups is more efficient in the open, exponentially grown films, while the hybridization efficiencies appeared to be high for all layer architectures. These results show the potential of such engineered gel-like structures to increase the detection of bio-relevant analytes in biosensing systems.
... A monolayer thin film of this material attaches to the sensor surface via electrostatic forces, and the surface can regenerate by changing the pH values to frustrate such interactions. [86] Analyte binding events lead to direct electrostatic effects that modulate the electrical characteristics of the FETs according to principles described previously. Screening of charged biomolecules by ions in the solution represents a key limitation of this sensing approach, known as Debye screening according to the Gouy Chapman Stern model (Figure 4A). ...
Opportunities for quantitative, real‐time monitoring of gases, ions, and biomolecules in the environment and in the human body motivate programs of fundamental and applied research on chemically selective sensors with fast response times. In this context, silicon field‐effect transistors are of considerable interest as label‐free, scalable platforms for detecting a variety of chemical and biological species. Herein, recent progress and research directions in this area are reviewed. The focus of this article is on operational parameters, device architectures, schemes for surface chemical functionalization, and methods for bio‐integration across a variety of use cases. The content includes strategies that combine Si with other functional materials to create hybrid structures for enhanced sensing performance. The final section highlights some remaining challenges and provides perspectives on the future of basic research and engineering development in this field.
... PLL-biotin(10%)-OEG(30%) (PLL-biotin) and PLL-OEG(35%) (PLL-OEG) were synthesized and characterized by the methods of Duan et al. 37 The modified PLLs were dissolved in PBS (10 mM, pH 7.2) with 10% glycerol at a concentration of 1.5 mg mL −1 . PLL-biotin and PLL-OEG were immobilized on clean aMZI and QCM chips by dropping the solution of the desired PLL to cover the sensing windows of the aMZI and whole QCM chip. ...
The sensitivity and performance of an asymmetric Mach-Zehnder interferometer (aMZI) were compared to quartz crys-tal microbalance with dissipation (QCM-D). The binding of streptavidin to sensor chips coated with poly-L-lysine (PLL), modified with biotin and oligoethyleneglycol (OEG) (PLL-biotin), was used to compare the binding signals obtained from both technologies. PLL-biotin proved to be an efficient method to add bioreceptors to both the QCM-D and aMZI chips. The final, saturated value of streptavidin binding was compared between the aMZI (253 ng cm-2) and QCM-D (460 ng cm-2). These values were then used to evaluate that 45% of the measured streptavidin mass in the QCM-D came from hydro-dynamically coupled water. Importantly, the signal-to-noise ratio of the aMZI was found to be 200 times higher than that of the QCM-D. These results indicate the potential of the aMZI platform for highly sensitive and accurate biosensing ap-plications.
... Moreover, the graphene has to be either transferred to a device-compatible substrate through wet chemical transfer steps or physical stamping processes [8,9], or the graphene needs to be patterned on a growth substrate into a device through atomic layer etching [10], focused ion beam patterning [11], or block copolymer lithography [12,13]. Although these techniques are capable of manufacturing high-resolution devices (<50 µm line resolution), they are energy intensive and low yield, and often require sophisticated cleanroom processing [14][15][16]. ...
Carbon nanomaterials such as graphene exhibit unique material properties including high electrical conductivity, surface area, and biocompatibility that have the potential to significantly improve the performance of electrochemical sensors. Since in-field electrochemical sensors are typically disposable, they require materials that are amenable to low-cost, high-throughput, and scalable manufacturing. Conventional graphene devices based on low-yield chemical vapor deposition techniques are too expensive for such applications, while low-cost alternatives such as screen and inkjet printing do not possess sufficient control over electrode geometry to achieve favorable electrochemical sensor performance. In this work, aerosol jet printing (AJP) is used to create high-resolution (∼40 μm line width) interdigitated electrodes (IDEs) on flexible substrates, which are then converted into histamine sensors by covalently linking monoclonal antibodies to oxygen moieties created on the graphene surface through a CO2 thermal annealing process. The resulting electrochemical sensors exhibit a wide histamine sensing range of 6.25–200 ppm (56.25 μM–1.8 mM) and a low detection limit of 3.41 ppm (30.7 μM) within actual tuna broth samples. These sensor metrics are significant since histamine levels over 50 ppm in fish induce adverse health effects including severe allergic reactions (e.g. Scombroid food poisoning). Beyond the histamine case study presented here, the AJP and functionalization process can likely be generalized to a diverse range of sensing applications including environmental toxin detection, foodborne pathogen detection, wearable health monitoring, and health diagnostics.
... 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). ...
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.
... We coated the inner surface of the capillary by applying a selfsynthesized PLL-g-OEG-Biotin 54,55 solution to the capillary inner surface. The cationic macromolecule Poly-L-Lysine (PLL) assembled on the negatively charged glass capillary surface, and oligo ethylene glycol (OEG) was used to block non-specific binding 54 . Successful functionalization of the capillary with biotin and reactivity of the immobilized biotin was demonstrated using subsequent binding of fluorescein isothiocyanate-labeled Streptavidin within 1-2 h as microscopically observed (Fig. 5b). ...
Dimensions and surface properties are the predominant factors for the applications of nanofluidic devices. Here we use a thin liquid film as a nanochannel by inserting a gas bubble in a glass capillary, a technique we name bubble-based film nanofluidics. The height of the film nanochannel can be regulated by the Debye length and wettability, while the length independently changed by applied pressure. The film nanochannel behaves functionally identically to classical solid state nanochannels, as ion concentration polarizations. Furthermore, the film nanochannels can be used for label-free immunosensing, by principle of wettability change at the solid interface. The optimal sensitivity for the biotin-streptavidin reaction is two orders of magnitude higher than for the solid state nanochannel, suitable for a full range of electrolyte concentrations. We believe that the film nanochannel represents a class of nanofluidic devices that is of interest for fundamental studies and also can be widely applied, due to its reconfigurable dimensions, low cost, ease of fabrication and multiphase interfaces. Dimensions and the surface properties of the nanochannels are vital to the functions and applications of nano-fluidic devices. Here Ma et al. studied the film thickness of a Bretherton bubble in a microcapillary and demonstrate the liquid film can be used for label-free biosensing.
... More recently, Duan and coworkers employed oligo (ethylene glycol) (OEG)-grafted PLL (PLLÀ OEG) for the fabrication of a bio-functionalized film on nano-bioFETs. [68] OEG and OEGÀ biotin moieties were grafted in different ratios to produce PLL-g-OEG-biotin containing different ligand densities. The total degree of functionalization of the PLL was varied to study the optimal functionalization to obtain both stability of the PLL on the surface and maximal adsorption of SAv. ...
In the study of multivalent interactions at interfaces, as occur for example at cell membranes, the density of the ligands or receptors displayed at the interface plays a pivotal role, affecting both the overall binding affinities and the valencies involved in the interactions. In order to control the ligand density at the interface, several approaches have been developed, and they concern the functionalization of a wide range of materials. Here, different methods employed in the modification of surfaces with controlled densities of ligands are being reviewed. Examples of such methods encompass the formation of self‐assembled monolayers (SAMs), supported lipid bilayers (SLBs) and polymeric layers on surfaces. Particular emphasis is given to the methods employed in the study of different types of multivalent biological interactions occurring at the functionalized surfaces and their working principles. Play it again, SAM: An overview of various surface functionalization methods that allow control over the ligand density is provided, with a focus on the study of multivalent interactions at two‐dimensional interfaces.
... The synthetic method of PLL-g-OEG 4 -Biotin is described in the previous published work. [26] 1 mg mL −1 PLL-g-OEG 4 -Biotin was mixed to PEDOT:PSS in 1:9 volume proportion. Once the nanochannels were fully filled and the solvent evaporates completely, the PDMS model was carefully removed. ...
The demand for fast and ultratrace biomarkers detection is increasing in bioanalytical chemistry. In this work, highly ordered nanowires array and sensor integration are achieved with nanoscale printing approach. Negatively charged poly(3,4‐ethylenedioxythiophene)–poly(styrenesulfonate) doped with positively charged PEGylated biotin‐derivatized polyelectrolytes results a direct biofunctionalization on the nanowire surface without multiple postmodification steps. It provides homogeneous dispersed biofunctional sites and nonfouling surface on the nanowires. The ordered nanowires array enables the immunosensor to detect biotargets quickly and ultrasensitively. The nanowires impedimetric immunosensor is demonstrated for specific biomarkers detection and achieved a minimum responsive concentration as low as 10 pg mL−1 for protein biomarker and 10 CFU mL−1 for pathogen. A kind of biotin functionalization ink is achieved by doping with PEGylated biotin‐derivatized polyelectrolytes which enables a direct biofunctionalization on the nanowire surface. Then an immunosensor based on highly ordered nanowires array is fabricated by nanoscale printing approach. This sensor can be a general platform for different materials and various bioanalytical applications with ultrahigh sensitivity, trace detection, and fast response.
... Antihuman IgGs were immobilized on the resonator surface through a PLL−PEG−biotin−SAV linker. 43 The main reason that we employed PLL−PEG−biotin−SAV for anti-IgG immobilization instead of direct conjugation of anti-IgG is to test the concentration effect on the same device by regenerating the sensor surface. Since the assemblies of the PLL−PEG−biotin−SAV on the resonator are driven by electrostatic interactions between the positively charged polymer and negatively charged surface, such assemblies can be regenerated easily by tuning the pH value of the buffer. ...
In designing bioassay systems for low-abundance biomolecule detection, most research focuses on improving transduction mechanisms while ignoring the intrinsically fundamental limitations in solution: mass transfer and binding affinity. We demonstrate enhanced biomolecular surface binding using an acoustic nano-electromechanical system (NEMS) resonator, as an on-chip biomolecular concentrator which breaks both mass transfer and binding affinity limitations. As a result, a concentration factor of 10⁵ has been obtained for various biomolecules. The resultantly enhanced surface binding between probes on the absorption surface and analytes in solution enables us to lower the limit of detection for representative proteins. We also integrated the biomolecular concentrator into an optoelectronic bioassay platform to demonstrate delivery of proteins from buffer/serum to the absorption surface. Since the manufacture of the resonator is CMOS-compatible, we expect it to be readily applied to further analysis of biomolecular interactions in molecular diagnostics.
... 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. ...
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.
... Surfaces covalently coated with such molecules can be used to conjugate thiol-, amine-or hydroxyl-containing ligands, for then attaching biomolecules to them. 24,25 One way to attach specific functional molecules to the GOPTSfunctionalized surface was demonstrated by covalently linking a ferrocene-labeled thiolated molecule, i.e., 6-(ferrocenyl)hexanethiol, via reaction of epoxy rings with sulfide terminations. ITO surfaces were first cleaned with acetone for 10 min and dichloromethane for another 10 min. ...
The electroactivity of nanostructured indium tin oxide (ITO) has been investigated for its further use in
applications such as sensing biological compounds by the analysis of redox active molecules. ITO films were
fabricated by using electron beam evaporation at different substrate temperatures and subsequently annealed for promoting their crystallization. The morphology of the deposited material was monitored by scanning electron microscopy, confirming the deposition of either thin films or nanowires, depending on the substrate temperature. Electrochemical surface characterization revealed a 45 % increase in the electroactive surface area of nanostructured ITO with respect to thin films, one third lower than the geometrical surface area variation determined by atomic force microscopy. ITO surfaces were functionalized with a model organic
molecule known as 6-(ferrocenyl)hexanethiol. The chemical attachment was done by means of a glycidoxy
compound containing a reactive epoxy group, the so-called 3-glycidoxypropyltrimethoxy-silane (GOPTS).
ITO functionalization was useful for determining the benefits of nanostructuration on the surface coverage of active molecules. Compared to ITO thin films, an increase in the total peak height of 140 % was observed
for as-deposited nanostructured electrodes, whereas the same measurement for annealed electrodes resulted
in an increase of more than 400 %. These preliminary results demonstrate the ability of nanostructured ITO
to increase the surface-to-volume ratio, conductivity and surface area functionalization, features that highly
benefit the performance of biosensors.
... Regeneration of the sensor surface would be highly beneficial for their use, since it 290 VOLUME 3, 2015 provides the possibility to measure analytes using the same high quality device and avoids the variations in device characteristics from one sensor to another. In a more recent work, Duan et al. demonstrated a simpler way to regenerate the sensor surface [94]. Devices were coated with biocompatible polyelectrolyte thin films consisting of poly-L-Lysine backbones grafted with oligo-ethylene-glycol (OEG) ( Figure 2D). ...
Silicon nanowire field-effect transistors (Si-NW FETs) have been demonstrated as a versatile class of potentiometric nanobiosensors for real time, label-free, and highly sensitive detection of a wide range of biomolecules. In this review, we summarize the principles of such devices and recent developments in device fabrication, fluid integration, surface functionalization, and biosensing applications. The main focus of this review is on CMOS compatible Si-NW FET nanobiosensors.
The future of medical diagnostics calls for portable biosensors at the point of care, aiming to improve healthcare by reducing costs, improving access, and increasing quality – what is called the ‘triple aim’. Developing point-of-care sensors that provide high sensitivity, detect multiple analytes, and provide real time measurements can expand access to medical diagnostics for all. Field-effect transistor (FET)-based biosensors have several advantages, including ultrahigh sensitivity, label-free and amplification-free detection, reduced cost and complexity, portability, and large-scale multiplexing. They can also be integrated into wearable or implantable devices and provide continuous, real-time monitoring of analytes in vivo, enabling early detection of biomarkers for disease diagnosis and management. This review analyzes advances in the sensitivity, parallelization, and reusability of FET biosensors, benchmarks the limit of detection of the state of the art, and discusses challenges and opportunities towards ultrasensitive, parallelized, and reusable FET biosensors for future healthcare applications.
Viral particles bind to receptors through multivalent protein interactions. Such high avidity interactions on sensor surfaces are less studied. In this work, three polyelectrolytes that can form biosensing surfaces with different interfacial characteristics in probe density and spatial arrangement were designed. Quartz crystal microbalance, interferometry and atomic force microscopy were used to study their surface density and binding behaviors with proteins and virus particles. A multivalent adsorption kinetic model was developed to estimate the number of bonds from the viral particles bound to the polyelectrolyte surfaces. Experimental results show that the heterogeneous 3D surface with jagged forest-like structure enhances the virus capture ability by maximizing the multivalent interactions. As a proof of concept, specific coronavirus detection was achieved in spiked swab samples. These results indicate the importance of both probe density and their spatial arrangement on the sensing performance, which could be used as a guideline for rational biosensing surface design.
Food Borne Diseases (FBD) are an emerging threat to human health worldwide. FBD are generally caused by foodborne pathogens, toxins they release and other contaminants. Various methods have been explored to identify and detect these in the food. Some of these methods utilize the conventional detection techniques that involve complicated sample pretreatment, are time-consuming, require advanced instruments that need trained personnel for its operation. This has led to the need to develop accurate, sensitive and rapid detection methods. Recently, nanosensors/nanobiosensors have been explored as alternative detection method that is more effective, rapid and have operational stability. This article highlights various nanomaterial-based sensors, their types, advantages and specificity towards the detection of food borne pathogens.
Bioelectronic devices have received the massive attention because of their huge potential to develop the core electronic components for biocomputing system. Up to now, numerous bioelectronic devices have been reported such as biomemory and biologic gate by employment of biomolecules including metalloproteins and nucleic acids. However, the intrinsic limitations of biomolecules such as instability and low signal production hinder the development of novel bioelectronic devices capable of performing various novel computing functions. As a way to overcome these limitations, nanomaterials have the great potential and wide applicability to grant and extend the electronic functions, and improve the inherent properties from biomolecules. Accordingly, lots of nanomaterials including the conductive metal, graphene, and transition metal dichalcogenide nanomaterials are being used to develop the remarkable functional bioelectronic devices like the multi-bit biomemory and resistive random-access biomemory. This review discusses the nanomaterial-based superb bioelectronic devices including the biomemory, biologic gates, and bioprocessors. In conclusion, this review will provide the interdisciplinary information about utilization of various novel nanomaterials applicable for biocomputing system.
Supramolecular materials, which rely on dynamic non-covalent interactions, present a promising approach to advance the capabilities of currently available biosensors. The weak interactions between supramolecular monomers allow for adaptivity and responsiveness of supramolecular or self-assembling systems to external stimuli. In many cases, these characteristics improve the performance of recognition units, reporters, or signal transducers of biosensors. The facile methods for preparing supramolecular materials also allow for straightforward ways to combine them with other functional materials and create multicomponent sensors. To date, biosensors with supramolecular components are capable of not only detecting target analytes based on known ligand affinity or specific host-guest interactions, but can also be used for more complex structural detection such as chiral sensing. In this Review, we discuss the advancements in the area of biosensors, with a particular highlight on the designs of supramolecular materials employed in analytical applications over the years. We will first describe how different types of supramolecular components are currently used as recognition or reporter units for biosensors. The working mechanisms of detection and signal transduction by supramolecular systems will be presented, as well as the important hierarchical characteristics from the monomers to assemblies that contribute to selectivity and sensitivity. We will then examine how supramolecular materials are currently integrated in different types of biosensing platforms. Emerging trends and perspectives will be outlined, specifically for exploring new design and platforms that may bring supramolecular sensors a step closer towards practical use for multiplexed or differential sensing, higher throughput operations, real-time monitoring, reporting of biological function, as well as for environmental studies.
The subphase adsorption behavior and the morphology of the trypsin enzyme, lysozyme enzyme, cytochrome C protein, and glucose oxidase present on the Langmuir monolayer of organo-modified single-wall carbon nanotubes (SWCNTs) were investigated. Furthermore, we estimated the maintenance characteristics of the three-dimensional structure of organo-modified SWCNT film-adsorbed biomolecules when exposed to a high-temperature environment. In this study, each biomolecule, which was an amphoteric electrolyte, was hydrophilized by mixed acid treatment and electrostatically adsorbed on the charged SWCNT surfaces at the air–water interface. An amide band derived from the adsorbent was confirmed by the infrared (IR) spectra of the biomolecule adsorbing organo-SWCNT multilayers. The atomic force microscopic image of the X-type monolayer of adsorbed biomolecules on organo-SWCNTs shows the adsorbent shape. The out-of-plane X-ray diffraction profiles of the biomolecule adsorbing organo-SWCNT multilayers confirmed the maintenance of the layered structure due to the presence of the adsorbent. After heat treatment, the secondary structure of the biomolecular adsorbing organo-SWCNT multilayers was maintained up to 100–150 °C, as confirmed by the IR spectrum.
Poly(α-l-lysine) (PLL) is a class of water-soluble, cationic biopolymer composed of α-l-lysine structural units. The previous decade witnessed tremendous progress in the synthesis and biomedical applications of PLL and its composites. PLL-based polymers and copolymers, till date, have been extensively explored in the contexts such as antibacterial agents, gene/drug/protein delivery systems, bio-sensing, bio-imaging, and tissue engineering. This review aims to summarize the recent advances in PLL-based nanomaterials in these biomedical fields over the last decade. The review first describes the synthesis of PLL and its derivatives, followed by the main text of their recent biomedical applications and translational studies. Finally, the challenges and perspectives of PLL-based nanomaterials in biomedical fields are addressed.
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.
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.
Biosensors and biomedical devices require antifouling surfaces to prevent the non-specific adhesion of proteins or cells, for example, when aiming to detect circulating cancer biomarkers in complex natural media (e.g., in blood plasma or serum). A mixed-charge polymer was prepared by the coupling of a cationic polyelectrolyte and an anionic oligopeptide through a modified “grafting-to” method. The poly-L-lysine (PLL) backbone was modified with different percentages (y%) of maleimide–NHS ester chains (PLL-mal(y%), from 13% to 26%), to produce cationic polymers with specific grafting densities, obtaining a mixed-charge polymer. The anionic oligopeptide structure (CEEEEE) included one cysteine (C) and five glutamic acid (E) units, which were attached to the PLL-mal(y%) polymers, preadsorbed on gold substrates, through the thiol–maleimide Michael-type addition. Contact angle and PM-IRRAS data confirmed monolayer formation of the modified PLLs. Antifouling properties of peptide–PLL surfaces were assessed in adsorption studies using quartz crystal microbalance with dissipation (QCM-D) and surface plasmon resonance imaging (SPRI) techniques. PLL-mal(26%)-CEEEEE showed the best antifouling performance in single-protein solutions, and the nonspecific adsorption of proteins was 46 ng cm−2 using diluted human plasma samples. The new PLL-mal(26%)-CEEEEE polymer offers a prominent low-fouling activity in complex media, with rapid and simple procedures for the synthesis and functionalization of the surface compared to conventional non-fouling materials.
Field-Effect Transistor sensors (FET-sensors) have been receiving increasing attention for biomolecular sensing over the last two decades due to their potential for ultra-high sensitivity sensing, label-free operation, cost reduction and miniaturisation. Whilst the commercial application of FET-sensors in pH sensing has been realised, their commercial application in biomolecular sensing (termed BioFETs) is hindered by poor understanding of how to optimise device design for highly reproducible operation and high sensitivity. In part, these problems stem from the highly interdisciplinary nature of the problems encountered in this field, in which knowledge of biomolecular-binding kinetics, surface chemistry, electrical double layer physics and electrical engineering is required. In this work, a quantitative analysis and critical review has been performed comparing literature FET-sensor data for pH-sensing with data for sensing of biomolecular streptavidin binding to surface-bound biotin systems. The aim is to provide the first systematic, quantitative comparison of BioFET results for a single biomolecular analyte, specifically Streptavidin, which is the most commonly used model protein in biosensing experiments, and often used as an initial proof-of-concept for new biosensor designs. This novel quantitative and comparative analysis of the surface potential behaviour of a range of devices demonstrated a strong contrast between the trends observed in pH-sensing and those in biomolecule-sensing. Potential explanations are discussed in detail and surface-chemistry optimisation is shown to be a vital component in sensitivity-enhancement. Factors which can influence the response, yet which have not always been fully appreciated, are explored and practical suggestions are provided on how to improve experimental design.
Nonspecific binding (NSB) is a general issue for surface based biosensors. Various approaches have been developed to prevent or remove the NSBs. However, these approaches either increased the background signals of the sensors or limited to specific transducers interface. In this work, we developed a hydrodynamic approach to selectively remove the NSBs using a microfabricated hypersonic resonator with 2.5 gigahertz (GHz) resonant frequency. The high frequency device facilitates to generate multiple controlled micro-vortices which then create cleaning forces at the solid-liquid interfaces. The competitive adhesive and cleaning forces have been investigated using the finite element method (FEM) simulation, identifying the feasibility of the vortices induced NSB removal. NSB proteins have been selectively removed experimentally both on the surface of the resonator and on other substrates which contact the vortices. Thus, the developed hydrodynamic approach is believed to be a simple and versatile tool for NSB removal and compatible to many sensor systems. The unique feature of the hypersonic resonator is that it can be used as a gravimetric sensor as well, thus a combined NSB removal and protein detection dual functional biosensor system is developed.
In this work, gigahertz solidly mounted resonators (SMRs) (2.5 GHz) were designed and fabricated to construct a novel particle-resonator system to achieve the biomolecular stiffness sensing in real time. The positive frequency shift of the system was used to estimate the stiffness of biomolecules connecting between the SMR and attached particles. The working principle was revealed by the mathematical analysis of the general block-spring model of the system. Further interpretations about the mechanism of such elastic interaction from the perspective of acoustic resonant modes of SMRs were given by finite element method. Biotin-streptavidin, antibody and antigen binding system were used as model molecular linkers to study the frequency shift varied with different particle diameters and particle densities. Different linker stiffness was realized by adjusting the concentrations of antigens connected with particles which form specific binding with antibodies immobilized on the SMR. The results fairly agree with the simulation results demonstrating the proposed particle-resonator system as an effective method to realize the real-time biomolecular stiffness detection.
A highly sensitive electrochemical immunosensor was developed by preventing electrode fouling and using a novel fusion protein of silica binding polypeptides (SBP)-protein G (ProG) created by recombinant DNA technology as a functional crosslinker for rapid and self-oriented immobilization of antibodies onto silica nanoparticles (SiNPs). Antibody immobilization onto the SiNPs by the SBP-ProG could rapidly be achieved without any chemical treatment. The immunosensor was fabricated through bonding of a partially gold-deposited cyclic olefin copolymer (COC) (top substrate) and gold patterned interdigitated array COC electrode (bottom substrate). To prevent electrode fouling, human immunoglobulin G (hIgG) was immobilized onto the ceiling inside the microchannel, instead of the bottom electrode. Alkaline phosphatase (AP)-labeled anti-hIgG was allowed to immunoreact with hIgG on the ceiling, followed by addition of an enzyme to generate an oxidative peak current. A three-fold increase in current was observed from the immunosensor without any electrode fouling compared with a control with the protein functionalized electrode. Also, the SiNPs facilely coated with AP-anti-hIgG via the SBP-ProG could increase the electrochemical signal up to 20% larger than that of the AP-anti-hIgG alone. Furthermore, this immunosensor was ultrasensitive with a detection limit of 0.68 pg/mL of a biomarker associated with prostate cancer.
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.
Micro/nano scale biosensors integrated with the local adsorption mask have been demonstrated to have a better limit of detection (LOD) and less sample consumptions. However, the molecular diffusions and binding kinetics in such confined droplet have been less studied which limited further development and application of the local adsorption method and imposed restrictions on discovery of new signal amplification strategies. In this work, we studied the kinetic issues via experimental investigations and theoretical analysis on microfabricated biosensors. Mass sensitive film bulk acoustic resonator (FBAR) sensors with hydrophobic Teflon film covering the non-sensing area as the mask were introduced. The fabricated masking sensors were characterized with physical adsorption of bovine serum albumin (BSA) and specific binding of antibody and antigen. Over an order of magnitude improvement on LOD was experimentally monitored. An analytical model was introduced to discuss the target molecule diffusion and binding kinetics in droplet environment, especially the crucial effects of incubation time, which has been less covered in previous local adsorption related literatures. An incubation time accumulated signal amplification effect was theoretically predicted, experimentally monitored and carefully explained. In addition, device optimization was explored based on the analytical model to fully utilize the merits of local adsorption. The discussions on the kinetic issues are believed to have wide implications for other types of micro/nano fabricated biosensors with potentially improved LOD.
Copyright © 2015 Elsevier B.V. All rights reserved.
Affinity biosensors use biorecognition elements and transducers to convert a biochemical event into a recordable signal. They provides the molecule binding information, which includes the dynamics of biomolecular association and dissociation, and the equilibrium association constant. Complementary metal oxide semiconductor-compatible silicon (Si) nanowires configured as a field-effect transistor (NW FET) have shown significant advantages for real-time, label-free and highly sensitive detection of a wide range of biomolecules. Most research has focused on reducing the detection limit of Si-NW FETs but has provided less information about the real binding parameters of the biomolecular interactions. Recently, Si-NW FETs have been demonstrated as affinity biosensors to quantify biomolecular binding affinities and kinetics. They open new applications for NW FETs in the nanomedicine field and will bring such sensor technology a step closer to commercial point-of-care applications. This article summarizes the recent advances in bioaffinity measurement using Si-NW FETs, with an emphasis on the different approaches used to address the issues of sensor calibration, regeneration, binding kinetic measurements, limit of detection, sensor surface modification, biomolecule charge screening, reference electrode integration and nonspecific molecular binding.
The performance of biosensors, i.e. the sensitivity, specificity, linearity, reusability, chemical stability, and reproducibility is critically dependent on the biofunctionalization of the sensor platform. The type(s) of linkers used for the immobilization of the capture probes and the exact immobilization protocol play a vital role in the overall performance of sensors. A variety of linker molecules have been used to biofunctionalize technologically important substrates (glass, gold, mica etc.). Amongst the different linkers, researchers have paid more attention to two dimensional architectures, e.g. silanes, polyaniline (PANI), alkanethiols, poly-L-lysine (PLL), etc. Despite extensive research and a large number of reports, researchers still face problems related to limited loading efficiency, limited accessibility of the probes, poor control over uniform spacing among the probes and a loss of functionality due to irregular orientation of the probes, all of which cause variability in the responses. Three dimensional gel based matrices have proved to be a better choice, except for the fact that the leaching of entrapped probe molecules has limited their use in developing sensor platforms. Taking into account the limitations of the two dimensional linker arrays and three dimensional gel matrices, supramolecular dendritic architectures have shown immense potential in designing and developing the sensor platforms. Dendrimers are well-defined, monodispersed, globular macromolecules constructed around a core unit. Different properties of dendrimers, i.e. their structural uniformity, globular shape, monodispersity, the existence of dendritic crevices, high functional group density, hydrophilicity, versatility to design dendrimer of different composition and their nanometric size can be exploited while developing high sensitivity biosensors. Researchers have demonstrated that these hyperbranched 3D molecules show enhanced sensitivity, reduced nonspecific binding, greater accessibility of the probe for the target analyte, high stability and low variability in their response. Hence, designing a sensor with a dendrimer as a linker is a successful approach to obtain superior sensor performance and minimize the overall cost of a sensor. In this article, we discuss various aspects of dendrimers from the point of view of sensor design, hoping that this review will excite more researchers into exploiting the exceptional properties of dendrimers in biosensor development.
Parameters controlling intrinsic stability and reactivity of organosilanols generated from alkoxysilanes in aqueous environments have been elucidated in several experiments. Data involving kinetics, equilibrium, phase separation, and bonding studies of alkyl and organofunctional alkoxysilanes are presented. The studies indicate that the rates of hydrolysis of alkoxysilanes are generally related to their steric bulk, but demonstrate that after rate-limiting hydrolysis of the first alkoxy group steric effects are much less important. Aqueous hydrolysis of alkylalkoxysilanes was studied to determine equilibrium constants and the extent of oligomerization up to phase separation. In the case of propyltrialkoxysilane, phase separation is coincident with the formation of tetramer. The equilibrium constant for esterification of silanols is Also, the performance properties of new water-borne silanes were evaluated and in most cases, their performance equalled or exceeded their traditional silane counterparts. keywords: silanol; coupling agents; organosilicon chemistry.
Monitoring the binding affinities and kinetics of protein interactions is important in clinical diagnostics and drug development because such information is used to identify new therapeutic candidates. Surface plasmon resonance is at present the standard method used for such analysis, but this is limited by low sensitivity and low-throughput analysis. Here, we show that silicon nanowire field-effect transistors can be used as biosensors to measure protein-ligand binding affinities and kinetics with sensitivities down to femtomolar concentrations. Based on this sensing mechanism, we develop an analytical model to calibrate the sensor response and quantify the molecular binding affinities of two representative protein-ligand binding pairs. The rate constant of the association and dissociation of the protein-ligand pair is determined by monitoring the reaction kinetics, demonstrating that silicon nanowire field-effect transistors can be readily used as high-throughput biosensors to quantify protein interactions.
The generation of surfaces and interfaces that are able to withstand protein adsorption is a major challenge in the design of blood-contacting materials for both medical implants and bioaffinity sensors. Poly(ethylene glycol)-derived materials are generally considered to be particularly effective candidates for the fabrication of protein-resistant materials. Most metallic biomaterials are covered by a protective, stable oxide film; converting such oxide surfaces, which are known to strongly interact with proteins, into noninteractive surfaces requires a specific design of the surface/interface architecture. A class of copolymers based on poly(L-lysine)g-poly(ethylene glycol) (PLL g-PEG) was found to spontaneously adsorb from aqueous solutions onto several metal oxide surfaces, such as TiO2, Si0.4Ti0.6O2 and Nb2O5, as measured by the in situ optical waveguide lightmode spectroscopy technique and by ex situ X-ray photoelectron spectroscopy. The resulting adsorbed layers are highly effective in reducing the adsorption both of blood serum and of individual proteins such as fibrinogen, which is known to play a major role in the cascade of events that lead to biomaterial-surface-induced blood coagulation and thrombosis. Adsorbed protein levels as low as <5 ng/cm(2) could be achieved for an optimized polymer architecture. The modified surfaces are stable to desorption under flow conditions at 37 degrees C and pH 7.4 in HEPES [4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid] and PBS (phosphate-buffered saline) buffers. The adsorbed layer of copolymer is thought to form a comblike structure at the surface, with positively charged primary amine groups of the PLL bound to the negatively charged metal oxide surface, while the hydrophilic and uncharged PEG side chains are exposed to the solution phase. Copolymer architecture is an important factor in the resulting protein resistance; it is discussed on the basis of packing-density considerations and the corresponding radii of gyration of the different PEG chain lengths studied. This surface functionalization technology is believed to be of value for use in both the biomaterial and biosensor areas, as the chosen macromolecules are biocompatible and the application is straightforward and cost-effective.
Silicon nanowire (Si NW)-based field effect transistors (FETs) have shown great potential as biosensors (bioFETs) for ultra-sensitive and label-free detection of biomolecular interactions. Their sensitivity depends not only on the device properties, but also on the function of the biological recognition motif attached to the Si NWs. In this study, we show that SiNWs can be chemically functionalized with Ni:NTA motifs, suitable for the specific immobilization of proteins via a short polyhistidine tag (His-tag) at close proximity to the SiNW surface. We demonstrate that the proteins preserve their function upon immobilization onto SiNWs. Importantly, the protein immobilization on the Si NWs is shown to be reversible after addition of EDTA or imidazole, thus allowing the regeneration of the bioFET when needed, such as in the case of proteins having a limited lifetime. We anticipate that our methodology may find a generic use for the development of bioFETs exploiting functional protein assays because of its high compatibility to various types of NWs and proteins.
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.
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.
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.
Boron-doped silicon nanowires (SiNWs) were used to create highly sensitive, real-time electrically based sensors for biological
and chemical species. Amine- and oxide-functionalized SiNWs exhibit pH-dependent conductance that was linear over a large
dynamic range and could be understood in terms of the change in surface charge during protonation and deprotonation. Biotin-modified
SiNWs were used to detect streptavidin down to at least a picomolar concentration range. In addition, antigen-functionalized
SiNWs show reversible antibody binding and concentration-dependent detection in real time. Lastly, detection of the reversible
binding of the metabolic indicator Ca2+ was demonstrated. The small size and capability of these semiconductor nanowires for sensitive, label-free, real-time detection
of a wide range of chemical and biological species could be exploited in array-based screening and in vivo diagnostics.
We report the selective and real-time detection of label-free DNA using an electronic readout. Microfabricated silicon field-effect sensors were used to directly monitor the increase in surface charge when DNA hybridizes on the sensor surface. The electrostatic immobilization of probe DNA on a positively charged poly-l-lysine layer allows hybridization at low ionic strength where field-effect sensing is most sensitive. Nanomolar DNA concentrations can be detected within minutes, and a single base mismatch within 12-mer oligonucleotides can be distinguished by using a differential detection technique with two sensors in parallel. The sensors were fabricated by standard silicon microtechnology and show promise for future electronic DNA arrays and rapid characterization of nucleic acid samples. This approach demonstrates the most direct and simple translation of genetic information to microelectronics.
Semiconducting nanowires have the potential to function as highly sensitive and selective sensors for the label-free detection of low concentrations of pathogenic microorganisms. Successful solution-phase nanowire sensing has been demonstrated for ions, small molecules, proteins, DNA and viruses; however, 'bottom-up' nanowires (or similarly configured carbon nanotubes) used for these demonstrations require hybrid fabrication schemes, which result in severe integration issues that have hindered widespread application. Alternative 'top-down' fabrication methods of nanowire-like devices produce disappointing performance because of process-induced material and device degradation. Here we report an approach that uses complementary metal oxide semiconductor (CMOS) field effect transistor compatible technology and hence demonstrate the specific label-free detection of below 100 femtomolar concentrations of antibodies as well as real-time monitoring of the cellular immune response. This approach eliminates the need for hybrid methods and enables system-scale integration of these sensors with signal processing and information systems. Additionally, the ability to monitor antibody binding and sense the cellular immune response in real time with readily available technology should facilitate widespread diagnostic applications.
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.
A multiwalled carbon nanotube (MWCNT)-based electrochemical biosensor is
developed for monitoring microcystin-LR (MC-LR), a toxic cyanobacterial toxin,
in sources of drinking water supplies. The biosensor electrodes are fabricated
using vertically well-aligned, dense, millimeter-long MWCNT arrays with a
narrow size distribution, grown on patterned Si substrates by water-assisted
chemical vapor deposition. High temperature thermal treatment (2500 ° C) in
an Ar atmosphere is used to enhance the crystallinity of the pristine materials,
followed by electrochemical functionalization in alkaline solution to
produce oxygen-containing functional groups on the MWCNT surface, thus
providing the anchoring sites for linking molecules that allow the immobilization
of MC-LR onto the MWCNT array electrodes. Addition of the monoclonal
antibodies specifi c to MC-LR in the incubation solutions offers the required
sensor specifi city for toxin detection. The performance of the MWCNT array
biosensor is evaluated using micro-Raman spectroscopy, including polarized
Raman measurements, X-ray photoelectron spectroscopy, cyclic voltammetry,
optical microscopy, and Faradaic electrochemical impedance spectroscopy. A
linear dependence of the electron-transfer resistance on the MC-LR concentration
is observed in the range of 0.05 to 20 μ g L − 1 , which enables cyanotoxin
monitoring well below the World Health Organization (WHO) provisional
concentration limit of 1 μ g L − 1 for MC-LR in drinking water.
Chemical surface modification of oxide-covered surfaces by silanization is a well-known technique in such fields as gas and liquid chromatography, electro-chemistry and immobilization of biomolecules. In the commonly used silanization technique the silane is reacted with the surfaces in a liquid phase. If strict anhydrous conditions do not prevail, however, this technique often results in polymerization, irreproducibility and instability of the silane films. We report here on a gas phase silanization of silicon surfaces at elevated temperatures. The method comprises a washing and surface activation step followed by silanization at about 0.5–1 Nm−2 and 80–190 °C depending on the type of silane. The silanized surfaces were characterized by ellipsometry, contact angle measurements and scanning electron microscopy, which revealed smooth, stable and reproducible silane films of monolayer character. A comparison of surfaces that were silanized in the gas phase with those that were silanized in the liquid phase was also made.
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.
This work describes n-type self-assembled monolayer field-effect transistors (SAMFETs) based on a perylene derivative which is covalently fixed to an aluminum oxide dielectric via a phosphonic acid linker. N-type SAMFETs spontaneously formed by a single layer of active molecules are demonstrated for transistor channel length up to 100 μm. Highly reproducible transistors with electron mobilities of 1.5 × 10−3 cm2 V−1 s−1 and on/off current ratios up to 105 are obtained. By implementing n-type and p-type transistors in one device, a complimentary inverter based solely on SAMFETs is demonstrated for the first time.
The kinetics of adsorption and competitive desorption of wild-type streptavidin (WT SA) and three genetically engineered mutants (S27A, N23E, and W120A) was studied at gold surfaces functionalized with mixed alkylthiolates, some terminated with biotin headgroups and the rest with oligo(ethylene oxide) using surface plasmon resonance (SPR). The saturation coverage of the protein varied strongly with surface biotin concentration (XBAT) and was independent of mutation (except at very low and very high XBAT, where a weak dependence was seen). Initial adsorption rates were nearly diffusion-limited except at extremely low XBAT, where the rate varied weakly between mutants in accordance with their differing strengths of binding to biotin. Initial sticking probabilities were estimated to be between 1−6 × 10-6 per collision with the surface. The adsorbed SA desorbs upon introduction of solution-phase biotin. For XBAT below 1%, the desorption rate constants of the SA variants closely follow their off-rate constants measured in homogeneous solution (which at 25 °C are WT = 4 × 10-6 sec-1, N23E = 1.6 × 10-3 sec-1, S27A = 1.2 × 10-3 sec-1, and W120A estimated to be ca. 23 s-1). This proves that SA is mainly bound to the surface by a single SA-biotin link at very low XBAT. Importantly, for XBAT between 10 and 40%, where desorption is 30- to >1000-fold slower and the saturation coverage maximizes, the ratios of off-rate constants between mutants (W120A/N23E and W120A/S27A) are approximately the square of their ratios for XBAT below 1%. This squaring strongly suggests that the dominant species at these coverages is doubly bounded SA (i.e., immobilized via two surface biotins). The kinetics are explained with a mechanism involving only two first-order rate constants, that is, for (1) the slow dissociation of any bond between an SA site and a surface-immobilized biotin and (2) the fast reforming of this bond in the special case that it was released from a doubly bonded SA whose other site is still linked to one surface-immobilized biotin. The rate constant for (2) is almost independent of the SA mutant, as it is for adsorption. For XBAT > 60%, the desorption rates again approach the singly bound SA values, and the ratios of rate constants for the SA variants drop to slightly less than below 1% biotin. This is due to the dominance of singly bonded SA, plus a contribution from nonspecific binding, consistent with structural studies of these alkylthiolate films.
This paper reports a study of the adsorption of four proteins-fibrinogen, lysozyme, pyruvate kinase, and RNAse A-to self-assembled monolayers (SAMs) on gold. The SAMs examined were derived from thiols of the structure HS(CH2)10R, where R was CH3, CH2OH, and oligo(ethylene oxide). Monolayers that contained a sufficiently large mole fraction of alkanethiolate groups terminated in oligo(ethylene oxide) chains resisted the kinetically irreversible, nonspecific adsorption of all four proteins. Longer chains of oligo(ethylene oxide) were resistant at lower mole fractions in the monolayer. Resistance to the adsorption of proteins increased with the length of the oligo(ethylene oxide) chain: the smallest mole fraction of chains that prevented adsorption was proportional to n-0.4, where n represents the number of ethylene oxide units per chain. Termination of the oligo(ethylene oxide) chains with a methoxy group instead of a hydroxyl group had little or no effect on the amount of protein adsorbed. The amount of pyruvate kinase that adsorbed to mixed SAMs containing hexa(ethylene oxide)-terminated chains depended upon the temperature. When the mole fraction of oligo(ethylene oxide) groups in the monolayer was below the level needed to prevent adsorption, more pyruvate kinase adsorbed to the monolayer at 37-degrees-C than at 25-degrees-C. No difference was observed between adsorption at 25 and 4-degrees-C.
Recent studies have demonstrated the ability of semiconducting nanowire (NW) field-effect transistors (FETs) to serve as highly sensitive label-free sensors for biochemicals, including small molecules, proteins, and nucleic acids. The nanoscale confinement of the channel current in concert with the large-surface area-to-volume ratio enables charged molecules bound to the surface to effectively gate the device. Functionalization of the NW surface with specific receptors therefore enables direct electronic detection of particular molecules of interest. The original work in the field relied on NWs grown by the chemical vapor deposition method, which require hybrid bottom-up fabrication processes for device realization. The lack of reproducibility with these techniques and the associated inability to leverage the central advantage of complementary MOSFETs, namely, very large scale integration, have recently led a number of groups to create NW sensors using only traditional top-down fabrication techniques. In this paper, we focus primarily on these most recent studies and discuss necessary future studies as dictated by experimental and theoretical considerations.
Polysilicon nanowire biosensors have been fabricated using a top-down process and were used to determine the binding constant of two inflammatory biomarkers. A very low cost nanofabrication process was developed, based on simple and mature photolithography, thin film technology, and plasma etching, enabling an easy route to mass manufacture. Antibody-functionalized nanowire sensors were used to detect the proteins interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α) over a wide range of concentrations, demonstrating excellent sensitivity and selectivity, exemplified by a detection sensitivity of 10 fM in the presence of a 100,000-fold excess of a nontarget protein. Nanowire titration curves gave antibody-antigen dissociation constants in good agreement with low-salt enzyme-linked immunosorbent assays (ELISAs). This fabrication process produces high-quality nanowires that are suitable for low-cost mass production, providing a realistic route to the realization of disposable nanoelectronic point-of-care (PoC) devices.
Five functional silanes--3-aminopropyltriethoxysilane (APTES), 3-aminopropyltrimethoxysilane (APTMS), N-(2-aminoethyl)-3-aminopropyltriethoxysilane (AEAPTES), N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEAPTMS), and N-(6-aminohexyl)aminomethyltriethoxysilane (AHAMTES)--were assessed for the preparation of hydrolytically stable amine-functionalized silica substrates. These can be categorized into three groups (G1, G2, and G3) based on the intramolecular coordinating ability of the amine functionality to the silicon center. Silanizations were carried out in anhydrous toluene as well as in the vapor phase at elevated temperatures. Aminosilane-derived layers prepared in solution are multilayers in nature, and those produced in the vapor phase have monolayer characteristics. In general, vapor-phase reactions are much less sensitive to variations in humidity and reagent purity, are more practical than the solution-phase method, and generate more reproducible results. Intramolecular catalysis by the amine functionality is found to be important for both silanization and hydrolysis. The primary amine group in the G1 silanes (APTES and APTMS) can readily catalyze siloxane bond formation and hydrolysis to render their silane layers unstable toward hydrolysis. The amine functionality in the G3 silane (AHAMTES) is incapable of intramolecular catalysis of silanization so that stable siloxane bonds between the silane molecules and surface silanols do not form easily. The secondary amine group in the G2 silanes (AEAPTES and AEAPTMS), on the other hand, can catalyze siloxane bond formation, but the intramolecular catalysis of bond detachment is sterically hindered. The G2 silanes are the best candidates for preparing stable amine-functionalized surfaces. Between the two G2 aminosilanes, AEAPTES results in more reproducible silane layers than AEAPTMS in the vapor phase due to its lower sensitivity to water content in the reaction systems.
Patchy polymer brushes contain nanoscale (5-15 nm) adhesive elements, such as polymer coils or nanoparticles, embedded at their base at random positions on the surface. The competition between the brush's steric (protein resistant) repulsions and the attractions from the discrete adhesive elements provides a precise means to control bioadhesion. This differs from the classical approach, where functionality is placed on the brush's periphery. The current study demonstrates the impact of poly(etheylene glycol) (PEG) brush architecture and ionic strength on fibrinogen adsorption on brushes containing embedded poly-l-lysine (PLL, 20K MW) coils or "patches". The consistent appearance of a fibrinogen adsorption threshold, a minimum loading of patches on the surface, below which protein adsorption does not occur, suggests multivalent protein capture: Adsorbing proteins simultaneously engage several patches. The surface composition (patch loading) at the threshold is extremely sensitive to the brush height and ionic strength, varying up to a factor of 5 in the surface loading of the PLL patches (~50% of the range of possible surfaces). Variations in ionic strength have a similar effect, with the smallest thresholds seen for the largest Debye lengths. While trends with brush height were the clearest and most dominant, consideration of the PEG loading within the brush or its persistence length did not reveal a critical brush parameter for the onset of adsorption. The lack of straightforward correlation on brush physics was likely a result of multivalent binding, (producing an additional dependence on patch loading), and might be resolved for univalent adsorption onto more strongly binding patches. While studies with similar brushes placed uniformly on a surface revealed that the PEG loading within the brush is the best indicator of protein resistance, the current results suggest that brush height is more important for patchy brushes. Likely the interactions producing brush extension normal to the interface act similarly to drive lateral tether extension to obstruct patches.
Charge-detecting biosensors have recently become the focal point of biosensor research, especially research onto organic thin-film transistors (OTFTs), which combine compactness, a low cost, and fast and label-free detection to realize simple and stable in vivo diagnostic systems. We fabricated organic pentacene-based bottom-contact thin-film transistors with an ultra-thin insulating layer of a cyclized perfluoro polymer called CYTOP (Asahi Glass Co., Tokyo, Japan) on SiO(2) for operation in aqueous media. The stability and sensitivity of these transistor sensors were examined in aqueous buffer media with solutions of variable pH levels after the passivation of perfluoro polymers with thicknesses ranging from 50 to 300 nm. These transistor sensors were further modified with an ultra-thin film (5 nm) functional layer for selective BSA/antiBSA detection in aqueous buffer media, demonstrating a detection capability as low as 500 nM of concentrated antiBSA. The dissociation constant from the antiBSA detection results was 2.1×10(-6)M. Thus, this study represents a significant step forward in the development of organic electronics for a disposable and versatile chemical and bio-sensing platform.
Within the past years there has been much effort in developing and improving new techniques for the nanoscale patterning of functional materials used in promising applications like nano(opto)electronics. Here a high-resolution soft lithography technique—nanomolding in capillaries (NAMIC)—is demonstrated. Composite PDMS stamps with sub-100 nm features are fabricated by nanoimprint lithography to yield nanomolds for NAMIC. NAMIC is used to pattern different functional materials such as fluorescent dyes, proteins, nanoparticles, thermoplastic polymers, and conductive polymers at the nanometer scale over large areas. These results show that NAMIC is a simple, versatile, low-cost, and high-throughput nanopatterning tool.
A simple dipping process has been used to prepare PEGylated surface gradients from the polycationic polymer poly(L-lysine), grafted with poly(ethylene glycol) (PLL-g-PEG), on metal oxide substrates, such as TiO(2) and Nb(2)O(5). PLL-g-PEG coverage gradients were prepared during an initial, controlled immersion and characterized with variable angle spectroscopic ellipsometry and x-ray photoelectron spectroscopy. Gradients with a linear change in thickness and coverage were generated by the use of an immersion program based on an exponential function. These single-component gradients were used to study the adsorption of proteins of different sizes and shapes, namely, albumin, immunoglobulin G, and fibrinogen. The authors have shown that the density and size of defects in the PLL-g-PEG adlayer determine the amount of protein that is adsorbed at a certain adlayer thickness. In a second step, single-component gradients of functionalized PLL-g-PEG were backfilled with nonfunctionalized PLL-g-PEG to generate two-component gradients containing functional groups, such as biotin, in a protein-resistant background. Such gradients were combined with a patterning technique to generate individually addressable spots on a gradient surface. The surfaces generated in this way show promise as a useful and versatile biochemical screening tool and could readily be incorporated into a method for studying the behavior of cells on functionalized surfaces.
In this perspective, we take a critical look at the research progress within the nanowire community for the past decade. We discuss issues on the discovery of fundamentally new phenomena versus performance benchmarking for many of the nanowire applications. We also notice that both the bottom-up and top-down approaches have played important roles in advancing our fundamental understanding of this new class of nanostructures. Finally we attempt to look into the future and offer our personal opinions on what the future trends will be in nanowire research.
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.
The usefulness of the hybrid materials of nanoparticles and biological molecules in many occasions depends on how well one can achieve a rational design based on specific binding and programmable assembly. Nonspecific binding between nanoparticles and biomolecules is one of the major barriers for achieving its utilities in a biological system. In this paper, we demonstrate a new approach to eliminate nonspecific interactions between nanoparticles and proteins by synthesizing ethylene glycol protected gold nanoparticles. We discovered that with the water content optimized in the range of 9-18% in the reaction mixture, di-, tri-, and tetra(ethylene glycol) protected gold nanoparticles Au-S-EGn (n = 2, 3, and 4) could be directly synthesized. These gold nanoparticles that are bonded with a uniform monolayer with defined length varying from 0.8 to 1.6 nm (from molecular modeling) have great stability in aqueous solutions with a high concentration of electrolyte and organic solutions. Using ion-exchange chromatography and gel electrophoresis, we demonstrated that these Au-S-EGn (n = 2, 3, or 4) nanoparticles have complete resistance to protein nonspecific interactions. These types of nanoparticles provide a fundamental starting material for designing hybrid materials composed of metallic nanoparticles and biomolecules.
Nanowire field effect transistors (NW-FETs) can serve as ultrasensitive detectors for label-free reagents. The NW-FET sensing mechanism assumes a controlled modification in the local channel electric field created by the binding of charged molecules to the nanowire surface. Careful control of the solution Debye length is critical for unambiguous selective detection of macromolecules. Here we show the appropriate conditions under which the selective binding of macromolecules is accurately sensed with NW-FET sensors.
Direct electrical detection of biomolecules at high sensitivity has recently been demonstrated using semiconductor nanowires. Here we demonstrate that semiconductor nanoribbons, in this case, a thin sheet of silicon on an oxidized silicon substrate, can approach the same sensitivity extending below the picomolar concentration regime in the biotin/streptavidin case. This corresponds to less than approximately 20 analyte molecules bound to receptors on the nanoribbon surface. The micrometer-size lateral dimensions of the nanoribbon enable optical lithography to be used, resulting in a simple and high-yield fabrication process. Electrical characterization of the nanoribbons is complemented by computer simulations showing enhanced sensitivity for thin ribbons. Finally, we demonstrate that the device can be operated both in inversion as well as in accumulation mode and the measured differences in detection sensitivity are explained in terms of the distance between the channel and the receptor coated surface with respect to the Debye screening length. The nanoribbon approach opens up for large scale CMOS fabrication of highly sensitive biomolecule sensor chips for potential use in medicine and biotechnology.
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